CN111973601B

CN111973601B - Application of cinnamyl amino quinazoline compound as EGFR (epidermal growth factor receptor) inhibitor in preparation of medicines

Info

Publication number: CN111973601B
Application number: CN201910423934.9A
Authority: CN
Inventors: 袁杰; 饶国武; 陈洁; 吴祎丛; 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: CN111973601A

Abstract

The invention discloses application of a cinnamoylamidoquinazoline compound shown as a formula (I) as an EGFR inhibitor in preparation of drugs for treating or preventing EGFR-mediated diseases or drugs for inhibiting EGFR. The compound provided by the invention has good inhibitory activity on wild type or mutant EGFR or EGFR mutant cancer cells.

Description

Application of cinnamyl amino quinazoline compound as EGFR (epidermal growth factor receptor) inhibitor in preparation of medicines

(I) technical field

The invention relates to an application of a cinnamoylamidoquinazoline compound as an EGFR inhibitor in preparation of drugs for treating or preventing EGFR-mediated diseases or drugs 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 cinnamoylamidoquinazoline compound as an EGFR inhibitor in preparing medicaments for treating or preventing EGFR-mediated diseases or medicaments for inhibiting EGFR.

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

the invention provides an application of a cinnamoylaminoquinazoline compound shown in a formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing EGFR (epidermal growth factor receptor) -mediated diseases or inhibiting EGFR (epidermal growth factor receptor),

examples of pharmaceutically acceptable salts of the cinnamoylaminoquinazoline 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 cinnamyl amino quinazoline compound (I) of the invention is used for treating EGFR^T790MTK has remarkable inhibitory effect, IC₅₀Is 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 superior inhibitory activity against NCI-H1975 cancer cells over existing drugs.

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

Preparation of Compound (II) was carried out by the method described in reference (CN 108014114A).

Example 1: preparation of cinnamyl amido quinazoline (I)

Adding 0.27 g (0.55mmol) of aminoquinazoline (II), 0.13 g (1.64mmol) of pyridine and 20 ml of tetrahydrofuran into a reaction bottle in sequence, dropwise adding 0.733 g (4.40mmol) of cinnamoyl chloride under the stirring condition at-10 ℃, after dropwise adding, performing TLC tracking detection (a developing agent is ethyl acetate/petroleum ether is 1: 1) under the stirring condition at-10 ℃, reacting for 12 hours under the condition of 10 ℃, filtering, evaporating the filtrate to remove the solvent, 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 to remove the solvent to obtain a mixture of dried concentrate and silica gel, filling the mixture into a column, and then performing column chromatography on the mixture according to 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 (I) (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 (I) 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 2: 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) to respectively treat 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).

Set 0, 0.03, 0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 μ MATP standard wells (all ATP concentrations above are final concentrations of the substance when the total volume in the standard wells 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 gefitinib in various concentrations (100, 200, 400, 800, 1000 nM). When preparing, polyglutamic acid and tyrosine (4:1) kinase substrate, ATP, kinase are added into each hole(EGFR^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK or EGFR^T790MTK) and compound (I) or gefitinib, 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 assay are shown in Table 1, and Table 1 shows that compound (I) and gefitinib, a positive drug, are shown to act on EGFR^wt-TK，EGFR^L858R-TK，EGFR^L858R/T790M-TK and EGFR^T790M-activity data of TK kinase. It can be seen from the data in the table that compound (I) has different degrees of inhibition of the four EGFR activities, wherein compound (I) inhibits EGFR^T790MThe TK inhibition is strongest, and the IC thereof₅₀Is superior to gefitinib which is a medicament on the market.

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

Example 3: in vitro test for anti-cancer Activity

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

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, 2. mu.M or 1. mu.M samples diluted with medium were added, 100. mu.L per well, 3 wells per concentration, and the mixture was incubated 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 Compound (I) against cancer cell lines

Claims

1. The application of a cinnamoylamidoquinazoline compound shown as a formula (I) or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing EGFR (epidermal growth factor receptor) mediated diseases, wherein the EGFR mediated diseases are cancers, and cancer cells of the cancers are HCC827, NCI-H1975 or MDA-MB-231 cells;

。