Preparation of 2,4-disubstituted quinazoline compounds and their anti-tumor applications

Abstract

The application belongs to the technical field of antitumor drugs, and particularly relates to a 2,4-disubstituted quinazoline compound for inhibiting EGFR, a preparation method and application. As a 2,4-disubstituted quinazoline compound for targeting EGFR L858R / T790M, the compound comprises a compound shown in the following formula or a pharmaceutically acceptable salt thereof. The compound or the pharmaceutically acceptable salt thereof has the activity of inhibiting EGFR L858R / T790M, can be used as an inhibitor of EGFR L858R / T790M, has the activity of resisting tumors, and can effectively inhibit the growth of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of antitumor pharmaceutical technology, and particularly relates to a 2,4-disubstituted quinazoline compound for inhibiting EGFR, its preparation method, and its application. Background Technology

[0002] Epidermal growth factor receptor (EGFR) is a member of the HER family. EGFR is the expression product of the proto-oncogene c-erbB-1 and is a transmembrane tyrosine kinase receptor. EGFR plays a crucial role in physiological processes such as cell growth, proliferation, and differentiation by influencing downstream signaling pathways such as PI3K / AKT. Loss of EGFR function or abnormal activity can lead to tumors, diabetes, immunodeficiency, and cardiovascular diseases. In particular, EGFR has been identified as a key target for non-small cell lung cancer.

[0003] Existing EGFR tyrosine kinase inhibitors have evolved to third-generation inhibitors with the advantage of binding to EGFR-sensitive mutations and the T790M mutation site; however, the emergence of drug resistance has hindered their ability to exert ideal anti-tumor effects. This invention designs and synthesizes a 2,4-disubstituted quinazoline inhibitor targeting EGFR L858R / T790M. This inhibitor selectively inhibits the EGFR L858R / T790M protein and downstream signaling pathways, and exhibits good anti-proliferative activity in the corresponding tumor cells.

[0004] Chinese patent CN 201010179803.X describes a 2,4-disubstituted quinazoline compound, its preparation method, pharmaceutical composition, and uses. The compound, its precursor, stereoisomers, and physiologically acceptable salts are represented by general formula (I): It primarily inhibits the Pin1 enzyme.

[0005] Pin1 inhibitors typically target the C-terminal catalytic domain, which specifically isomerizes the pSer / Thr-Pro amide bond, inducing conformational and functional changes in the protein. Therefore, these inhibitors can disrupt pin1 function. Another type of inhibitor targets the N-terminal WW domain of pin1, preventing the WW domain from binding to substrates containing the pSer / Thr-Pro peptide, thus rendering pin1 ineffective.

[0006] EGFR inhibitors typically target the kinase domain located inside the cell, which is closely related to the kinase's own activity. These compounds inhibit EGFR phosphorylation by targeting ATP binding sites or allosteric sites within them, thereby affecting downstream signaling pathways and related functions.

[0007] Pin1 inhibitors and EGFR inhibitors target different structural domains. Pin1 inhibitors target the catalytic and WW domains of the Pin1 protein, while EGFR inhibitors target the intracellular kinase domain of the transmembrane protein EGFR. Furthermore, these two types of inhibitors act differently. Pin1 inhibitors work by preventing Pin1 from binding to substrates or disrupting the function of its isomerized pSer / Thr-Pro substrate, while EGFR inhibitors inactivate EGFR by inhibiting its phosphorylation, thus preventing its activation of downstream substrates.

[0008] Therefore, inhibitors that can suppress the effects of EGFR are needed. Summary of the Invention

[0009] To address the above technical problems, this invention provides a 2,4-disubstituted quinazoline compound for inhibiting EGFR, its preparation method, and its application, serving as a 2,4-disubstituted quinazoline inhibitor targeting EGFR L858R / T790M. It exhibits good anti-proliferative activity in corresponding tumor cells that highly express EGFR L858R / T790M.

[0010] The present invention provides compounds as shown below or pharmaceutically acceptable salts thereof:

[0011]

[0012] The present invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0013] Furthermore, the antitumor drug is preferably an inhibitor targeting EGFR L858R / T790M.

[0014] The antitumor drug is preferably an antitumor drug, and the corresponding tumor is a tumor with EGFR L858R / T790M overexpression characteristics.

[0015] The compounds prepared by this invention, or their pharmaceutically acceptable salts, can act as inhibitors targeting EGFR L858R / T790M, exhibiting antitumor activity and effectively inhibiting the growth of cancer cells. The compounds of this invention have inhibitory effects on tumor cells exhibiting EGFR L858R / T790M overexpression, particularly showing good inhibitory effects on non-small cell lung cancer. Attached Figure Description

[0016] Figure 1 The results of the antiproliferative activity test of the compound shown in Formula I against NCI-H1975, HCC827, A549, and A431 cells are as follows. Detailed Implementation

[0017] The present invention provides compounds as shown below or pharmaceutically acceptable salts thereof:

[0018]

[0019] The present invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0020] Furthermore, the antitumor drug is preferably an inhibitor targeting EGFR L858R / T790M.

[0021] The antitumor drug is preferably an antitumor drug, and the corresponding tumor is a tumor with EGFR L858R / T790M overexpression characteristics.

[0022] The present invention also provides a pharmaceutical composition comprising an effective dose of the above-described compound or a pharmaceutically acceptable salt thereof.

[0023] The compounds of the present invention can be formulated into the following forms by methods known in the art: tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely dispersed powders or aerosols or sprays for inhalation, and sterile aqueous or oily solutions or suspensions or sterile emulsions for parenteral (including intravenous, intramuscular, or infusion) administration. Liquid formulations can be prepared using sterile water or a water-propylene glycol solution as a solvent, and the active ingredients can also be formulated in an aqueous polyethylene glycol solution. Aqueous solutions for oral administration can be prepared by dissolving the active ingredients in water and adding suitable colorants, flavoring agents, stabilizers, and thickeners as needed. Aqueous suspensions for oral use can be prepared by dispersing the finely dispersed active ingredients together with a viscous substance in water, such as natural synthetic gums, resins, methylcellulose, carboxymethylcellulose, and other suspending agents known in the pharmaceutical field.

[0024] The pharmaceutical composition may be in unit dose form. In these forms, the composition is divided into unit doses containing an appropriate amount of the active ingredient. The unit dose form may be a packaged formulation comprising a portion of the formulation, such as boxed tablets, capsules, and powders in tubular vials or ampoules. The unit dose form may also be capsules, sachets, or tablets, or any of these packaging forms in appropriate quantities.

[0025] The active ingredient of the pharmaceutical composition of the present invention may be the compound of the present invention alone, or it may be combined with other antitumor compounds as the active ingredient.

[0026] In the treatment of tumors, the pharmaceutical compositions of the present invention can be used in combination with other antitumor drugs. For example, they can be used in combination with antiproliferative / antitumor drugs, cell growth inhibitors, anti-invasion drugs, growth factor function inhibitors, anti-angiogenic agents, and vascular damaging agents used in medical oncology.

[0027] In the treatment of tumors, such combination therapy can be achieved by administering various therapeutic components simultaneously, sequentially, or individually. Such combination products utilize compounds of the present invention within their effective dose range and other pharmaceutically active agents within their permitted dose range.

[0028] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0029] Example 1: Synthesis of the compound

[0030] The compound was synthesized using the following reaction:

[0031]

[0032] (i) DIPEA, i-PrOH, 80°C; (ii) TEA, THF, 0-25°C; (iii) TFA, 2-butanol, 80°C; (iv) K2CO3, DMF, 80°C; (v) Ranny-Ni, Hydrazine hydrate, MeOH, 0-25°C; (vi) TEA, THF, 0-25°C.

[0033] 1. General method for synthesizing intermediates 1-5

[0034] 2,4-Dichloroquinazoline (1.99 g, 10.0 mmol) was dissolved in 60 mL of isopropanol and stirred at room temperature. Then, p-phenylenediamine (0.86 g, 8.0 mmol) and DIPEA (6.61 mL, 40.0 mmol) were added sequentially. The reaction mixture was then heated to 80 °C and reacted for four hours. After TLC detection of complete reaction, the mixture was filtered under reduced pressure to obtain a yellow solid. The crude product was separated by column chromatography (petroleum ether / ethyl acetate, 2 / 1) to give intermediate 1 (2.11 g, yield 78%).

[0035] Intermediate 1 (2.11 g, 7.8 mmol) was dissolved in 30 mL of tetrahydrofuran and stirred in an ice bath. p-Fluorophenylacetyl chloride (1.88 g, 10.9 mmol) was added, followed by dropwise addition of triethylamine (1.58 g, 15.6 mmol). The mixture was then heated to 25 °C and reacted for 2 hours. After TLC detection to confirm complete reaction, the mixture was concentrated under reduced pressure. The crude product was separated by column chromatography (petroleum ether / ethyl acetate, 2 / 1) to obtain intermediate 2 (1.78 g, yield 56%).

[0036] Intermediate 2 (0.41 g, 1 mmol), 4-fluoro-3-nitroaniline (0.12 g, 0.8 mmol), and trifluoroacetic acid (222 μL, 3 mmol) were dissolved in sec-butanol (6 mL). The reaction mixture was then heated to 80 °C and stirred for 4 hours. After the reaction was complete as indicated by TLC, intermediate 3 (0.29 g, yield 56%) was obtained by column chromatography (dichloromethane / methanol, 200 / 1).

[0037] Intermediate 3 (0.29 g, 0.56 mmol), N-methylpiperazine (0.07 g, 0.73 mmol), and potassium carbonate (0.28 g, 2 mmol) were dissolved in sec-butanol (6 mL). The reaction mixture was then heated to 100 °C and stirred for 12 hours. After the reaction was complete as indicated by TLC, intermediate 4 (0.27 g, 80% yield) was obtained by column chromatography (dichloromethane / methanol, 100 / 1).

[0038] Intermediate 4 (0.27 g, 0.46 mmol), hydrazine hydrate (58 μL, 1.87 mmol), and Raney nickel (0.06 g, 0.94 mmol) were added to methanol and reacted under ice bath conditions. When the reaction was complete as detected by TLC, the mixture was filtered under reduced pressure, concentrated, and purified by column chromatography to obtain intermediate 5 (0.22 g), a yellow solid, with a yield of 82%.

[0039] 2. Synthesis method of compound 1

[0040] Acryloyl chloride (58 μL, 0.76 mmol) and TEA (158 μL, 1.14 mmol) were added to a solution of tetrahydrofuran (4 mL) containing intermediate 5 (0.22 g, 0.38 mmol). The mixture was stirred in an ice bath, and TLC analysis showed that the reaction was complete. The solution was concentrated under reduced pressure and separated by column chromatography (dichloromethane / methanol, 100 / 1) to give a yellow solid compound 1 (0.17 g, 70% yield).

[0041] Compound 1, yellow solid, 70% yield; 1H NMR (400MHz, DMSO-d6) δ10.20 (s, 1H), 9.49 (s, 1H), 9.10 (s, 1H), 9.02 (s, 1H), 8.47 (s, 1H), 8.37 (d, J=8.2Hz, 1H), 7. 92 (d, J=8.6Hz, 2H), 7.76 (d, J=6.2Hz, 1H), 7.63 (dd, J=11.4, 8.1Hz, 3H), 7.46 (d, J=8.3Hz, 1H), 7.40 (dd, J=8.5, 5.7 Hz, 2H), 7.25 (t, J=7.6Hz, 1H), 7.17 (t, J=8.9Hz, 2H), 7.10 (d, J=8.8Hz, 1H), 6.59 (dd, J=17.0, 10.3Hz, 1H), 6.24 (d, J=15.2Hz, 1H), 5.76 (d, J=11.9Hz, 1H), 3.66 (s, 2H), 3.30 (dd, J=8.3, 2.8Hz, 4H), 2.79 (t, J=4.7Hz, 4H), 2.25 (s, 3H). 13 C NMR (101MHz, DMSO) δ169.2, 162.8, 160.4, 158.4, 156.9, 152.1, 137.8, 135.4, 135.2, 133.3, 132.7, 132. 6, 131.5, 131.4, 126.8, 126.1, 122.8, 122.1, 120.2, 119.7, 115.6, 115.3, 112.3, 55.5, 52.2, 46.3, 42.7.

[0042] Experimental Example 1: Antiproliferative assay of compound 1 against NCI-H1975, HCC827, A549, and A431 cells.

[0043] Antiproliferative activity assays were performed in NCI-H1975, HCC827, A549, and A431 cells:

[0044] H1975, HCC827, A549, and A431 cells in logarithmic growth phase were seeded into 96-well plates (approximately 5 × 10³ cells per well). After 24 hours of culture and cell adhesion, specific concentrations (0-40 μM) of each compound were added and incubated for another 24 hours. MTT was dissolved in PBS to a final concentration of 0.5%, and then 20 μL of MTT solution was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator in the dark for 3-4 hours. The culture medium was then discarded, and 150 μL of DMSO was added to each well. The OD values ​​at 490 nM were measured using a microplate reader, and the effects of each compound on cell proliferation were analyzed using SPSS to assess the antiproliferative activity of each compound.

[0045] NCI-H1975, HCC827, A549, and A431 cells were treated with compound 1 from this invention, and the half-maximal inhibitory concentration (IC50) against different tumor cell types was determined. The corresponding OD values ​​were measured using a microplate reader, and the antiproliferative activity was evaluated using the MTT assay.

[0046] See results Figure 1 ,from Figure 1 It can be clearly seen that this compound has significant and strong anti-proliferative activity against cells expressing EGFR L858R / T790M, with half-maximal inhibitory concentrations (IC50) of 2.76±0.32 μM, 3.66±0.13 μM, 15.0±1.45 μM, and 14.1±1.02 μM against NCI-H1975, HCC827, A549, and A431 cells, respectively.

[0047] Based on the above experiments, we obtained an inhibitor that can effectively suppress the anti-cancer effect of EGFR L858R / T790M. This type of inhibitor can provide a good method for treating various tumors that overexpress EGFR L858R / T790M, including non-small cell lung cancer. The development of such inhibitors has a very broad research prospect.

Claims

1. A 2,4-disubstituted quinazoline compound for inhibiting EGFR, characterized in that: Provide the general structural formula of the compound shown below or a pharmaceutically acceptable salt thereof: 。 2. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug, wherein the tumor cells are NCI-H1975 or HCC827 cells.

3. The use according to claim 2, characterized in that: The anti-tumor drug is an inhibitor targeting EGFR L858R / T790M.

4. The use according to claim 3, characterized in that: The antitumor drug is a drug for treating tumors that exhibit EGFR L858R / T790M overexpression.

5. A pharmaceutical composition, characterized in that: It is a formulation comprising an effective dose of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

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