CN111973602A - Application of morpholinyl acetamido quinazoline compound as EGFR (epidermal growth factor receptor) inhibitor - Google Patents

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 morpholinoacetamidoquinazolines as EGFR inhibitors

(1) Technical field

The invention relates to a quinazoline compound and application thereof, in particular to a morpholinoacetamidoquinazoline compound used as an EGFR inhibitor in the preparation of a drug for treating or preventing EGFR-mediated diseases or a drug for inhibiting EGFR Applications.

(2) Background technology

Quinazoline compounds have many good biological activities and are widely used in the field of medicine, especially some quinazoline derivatives with special structures have obvious antiviral activity, antibacterial activity, antitumor activity, etc. Some types of compounds have been marketed as antitumor drugs. For example, Gefitinib (Gefitinib), Erlotinib (Erlotinib), and Lapatinib (Lapatinib), which are marketed for the treatment of cancer, all belong to quinazoline 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, ChemMedChem 2013, 8, 136-148; A. Garofalo, A. Farce, S. Ravez, A. Lemoine, P. Six, P. Chavatte, L. Goossens, P. Depreux, J . Med. Chem. 2012, 55, 1189-1204). Of course, most quinazoline compounds do not have antitumor activity.

EGFR is an epidermal growth factor receptor on the surface of cell membranes, which can effectively inhibit tumor growth and proliferation by inhibiting the active expression of protein tyrosine kinases. EGFR is a constitutively expressed component of many normal epithelial tissues, such as skin and hair follicles, and is overexpressed or overexpressed in most solid tumors. For example, the expression rate of EGFR in lung cancer reaches 40-80%. Therefore, selectively inhibiting EGFR and interfering with its mediated signal transduction pathway can achieve the purpose of treating lung cancer. The early results of EGFR inhibitors are encouraging, and most patients with activating mutations in NSCLC respond well to early treatment. But patients developed resistance to treatment with the first epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) within months. The most common mechanism of acquired resistance is the secondary T790M (threonine substitution methionine) target-controlled mutation in exon 20, which accounts for 60% of all mutations. The emergence of drug resistance not only reduces the sensitivity of patients to drugs, but also greatly reduces the quality of life of cancer patients.

To overcome drug resistance caused by the T790M mutation, several second- and third-generation irreversible EGFR-TKIs have been developed, but they are less selective. Therefore, research and development of novel EGFR inhibitors that selectively inhibit T790M mutation and overcome clinical drug resistance have important clinical significance and application prospects.

(3) Contents of the invention

The object of the present invention is to provide a new type of quinazoline compound-morpholinoacetamidoquinazoline compound as an EGFR inhibitor in the preparation of a drug for treating or preventing EGFR-mediated diseases or a drug for inhibiting EGFR.

For realizing the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

The present invention provides a morpholinoacetamidoquinazoline compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing EGFR-mediated diseases or a drug for inhibiting EGFR application,

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 with bases such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N - Inorganic and organic base salts of methylglucamine.

The EGFR described in the present invention includes but is not limited to: EGFR ^wt -TK, EGFR ^L858R -TK, EGFR ^L858R/T790M -TK and EGFR ^T790M -TK kinases. The morpholinoacetamidoquinazoline compound (I) of the present invention has a significant inhibitory effect on EGFR ^wt -TK, EGFR ^L858R -TK and EGFR ^T790M -TK, especially the IC ₅₀ of EGFR ^T790M -TK is obvious than gefitinib.

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

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

(4) Specific implementation methods

The present invention will be further described with reference to specific embodiments. The following embodiments are intended to illustrate the present invention, but not to limit the present invention in any way.

For comparison, compound (II) was prepared in the present invention. The preparations of compounds (III) and (IV) were obtained by referring to the method in the literature (CN108014114A).

Example 1: Preparation of morpholinoacetamidoquinazoline (I)

3.25 g (5.73 mmol) of chloroacetamidoquinazoline (IV), 0.598 g (6.86 mmol) of morpholine, 3.626 g (45.84 mmol) of pyridine, and 32.5 ml of methanol were added to a 50-ml reaction flask in turn, and heated to 40° C. , TLC tracking detection (developing solvent is ethyl acetate/petroleum ether=1:1 (v/v)), stirring the reaction for 10 hours, closing the reaction, the reaction solution is evaporated to remove the solvent, and 10 ml of ethyl acetate is added to the obtained concentrate Dissolve it to obtain a dissolving solution, add 1.5 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) to the dissolving solution, and after mixing, evaporate the solvent to obtain a mixture of dry concentrate and silica gel. Pack the column, and then use the petroleum ether/ethyl acetate mixed solution with a volume ratio of 1:10 as the eluent, elution, and TLC tracking detection (developing solvent is ethyl acetate/petroleum ether=1:1 (v/v) ), according to TLC detection, collect the eluate containing the compound shown in formula (I) (Rf value is 0.5), the collected solution is concentrated, and dried at 50 ° C to obtain the off-white solid product shown in formula (I), the yield is 65%, Melting point 122 ~ 125 ℃. ¹ H NMR (500MHz, CDCl ₃ )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 cinnamoamidoquinazoline (II)

0.27 g (0.55 mmol) of aminoquinazoline (III), 0.13 g (1.64 mmol) of pyridine, and 20 ml of tetrahydrofuran were successively added to the reaction flask, and 0.733 g (4.40 mmol) of cinnamoyl chloride was added dropwise with stirring at -10°C, After dripping, TLC tracking detection (developing solvent is ethyl acetate/petroleum ether=1:1), reacted at -10°C for 12 hours, filtered, the filtrate was evaporated to remove the solvent, and 10 ml of ethyl acetate was added to the concentrate to dissolve it, A dissolving solution is obtained, 0.60 g of column chromatography silica gel (300-400 mesh column chromatography silica gel) is added to the dissolving solution, after mixing, the solvent is evaporated to obtain a mixture of dry concentrate and silica gel, the mixture is packed into a column, and then Take petroleum ether/ethyl acetate mixed solution with a volume ratio of 1:10 as the eluent, elution, and TLC tracking detection (developing solvent is ethyl acetate/petroleum ether=1:1 (v/v)), according to TLC The eluate containing the compound represented by formula (II) (Rf value is 0.5) was detected and collected, the collected solution was concentrated, and dried at 50 °C to obtain the off-white solid of cinnamoamidoquinazoline represented by formula (II), with a yield of 68% , the melting point of 222 ~ 224 ℃. ¹ H NMR (500 MHz, CDCl ₃ ) δ: 3.28-3.34 (m, 1H), 3.54 (dt, J=3.5, 15.0 Hz, 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 ^TM chemiluminescence kinase activity detection kit is a kit for quantitatively detecting kinase activity by measuring the remaining amount of ATP in the solution after the kinase reaction by chemiluminescence method. In this experiment, Kinase-Lumi ^TM chemiluminescence kinase activity detection kit (Beyotime) was used to test compounds (I) and (II) at room temperature for EGFR ^wt -TK, EGFR ^L858R -TK, EGFR ^L858R/T790M -TK, respectively. and inhibitory activity of EGFR ^T790M -TK kinase.

The following are the recommended assay systems for 96-well plates.

(1) Preparation of ATP standard curve

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

Set 0, 0.03, 0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5, 10 μM ATP standard wells (all ATP concentrations above are based on the final concentration of the substance when the total volume in the standard well reaches 100 μL). When preparing, firstly, 50 μL per well, ATP was diluted with reaction buffer. Then add 50 μL Kinase-Lumi ^TM chemiluminescence kinase detection reagent and mix well. The reaction was carried out at room temperature (about 25°C) for 10 minutes, and then chemiluminescence detection was carried out with a multi-function microplate reader to prepare an ATP standard curve.

(2) Sample detection

Sample wells were configured so that each well contained 0.1 μg/mL polyglutamic acid and tyrosine (4:1) kinase substrate, 5 μM ATP and 10 μg/L kinase (EGFR ^wt -TK, EGFR ^L858R -TK, EGFR ^L858R/T790M -TK or EGFR ^T790M -TK), different concentrations (100, 200, 400, 800, 1000 nM) of compound (I) or compound (II) or gefitinib or lapatinib . When preparing, add polyglutamic acid and tyrosine (4:1) kinase substrate, ATP, kinase (EGFR ^wt -TK, EGFR ^L858R -TK, EGFR ^L858R/T790M -TK or EGFR ^T790M -TK to each well first) ) and compound (I) or compound (II) or gefitinib or lapatinib, add reaction buffer to dilute to a total volume of 50 μL; then add 50 μL Kinase-Lumi ^TM chemiluminescence kinase detection reagent, and mix well. The reaction was carried out at room temperature (about 25°C) for 10 minutes. Chemiluminescence detection was then performed with a multi-function microplate reader. The amount of ATP remaining in the sample wells was calculated from the standard curve, and the enzymatic activity was subsequently calculated from the definition of enzymatic activity. The _IC50 value was finally calculated.

The results of the in vitro kinase activity experiments are shown in Table 1. Table 1 shows that compounds (I) and (II) and the positive drugs gefitinib and lapatinib have positive effects on EGFR ^wt -TK, EGFR ^L858R -TK, EGFR ^L858R/ Activity data for ^T790M -TK and EGFR ^T790M -TK kinases. From the data in the table, it can be seen that compounds (I) and (II) have different degrees of inhibition on the activity of EGFR ^wt -TK, among which compound (I) has the strongest inhibitory effect on EGFR ^wt -TK with an IC ₅₀ of 25.8 nM , better than the marketed drug lapatinib. In addition, compound (I) has no obvious inhibitory effect on EGFR ^L858R/T790M -TK, but has a strong inhibitory effect on the activity of EGFR ^T790M -TK. Its IC ₅₀ is 36.2nM, which is better than the marketed drug gefitinib Ni. Therefore, in vitro kinase activity experiments showed that compound (I) had significant inhibitory effect on EGFR ^wt -TK, EGFR ^L858R -TK and EGFR ^T790M -TK, especially the _IC50 of EGFR ^T790M -TK was significantly better than gefitinib.

Table 1. Inhibitory activity of compounds (I) and (II) on EGFR

Example 4: In vitro test of anticancer activity

The prepared compounds (I) and (II) were tested for the biological activity of HCC827, NCI-H1975 and MDA-MB-231 cancer cell lines, respectively.

Test method: Tetrazolium salt reduction method (MTT method).

Cell lines: HCC827, NCI-H1975, MDA-MB-231. The above tumor cell lines were purchased from the Cell Bank of Shanghai Academy of Biological Sciences, Chinese Academy of Sciences.

The experimental steps are as follows:

(1) Sample preparation: For soluble samples, dissolve each 1 mg with 40 μL DMSO, take 2 μL and dilute it with 1000 μL medium to make the concentration 100 μg/mL, and then serially dilute with medium to the use concentration.

(2) Cell culture

① Preparation of medium: Each 1000 mL of DMEM medium (Gibco) contains 800,000 units of penicillin, 1.0 g of streptomycin, and 10% inactivated fetal bovine serum.

② Cell culture: The tumor cells were inoculated into the medium, cultured in a 37°C, 5% CO ₂ incubator, and passaged for 3-5 days.

③ Determination of the inhibitory effect of samples on tumor cell growth

The 5th passage cells were digested with EDTA-trypsin digestion solution, diluted with medium to 1×10 ⁶ /mL, added to a 96-well cell culture plate, 100 μL per well, and placed in a 37°C, 5% CO ₂ incubator nourish. 24h after inoculation, add 30 μM, 15 μM, 7.5 μM, 4.0 μM, 2 μM or 1 μM samples diluted with culture medium, 100 μL per well, 3 wells for each concentration, and culture in a 37°C, 5% CO ₂ incubator, After 36 h, add 20 μL of 5 mg/mL MTT solution to the cell culture wells, incubate at 37 °C for 3 h, add DMSO, 150 μL per well, shake with a shaker to completely dissolve the formazan, and compare the cells with a microplate reader at a wavelength of 490 nm. color. Taking the cells cultured in the medium containing the same concentration of DMSO without the sample under the same conditions as a control, the IC ₅₀ of the sample on the growth of tumor cells was calculated.

The test results are shown in Table 2:

Table 2. In vitro antitumor activity of compounds (I) and (II) on cancer cell lines

Claims (6)

1. the application of a morpholinoacetamidoquinazoline compound shown in formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating or preventing EGFR-mediated diseases or a medicine for inhibiting EGFR,

2. The use according to claim 1, wherein the EGFR-mediated disease is cancer. 3. The application of claim 2, wherein the cancer is selected from one of the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, prostate cancer, glial Cell tumor, ovarian cancer, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma and solid tumors. 4. The use according to claim 3, wherein the cancer is EGFR-mediated non-small cell lung cancer or breast cancer. 5. The use according to claim 4, wherein the cancer cells are HCC827, NCI-H1975 or MDA-MB-231 cells. 6. application as described in one of claim 1-3 is characterized in that: described EGFR is selected from one of following: EGFR ^wt -TK, EGFR ^L858R -TK, EGFR ^L858R/T790M -TK, EGFR ^T790M -TK .