Inhibitor compound and its application and medicine for treating cancer

Abstract

The present invention belongs to the field of drug synthesis technology, and specifically relates to an inhibitor compound and its application and a drug for treating cancer. The present invention provides an inhibitor compound or a pharmaceutically acceptable salt that can be used to prepare an EGFR kinase inhibitor and a drug for treating or preventing cancer. The present invention also provides a drug for treating cancer comprising the inhibitor compound or a pharmaceutically acceptable salt. The compound or drug provided by the present invention is highly effective and low in toxicity, not only having high anti-tumor activity and effectively inhibiting cancer cell growth, but also maintaining strong brain penetration properties, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug synthesis, and in particular relates to an inhibitor compound and its application and a drug for treating cancer. Background Art

[0002] Epidermal growth factor receptor (EGFR), a proto-oncogene expression product, is a transmembrane glycoprotein composed of an extracellular ligand-binding region, a transmembrane region, and an intracellular region with a tyrosine kinase domain. When the ligand binds to the extracellular region of EGFR, it can activate downstream signaling pathways such as PI3K-AKT and MAPK-Erk, leading to physiological processes such as cell growth, proliferation, and differentiation. However, when EGFR is functionally deficient or its activity is abnormal, it will continuously activate genes associated with tumor proliferation and differentiation, thereby inducing the formation and development of tumors such as lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, and glioblastoma.

[0003] Small molecule targeted EGFR tyrosine kinase inhibitors can act on the intracellular kinase region of EGFR. They can prevent EGFR autophosphorylation by competing with adenosine triphosphate molecules for the binding sites of tyrosine kinase residues, thereby blocking the transmission of EGFR signaling molecules and achieving the effect of inhibiting cancer cell proliferation.

[0004] Gefitinib and erlotinib, as reversible quinazoline EGFR inhibitors, cannot completely inhibit the growth of tumor cells. The EGFR mutations that occur cause tumor cells to develop drug resistance. Also, because these inhibitors cannot effectively cross the blood-brain barrier, they show limited effect in the treatment of patients with non-small cell lung cancer with brain metastases.

[0005] Afatinib and dacomitinib are irreversible quinazoline EGFR inhibitors that can inhibit the phosphorylation of EGF, HER-2 and HER-4 receptors and their subsequent kinase activity. However, cancer patients may experience adverse reactions such as rash and diarrhea during use.

[0006] Zolitinib, as a new generation of irreversible EGFR tyrosine kinase inhibitor, can inhibit the transmission of signal pathways by forming covalent bonds with the tyrosine kinase binding domain, thereby inhibiting the growth of tumor cells. However, it also has adverse reactions such as abnormal liver function. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides an inhibitor compound with strong activity, low toxicity, and the ability to more effectively inhibit tumor growth. At the same time, the inhibitor compound can maintain strong brain penetration properties, enabling it to exert a better therapeutic effect on cancers that have metastasized to the central nervous system, especially those that have metastasized to the brain, as well as cancers that cause leptomeningeal metastasis.

[0008] The present invention provides the following inhibitor compounds or pharmaceutically acceptable salts thereof:

[0009]

[0010] The present invention also provides the use of the above inhibitor compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing cancer.

[0011] The present invention also provides the use of the above inhibitor compound or a pharmaceutically acceptable salt thereof in the preparation of an EGFR kinase inhibitor.

[0012] The present invention also provides a drug for treating cancer, which contains the above inhibitor compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0013] Furthermore, the pharmaceutically acceptable salt is any one or more of the phosphate, camphorsulfonate, hydrobromide, hydrofluoride, sulfate, nitrate, formate, acetate, propionate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate, methanesulfonate, trifluoromethanesulfonate, toluenesulfonate, and benzenesulfonate of the inhibitor compound.

[0014] Furthermore, the pharmaceutically acceptable salt is the hydrochloride of the inhibitor compound.

[0015] Furthermore, the cancer is lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, glioblastoma, solid tumor, non-small cell lung cancer, papillary renal cell carcinoma or melanoma.

[0016] Furthermore, the inhibitor compound or a pharmaceutically acceptable salt thereof is the main active ingredient.

[0017] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0018] Compared with the existing technology, this solution has the following beneficial effects:

[0019] 1. The present invention provides BBB-7 and BBB-8 compounds that are structurally different from existing quinazoline EGFR inhibitors;

[0020] 2. The BBB-7 and BBB-8 compounds provided by the present invention, or pharmaceutically acceptable salts thereof, are highly effective, low-toxic EGFR inhibitors that not only have high anti-tumor activity and effectively inhibit cancer cell growth, but also have significant inhibitory effects on a variety of tumor cells. Furthermore, they maintain strong brain penetration and can effectively treat cancers that have metastasized to the central nervous system, especially those that have metastasized to the brain, as well as cancers that cause leptomeningeal metastases. These compounds have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the general structural formula of BBB compounds;

[0022] Figure 2 The figure shows the results of tumor growth inhibition in mice in the control group after 21 days of feeding;

[0023] Figure 3 The figure shows the results of tumor growth inhibition in mice in the ametinib group after 21 days of feeding;

[0024] Figure 4 The figure shows the results of tumor growth inhibition in mice in the Zolitinib group after 21 days of feeding;

[0025] Figure 5 The figure shows the results of tumor growth inhibition in mice in the BBB-7 group after 21 days of feeding;

[0026] Figure 6 The figure shows the results of tumor growth inhibition in mice in the BBB-8 group after 21 days of feeding;

[0027] Figure 7 The graph shows the brain photon flux results of mice in the control group and experimental group after 7 days of feeding;

[0028] Figure 8 The graph shows the brain photon flux results of mice in the control group and experimental group after 14 days of feeding;

[0029] Figure 9 The graph shows the brain photon flux results of mice in the control group and experimental group after feeding for 21 days;

[0030] Figure 10 The chemical structure diagram of the BBB series of compounds;

[0031] Figure 11 The following is the staining result of mouse organ tissue after feeding for 21 days. DETAILED DESCRIPTION

[0032] The present invention is further illustrated by the following examples, which are intended to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the present invention.

[0033] Preparation of BBB-7 compounds

[0034] Step 1: Preparation of BBB-3

[0035]

[0036] To a solution of 4-chloro-7-methoxyquinazolin-6-yl acetate (BBB-1, 4 g) in acetonitrile (160 mL) was added 3-bromo-2-fluoroaniline (BBB-2, 3.04 g). The reaction mixture was heated to reflux under stirring for 4 hours and then cooled to room temperature. After filtration, the filter cake was washed with acetonitrile (50 mL) and dried in vacuo to give the HCl salt of 5 (6 g, 86%) as a white solid.

[0037] Step 2: Preparation of BBB-4

[0038]

[0039] Potassium carbonate (4.12 g) was added to a solution of the HCl salt of BBB-3 (6 g) in methanol (40 mL), and the reaction mixture was stirred at 10°C for 2 hours. After filtration, the solid was washed with methanol (20 mL), and the filtrate was concentrated in vacuo to a residue and treated with methyl tert-butyl ether (40 mL). The resulting mixture was filtered and the solid was dried at 40°C to give the potassium salt of compound BBB-4 (4.8 g, 92%).

[0040] Step 3: Preparation of BBB-5

[0041]

[0042] BBB-4 (346 mg), acid chloride (270 mg) and potassium carbonate (276 mg) were added to the reaction flask, and then dry N, N-dimethylformamide (7 mL) was added. The mixture was stirred at room temperature overnight. The reaction was completed by TLC. The mixture was poured into water (20 mL) and filtered. The filter cake was dried under vacuum to obtain the crude product BBB-5 (0.58 g) as a yellow solid. No purification was required and the next step was directly carried out.

[0043] Step 4: Preparation of BBB-6

[0044]

[0045] A solution of hydrogen chloride in 1,4-dioxane (4 M, 2 mL) was added to a solution of BBB-5 (0.58 g) in methanol (2 mL), and the mixture was stirred at room temperature for 1 hour and then concentrated. The residue was diluted with water (5 mL) and neutralized with saturated sodium bicarbonate solution to pH 7. After filtration, the collected solid was treated with toluene (10 mL) and the toluene was evaporated under reduced pressure. This process was repeated twice, and the crude product BBB-6 (412 mg, yellow solid, 86% yield) was used without further purification.

[0046] Step 5: Preparation of BBB-7

[0047]

[0048] Sodium cyanoborohydride (41 mg) was added to a mixture of BBB-6 (147 mg) and acetaldehyde (28.3 mg) in methanol (2 mL). The reaction mixture was stirred at room temperature overnight and then concentrated in vacuo. The residue was treated with water and extracted with ethyl acetate (3×8 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the product was purified by column chromatography with dichloromethane: methanol 100:1 to 15:1.

[0049] 1 H NMR (400MHz, DMSO-d6) δ9.73 (s, 1H), 8.48 (s, 1H), 8.23 ​​(s, 1H), 7.60 (t, J = 7.2 Hz,1H),7.56(t,J=7.5Hz,1H),7.34(s,1H),7.22(t,J=8.0Hz,1H),4.33(br,1H ),3.95(s,3H),3.85(br.1H),3.23(br.1H),2.90(d,J=11.2Hz,1H),2.78(d,J =11.2Hz,1H),2.39-2.30(m,2H),2.11(m,1H),1.92(t,J=11.2Hz,1H),1.34(br 3H), 1.03 (t, J = 7.1 Hz, 3H).

[0050] 13 C NMR(101MHz,DMSO-d6)δ158.1,156.7,155.2,153.8(d,JCF=247Hz),152.9,150.4,140.4,130.7,128.3(d,JCF=13H z), 128.1, 126.0 (d, JCF = 4Hz), 116.9, 109.2 (d, JCF = 20Hz), 108.8, 108.5, 57.2, 56.9, 52.8, 52.0, 48.5, 16.8, 12.4.

[0051] Preparation of BBB-8 compound

[0052] Step 1: Preparation of BBB-3

[0053]

[0054] To a solution of 4-chloro-7-methoxyquinazolin-6-yl acetate (BBB-1, 4 g) in acetonitrile (160 mL) was added 3-bromo-2-fluoroaniline (BBB-2, 3.04 g). The reaction mixture was heated to reflux under stirring for 4 hours and then cooled to room temperature. After filtration, the filter cake was washed with acetonitrile (50 mL) and dried in vacuo to give the HCl salt of 5 (6 g, 86%) as a white solid.

[0055] Step 2: Preparation of BBB-4

[0056]

[0057] Potassium carbonate (4.12 g) was added to a solution of the HCl salt of BBB-3 (6 g) in methanol (40 mL), and the reaction mixture was stirred at 15°C for 2 hours. After filtration, the solid was washed with methanol (20 mL), and the filtrate was concentrated in vacuo to a residue and treated with methyl tert-butyl ether (40 mL). The resulting mixture was filtered and the solid was dried at 45°C to give the potassium salt of compound BBB-4 (4.8 g, 92%).

[0058] Step 3: Preparation of BBB-5

[0059]

[0060] BBB-4 (346 mg), acid chloride (270 mg) and potassium carbonate (276 mg) were added to the reaction flask, and then dry N, N-dimethylformamide (7 mL) was added. The mixture was stirred at room temperature overnight. The reaction was completed by TLC. The mixture was poured into water (20 mL) and filtered. The filter cake was dried under vacuum to obtain the crude product BBB-5 (0.58 g) as a yellow solid. No purification was required and the next step was directly carried out.

[0061] Step 4: Preparation of BBB-6

[0062]

[0063] Hydrogen chloride 1,4-dioxane solution (4M, 2mL) was added to a solution of BBB-5 (0.58g) in methanol (2mL), and the mixture was stirred at room temperature for 1 hour and then concentrated. The residue was diluted with water (5mL) and neutralized with saturated sodium bicarbonate solution to pH 7. After filtration, the collected solid was treated with toluene (10mL) and the toluene was evaporated under reduced pressure. This process was repeated twice, and the crude product BBB-6 (412mg, yellow solid, 86% yield) was used without further purification.

[0064] Step 5: Preparation of BBB-8

[0065]

[0066] Sodium cyanoborohydride (41 mg) was added to a mixture of BBB-6 (147 mg) and paraformaldehyde (19.3 mg) in methanol (2 mL). The reaction mixture was stirred at room temperature overnight and then concentrated in vacuo. The residue was treated with water and extracted with ethyl acetate (3 × 8 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, the product was purified by column chromatography using dichloromethane: methanol 100:1 to 15:1.

[0067] 1 H NMR(400MHz,DMSO-d6)δ9.73(s,1H),8.48(s,1H),8.23(s,1H),7.60(ddd,J=8.6,6.1,1 .6Hz,1H),7.56(m,1H),7.34(s,1H),7.22(t,J=8.0Hz,1H),3.95(s,3H),4.50-4.17(br 1H),4.00-3.70(br,1H),3.40-3.17(br,1H),2.81(d,J=11.2Hz,1H),2.68(d,J=1 1.2Hz, 1H), 2.21 (s, 3H), 2.11 (dd, J = 11.7, 4.0Hz, 1H), 1.91 (m, 1H), 1.35 (br, 3H).

[0068] 13 C NMR(101MHz,DMSO-d6)δ158.1,156.7,155.2,153.8(d,JCF=247Hz),153.0,150.4,140.4,130.7,128.3(d,JCF=1 3Hz), 128.1, 126.0 (d, JCF = 4Hz), 116.9, 109.1 (d, JCF = 20Hz), 108.8, 108.5, 59.5, 56.9, 55.0, 48.4, 46.4, 16.6.

[0069] Experimental study on drug inhibition of lung cancer transplanted tumors in brain

[0070] Balb / c-nude mice (5 weeks old) were fasted and anesthetized with isoflurane before surgery. The mice were placed in a prone position on a stereotaxic apparatus. After routine disinfection, the skull was carefully drilled through the right parietal lobe (3 mm lateral to the sagittal suture and 1 mm in front of the coronal suture) using a 1 mm diameter dental drill. The prepared PC-9-LUC cell suspension (6 × 10 4 / μl) of 5μl, slowly advance the needle 3.5mm vertically into the skull plate, retreat 0.5mm, and slowly (about 1μl / min) inject 3μl of cell suspension. After leaving the needle in place for 1 minute, slowly withdraw the needle, rinse the surgical field with normal saline, suture the scalp, and feed the mice as usual; on the 7th, 14th, and 21st days, small animal in vivo imaging technology was used to track the size of brain tumors in the blank group and experimental (15mg / kg) groups (ametinib group, zolitinib group, BBB-7 group, and BBB-8 group), and the drug efficacy was reflected by the size of the fluorescence signal value, that is, the stronger the fluorescence signal, the more tumor cells there are, and also the larger the tumor volume.

[0071] Figure 2 The figure shows the results of tumor growth inhibition in blank group mice after 21 days of feeding. Figure 3 The figure shows the results of tumor growth inhibition in mice in the Ametinib group after 21 days of feeding. Figure 4 The figure shows the results of tumor growth inhibition in mice in the Zolitinib group after 21 days of feeding. Figure 5 The figure shows the results of tumor growth inhibition in mice in the BBB-7 group after 21 days of feeding. Figure 6 The figure shows the results of tumor growth inhibition in mice in the BBB-8 group after 21 days of feeding; Figure 7 The following is the result of brain photon flux of mice in the blank group and experimental group after feeding for 7 days. Figure 8 The following is the result of brain photon flux of mice in the blank group and experimental group after feeding for 14 days. Figure 9 The graph shows the brain photon flux results of mice in the blank group and experimental group after feeding for 21 days.

[0072] like Figures 2 to 6 As shown, after 21 days of drug treatment, the fluorescence signal intensity of the experimental group mice was weaker than that of the blank group mice, indicating that all four groups of drugs can inhibit the growth of lung cancer brain transplanted tumors in mice.

[0073] During the feeding and drug sampling period, the photon flux values ​​of the mouse brain were further analyzed, e.g. Figures 7 to 9As shown, the brain photon flux of the blank group mice increased with the increase of inoculation time, indicating that the tumor grew well, and the brain photon flux of the experimental group mice decreased with the increase of inoculation time, indicating that taking drugs can inhibit tumor growth.

[0074] Among the four groups of drugs, ametinib is an irreversible third-generation EGFR tyrosine kinase inhibitor, which has been approved for marketing in my country in 2020. T790M has higher selectivity and stronger blood-brain barrier penetration, and can inhibit tumor proliferation by competitively binding to the tyrosine region of EGFR. Zolitinib is an EGFR inhibitor that can effectively penetrate the central nervous system and has excellent blood-brain barrier permeability. Clinical studies have shown that zolitinib can achieve drug concentrations in the brain equivalent to plasma, effectively inhibiting brain tumor growth, reducing brain tumor area, and preventing brain tumor formation. In the pharmacodynamic inhibition experiment of the present invention, compared with the ametinib and zolitinib groups, the brain photon flux values ​​of mice in the BBB-7 and BBB-8 groups were lower, indicating that the BBB-7 and BBB-8 drugs prepared by the present invention have better inhibitory effects on brain transplanted lung cancer tumors in mice than ametinib and zolitinib. In addition, compared with the BBB-7 group, the tumor inhibition level of mice in the BBB-8 group was significantly increased (on the 7th day, p < 0.01; on the 14th day, p < 0.001; on the 21st day, p < 0.05).

[0075] Data from the lung cancer brain transplant tumor model obtained in the experiment showed that compared with ametinib and zolitinib, the BBB-7 and BBB-8 compounds prepared by the present invention can better inhibit tumor growth in mice. Among them, BBB-8 has a stronger tumor inhibition effect on mice, and the tumors in the mice hardly grow.

[0076] Experimental study on the effects of BBB series drugs on cancer cell proliferation

[0077] By testing the effects of BBB series drugs on cancer cell growth, we further evaluated the inhibitory effect of BBB series compounds on cancer cell proliferation:

[0078] First of all, Figure 1 The BBB compound structure formula was modified in various ways to obtain a series of BBB compounds. The obtained BBB series compounds were then added to human lung cancer PC-9 cells at different concentrations (25nM, 50nM, 100nM, 200nM, and 400nM in DMSO) and incubated for 72h. Finally, the absorbance at 450nm was detected in a microplate reader using Cell Counting Kit-8 (CCK-8) reagent to obtain the OD value and the IC 50 .

[0079] Figure 10The chemical structure diagram of BBB series compounds is shown in the figure. Figure 1 The general structural formula of the compound is modified to obtain structural changes of various compounds, wherein R1 and R2 can be independently selected from alkyl, cycloalkyl, heterocycloalkyl, heteroaryl, etc.

[0080] Table 1 shows the effects of BBB series compounds on cancer cell proliferation. After treating human lung cancer cells PC-9 with the above-synthesized BBB series compounds, the corresponding IC values ​​shown in the table were obtained. 50 value.

[0081] The results in Table 1 show the structural formulas of BBB compounds and the measured IC 50 There is a strong uncertainty between the values. For example, in the structural formula of BBB-18 compound, R1 is ethyl and R2 is propyl, while in the structural formula of BBB-19 compound, R1 is ethyl and R2 is butyl. The two differ in structure by only one methylene group, but the IC value of BBB-18 is 50 The IC value of BBB-19 was 77.39 nM. 50 The value is 19.99nM, which is nearly 3 times different. In the structural formula of BBB-13 compound, the R group is a heterocyclic ring, while in the structural formula of BBB-16 compound, the R group is a straight-chain alkane. The two are very different in structure, but the IC value of BBB-13 is 50 The IC value of BBB-16 was 38.35 nM. 50 The value is 36.21nM, and the numerical difference is very small.

[0082] IC of BBB-7 and BBB-8 prepared by the present invention 50 IC values ​​relative to other synthesized BBB compounds 50 The lower values ​​indicate that the chemical structures of BBB-7 and BBB-8 compounds have better inhibitory effects on cancer cell proliferation, among which BBB-8 has the best inhibitory effect.

[0083] Table 1: IC values ​​of BBB series compounds 50

[0084]

[0085] Experiment on the effects of BBB series drugs on mouse organs

[0086] HE staining was used to evaluate the effects of the BBB series of drugs on the heart, liver, spleen, lung, and kidney of mice. After 21 days of feeding with the BBB series of drugs, the heart, liver, spleen, lung, and kidney of mice were obtained, embedded and sectioned, stained with hematoxylin, and finally observed under a microscope.

[0087] Figure 11This is the result of mouse organ tissue staining. As shown in the figure, after the mice were fed with the BBB series of drugs for treatment, the mice's heart, liver, spleen, lungs, and kidneys were not damaged, and the side effects of the BBB series of drugs were relatively small.

[0088] From the above experiments, it can be seen that the inhibitor compound provided by the present invention can be used to reduce or inhibit the activity of EGFR kinase or mutant EGFR kinase in cells or subjects, thereby preventing or treating cell proliferative or EGFR-related diseases. Therefore, BBB-7 and BBB-8 compounds can be prepared into corresponding drugs.

[0089] Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. An inhibitor compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the inhibitor compound is:

2. An inhibitor compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the inhibitor compound is:

3. The inhibitor compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt is any one or more of the phosphate, camphorsulfonate, hydrobromide, hydrofluoride, sulfate, nitrate, formate, acetate, propionate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate, methanesulfonate, trifluoromethanesulfonate, toluenesulfonate, and benzenesulfonate of the inhibitor compound.

4. The inhibitor compound or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salt is the hydrochloride salt of the inhibitor compound.

5. Use of the inhibitor compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: Used in the preparation of drugs for treating or preventing cancer; the cancer is lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, glioblastoma, solid tumor, non-small cell lung cancer, papillary renal cell carcinoma or melanoma.

6. Use of the inhibitor compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: Used in the preparation of EGFR kinase inhibitors.

7. A drug for treating cancer, characterized in that: The medicament contains the inhibitor compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

8. The drug for treating cancer according to claim 7, characterized in that: The main active ingredient is the inhibitor compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

9. The drug according to claim 8, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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