WO2018059022A1 - Tyrosine kinase inhibitor and application thereof - Google Patents

Abstract

A salt compound represented by the following general formula (I), a tyrosine kinase inhibitor comprising the salt compound, and an application of the compound for preparing a drug for treating cancers. The tyrosine kinase inhibitor can inhibit the biological activities of multiple signal transduction kinase, such as C-MET, VEGF, and KDR, can effectively inhibit cell proliferation, has a good therapeutic effect on multiple diseases such as cancers, and in particular, has a significant therapeutic effect on lung cancer, gastric cancer, ovarian cancer, malignant glioma and the like, and thus has broad application prospects.

Description

Tyrosine kinase inhibitors and their applications Technical field

The invention relates to the technical field of medicine, in particular to a tyrosine kinase inhibitor and application thereof.

Background technique

Protein kinase is a phosphotransferase that transfers the gamma phosphate of ATP to a specific amino acid residue to achieve phosphorylation of the protein, thereby achieving its physiological and biochemical functions.

Protein kinases have important functions in information transmission. Abnormal protein kinases are unable to transmit normal signals and may cause pathological changes such as tumor cell proliferation, cell death, inflammation, cardiovascular disease and other protein kinases. Protein kinases fall into two main categories: protein tyrosine kinase PTKs and receptor tyrosine kinase RTKs. ROS1/C-MET in MET family protein kinase is an important sub-family of RTPs, also known as hHGFR and RON; ROS1/C-MET can play an important role in the growth and metabolism of the starting tumor cells. Target of clinical research.

Therefore, ROS1/C-MET kinase inhibitors, especially tyrosine kinase inhibitors of small molecule compounds, are urgently needed in the field of biomedical technology.

Summary of the invention

The technical problem to be solved by the present invention is to provide a tyrosine kinase inhibitor capable of inhibiting various tyrosines involved in signal transduction such as C-MET, VEGF, KDR, RON, KIT, PDGF, FGF, SRC, and the like. The activity of acid kinase can effectively inhibit the proliferation of tumor cells and achieve better results in clinical cancer treatment.

In order to solve the above technical problem, the present invention is implemented by the following technical solutions:

In one aspect of the invention, there is provided a salt compound of the formula (I),

among them,

R1 is selected from the group consisting of halogen, halogen anion, lower halogenated alkane, lower alkane, lower alkene, lower alkyne, substituted saturated or unsaturated cycloalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, substituted or Not substituted a cyclic group, a substituted or unsubstituted heterocycloalkyl group, or an empty group;

Y is selected from C, O, N, S or is empty;

X is selected from the group consisting of carbonate, sulfate, sulfite, hydrogen sulfate, phosphate, phosphite, hydrogen phosphate, dihydrogen phosphate, molybdate, chlorate, or empty.

Preferably, the compound of formula (I) comprises a compound of the following specific structure:

In another aspect of the invention, there is also provided a pharmaceutical composition comprising a safe and effective amount of the above compound and a pharmaceutically acceptable carrier.

The above acceptable carriers are non-toxic, can aid in administration and have no adverse effect on the therapeutic effect of the compound. Such carriers can be any solid excipient, liquid excipient, semi-solid excipient or a gaseous excipient in an aerosol composition which is generally available to those skilled in the art. Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, stearyl glyceryl ester, sodium chloride , anhydrous skim milk, etc. The liquid and semi-solid excipients may be selected from the group consisting of glycerin, propylene glycol, water, ethanol, and various oils. Including oils derived from petroleum, animal, plant or synthetic oils, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc., preferred liquid carriers, especially for injectable solutions, including water, saline, aqueous dextrose and alcohol. It is also possible to add other adjuvants such as flavoring agents, sweeteners and the like to the composition.

The compounds of the invention are administered in a therapeutically effective amount, which may be administered orally, systemically (e.g., through the skin, nasally, or with a suppository) or parenterally (e.g., intramuscularly, intravenously or subcutaneously). ). The preferred mode of administration is oral, which can be adjusted depending on the extent of the disease.

The actual amount of application (i.e., active ingredient) of a compound of the invention depends on a number of factors, such as the severity of the condition to be treated, the age and relative health of the subject being treated, the potency of the compound employed, the route and form of administration, and the like. factor.

The various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional methods in the pharmaceutical arts. For example, the compound is mixed with one or more carriers and then formulated into a desired dosage form such as a tablet, a pill, a capsule, a semisolid, a powder, a sustained release dosage form, a solution, a suspension, a formulation, a gas Aerosol and so on.

In another aspect of the invention, there is also provided a tyrosine kinase inhibitor comprising the above salt compound.

The tyrosine kinases include C-MET, VEGF, KDR, RON, KIT, PDGF, FGF, SRC kinase.

In another aspect of the invention, there is also provided the use of a salt compound as described above for the manufacture of a medicament for the treatment of cancer.

Since tyrosine kinase is a currently effective antitumor drug target, and the salt compound of the present invention has significant tyrosine kinase inhibitory activity, it has been confirmed by experiments that these compounds have an inhibitory effect on proliferation of various cancer cells, and therefore The inventive salt compounds are suitable for the treatment of various cancers. Especially for lung cancer, gastric cancer, ovarian cancer, colon cancer, malignant glioma has a better therapeutic effect.

In another aspect of the invention, there is also provided the use of a salt compound as described above for the manufacture of a medicament for the treatment of inflammation.

The salt compound of the invention has good biological activity with various signal transduction kinases such as C-MET, VEGF, KDR, RON, KIT, PDGF, FGF, SRC, etc., and is associated with various signal transduction pathways, and thus A variety of diseases have therapeutic effects, such as cancer, inflammation, lymphedema, diabetes, and the like.

The tyrosine kinase inhibitor of the present invention can inhibit the biological activities of various signal transduction kinases such as C-MET, VEGF, and KDR, can effectively inhibit cell proliferation, and has a good therapeutic effect on various diseases such as cancer, especially Lung cancer, gastric cancer, ovarian cancer, malignant glioma and the like have significant therapeutic effects, and the application prospect is very broad.

DRAWINGS

The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

Figure 1 is a graph showing the fitting inhibition of proliferation of human lung adenocarcinoma cell line HCC78 by compound 6 of Example 7 of the present invention;

Figure 2 is a graph showing the fitting inhibition of the proliferation inhibition of human malignant glioma cell line U87MG by the compound 6 of Example 7 of the present invention;

Figure 3 is a graph showing the fitting inhibition of the proliferation inhibition of human umbilical vein endothelial cells HUVEC by the compound 6 of Example 7 of the present invention;

Figure 4 is a comparison of the proliferation inhibition of human malignant glioma cell line U87MG by compounds 2 and 4 of Example 7 of the present invention. line graph;

Figure 5 is a graph showing the fitting inhibition of proliferation inhibition of human gastric cancer cells MKN-45 by Compounds 2 and 4 of Example 7 of the present invention;

Figure 6 is a graph showing the fitting inhibition of proliferation inhibition of human lung adenocarcinoma cells HCC78 by Compounds 2 and 4 of Example 7 of the present invention;

Figure 7 is a graph showing the fitting inhibition of proliferation of human ovarian cancer cell line SK-OV-3 by the compounds 2, 4, and 6 of Example 7 of the present invention;

Figure 8 is a graph showing the fitting inhibition of proliferation of human colon cancer cells HCT116 by the compounds 2, 4, and 6 of Example 7 of the present invention;

Figure 9 is a graph showing the fitting inhibition of proliferation of human lung adenocarcinoma cell line A549 by the compounds 2, 4, and 6 of Example 7 of the present invention.

detailed description

Example 1 Synthesis of a tyrosine kinase inhibitor p-toluenesulfonate compound

Compound 1 (N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl) ]-N'-(4-Fluorophenyl)cyclopropane-1,1-dimethylformamide) (632 mg, 1 mmol) was dissolved in tetrahydrofuran (10 mL). After ice-water bath to internal temperature 0 ° C, p-toluenesulfonic acid was added. (173 mg, 1 mmol), reacted at room temperature for 2 h. After completion of the reaction, filtered to give a white solid N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propane ]]oxy]quinolin-4-yl]oxy]phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dimethylformamide p-toluenesulfonate (Compound 2) 650 mg, yield 81%.

^{1 H NMR (500MHz, DMSO-} d 6): δ10.41 (s, 1H), 9.99 (s, 1H), 9.48 (brs, 1H), 8.50 (d, J = 5Hz, 1H), 7.92 (dd, 1H), 7.52-7.65 (m, 4H), 7.40-7.48 (m, 4H), 7.10-7.18 (m, 4H), 6.46 (d, J = 5.5 Hz, 1H), 4.26 (t, 2H), 4.10 (d, 2H), 4.10 (m, 1H), 3.97 (s, 3H), 3.66 (t, 2H), 3.60 (m, 2H), 3.15 (m, 2H), 2.21-2.36 (m, 5H), 1.45-1.51 (m, 4H).

LC-MS (ESI) m.

Example 2 Synthesis of a tyrosine kinase inhibitor glutamate compound

Compound 1 (N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl) ]-N'-(4-Fluorophenyl)cyclopropane-1,1-dimethylformamide) (632 mg, 1 mmol) dissolved in tetrahydrofuran / water (10 mL, 1:1), iced water to 0 ° C L-glutamic acid (147 mg, 1 mmol) was added and reacted at room temperature for 8 h. After completion of the reaction, the white solid N-[3-fluoro-4-[[6-methoxy-7-[[3-(? Phenyl-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dimethylamide glutamate (compound) 3) 450 mg, yield 58%.

LC-MS (ESI) m.

Example 3 Synthesis of a tyrosine kinase inhibitor n-butyl phosphate compound

Compound 1 (N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl) ]-N'-(4-Fluorophenyl)cyclopropane-1,1-dimethylformamide) (632mg, 1mmol) was dissolved in dichloromethane (10mL), and then added to n-butyl carbonate in an ice-water bath to an internal temperature of 0 °C. Phosphoric acid (154 mg, 1 mmol) was reacted for 4 h at room temperature. After completion of the reaction, filtered to give a white solid N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)) Propyl]oxy]quinolin-4-yl]oxy]phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dimethylformamide n-butyl phosphate (Compound 4) 650 mg, yield 83%.

^{1 H NMR (500MHz, DMSO-} d 6): δ10.43 (s, 1H), 9.94 (s, 1H), 9.20 (brs, 1H), 8.51 (d, J = 5Hz, 1H), 7.92 (dd, 1H), 7.52-7.65 (m, 4H), 7.40-7.48 (m, 4H), 7.10-7.18 (m, 4H), 6.46 (d, J = 5.5 Hz, 1H), 4.21 (t, 2H), 4.15 (d, 2H), 4.08 (m, 1H), 3.77 (m, 2H), 3.66 (t, 2H), 3.60 (m, 2H), 3.15 (m, 2H), 2.21-2.36 (m, 5H), 1.45-1.51(m,4H), 1.23(m,4H).1.10(t,3H),

LC-MS (ESI) m.

Example 4 Synthesis of a tyrosine kinase inhibitor malate compound

Compound 1 (N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl) ]-N'-(4-Fluorophenyl)cyclopropane-1,1-dimethylformamide) (632 mg, 1 mmol) was dissolved in tetrahydrofuran (10 mL). After ice-water bath to an internal temperature of 0 ° C, malic acid (134 mg) was added. , 1 mmol), reacted at room temperature for 8 h, after completion of the reaction, filtered to give a white solid N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propyl] Oxy]quinolin-4-yl]oxy]phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dimethyl Amide malate (Compound 5) 550 mg, yield 72%.

LC-MS (ESI) m.

Example 5 Synthesis of a tyrosine kinase inhibitor alendronate compound

Compound 1 (N-[3-fluoro-4-[[6-methoxy-7-[[3-(morpholin-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl) ]-N'-(4-Fluorophenyl)cyclopropane-1,1-dimethylformamide) (632 mg, 1 mmol) was dissolved in N,N-dimethylformamide (10 mL). After °C, alendronic acid (249 mg, 1 mmol) was added and reacted at room temperature for 8 h. After completion of the reaction, the white solid N-[3-fluoro-4-[[6-methoxy-7-[[3-( Morpholin-4-yl)propyl]oxy]quinolin-4-yl]oxy]phenyl]-N'-(4-fluorophenyl)cyclopropane-1,1-dimethylamide alendronate (Compound 6) 150 mg, yield 17%.

LC-MS (ESI) m.

Example 6 Detection of tyrosine kinase inhibitory activity

The salt compounds of the above examples were applied to the detection and screening of C-MET and KDR kinase inhibitory activities.

Method

(1) Add 4 μL of the prepared kinase buffer solution to the 384-well plate, or 4 μL of the kinase solution (100% inhibition control); add 2 μL of the compound to the well, or 2 μL of the buffer containing no compound (0% inhibition control) ); all of the above samples or controls are set to 2 duplicate wells.

(2) Incubation at 25 ° C for 5 min;

(3) adding 2 μL of ATP/substrate/MgCl ₂ /MnCl ₂ /SEB/DTT mixture to the well;

(4) Centrifuge at 1000 rpm for 1 min, incubate at 30 ° C for 30 min;

(5) adding 8 μL of a mixture of XL-665/antibody to the well;

(6) Incubating at 25 ° C for 1 h;

(7) The PHERAstar FS instrument reads the 665 nm and 620 nm signals;

(8) The data was analyzed according to the kit instructions and the IC50 was calculated using GraphPad Prism5 fit.

Ratio=665nm/620nm

2. Experimental results

The results of the detection of each of the test compound and the reference compound are summarized in Tables 1 and 2 below, wherein the control compound is the existing C-MET kinase inhibitor Foretinib (the structural formula is as follows). The chemical structural formulas of For-Oxide and For-Methyl are also as follows.

Table 1 shows the inhibitory activity of compound 6 on C-MET

Table 2 inhibitory activity of compound 6 on KDR

Table 3 Inhibitory activity of compounds 2 and 4 on C–Met/KDR

As is clear from the data of Tables 1-3, the salt compound of the above-described examples of the present invention has an inhibitory activity against tyrosine kinases such as C-MET and KDR.

Example 7 inhibition test for tumor cell proliferation

Method

(1) Cell culture: cells such as human lung adenocarcinoma cell line HCC78, human malignant glioma cell line U87MG, human gastric cancer cell line MKN-45, human umbilical vein endothelial cell line HUVEC, human lung adenocarcinoma cell line A549, etc. The medium was cultured under the conditions of 37 ° C, 5% CO ₂ .

(2) Cell seeding: Take cells in a good condition in the exponential growth phase, add appropriate amount of trypsin to digest the cells, collect the cells for centrifugation, and discard the supernatant. The cells were resuspended in serum-containing medium, then counted and cell suspension was seeded at 3000/well in 96-well plates at 90 μL/well. The plate was transferred to a constant temperature CO ₂ incubator and incubated at 37 ° C, 5% CO ₂ and saturated humidity for 24 hours.

(3) The test compound was added: 10 μL/well, cultured for 72 hours, and each group was provided with 3 parallel wells.

(4) Determination of results: After 72 hours of compound action, 10 μL/well of CCK8 was added, and the cells were incubated in an incubator for an appropriate period of time, and the absorbance was measured at 450 nm.

2. Experimental results

The test results of each test compound are summarized in Table 4-6 below:

Table 4 shows the inhibition of proliferation of human lung adenocarcinoma cell line HCC78, human malignant glioma cell line U87MG, human umbilical vein endothelial cell line HUVEC, and human gastric cancer cell line MKN-45 (corresponding fitting curve is shown in Figure 1-3)

Table 5 shows the proliferation inhibition results of compounds 2 and 4 on human malignant glioma cell line U87MG, human gastric cancer cell line MKN-45, and human lung adenocarcinoma cell line HCC78 (corresponding fitting curve is shown in Fig. 4-6)

Table 6 shows the inhibition of proliferation of human ovarian cancer cell line SK-OV-3, human colon cancer cell line HCT116, and human lung adenocarcinoma cell line A549 by compound 2, 4, and 6 (corresponding fitting curves are shown in Fig. 7-9).

From the data of the above Table 4-6, the salt compound of the present invention can effectively inhibit human lung adenocarcinoma cells, human gastric cancer cells, human colon cancer cells, and humans by inhibiting the activity of tyrosine kinases such as C-MET and KDR. The proliferation of various cancer cells such as ovarian cancer cells and human malignant glioma cells is particularly suitable for the treatment of cancer.

The above-mentioned embodiments are merely illustrative of the embodiments of the present invention, and the description thereof is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be subject to the appended claims.

Claims (11)

a salt compound having the formula (I),

among them, R1 is selected from the group consisting of halogen, halogen anion, lower halogenated alkane, lower alkane, lower alkene, lower alkyne, substituted saturated or unsaturated cycloalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryl, substituted or Unsubstituted heterocyclic group, substituted or unsubstituted heterocycloalkyl group, or empty; Y is selected from C, O, N, S or is empty; X is selected from the group consisting of carbonate, sulfate, sulfite, hydrogen sulfate, phosphate, phosphite, hydrogen phosphate, dihydrogen phosphate, molybdate, chlorate, or empty. The salt compound according to claim 1, wherein the compound of the formula (I) comprises:

A pharmaceutical composition comprising a safe and effective amount of the salt compound of claim 1 and a pharmaceutically acceptable carrier. A tyrosine kinase inhibitor comprising the salt compound of claim 1 or 2. The tyrosine kinase inhibitor according to claim 4, wherein the tyrosine kinase comprises C-MET, VEGFR, KDR, RON, KIT, PDGF, FGF, SRC kinase. Use of the salt compound according to claim 1 or 2 for the preparation of a medicament for treating cancer. The use according to claim 6, wherein the cancer comprises lung cancer, gastric cancer, ovarian cancer, colon Cancer, pancreatic cancer, esophageal adenocarcinoma, malignant glioma. Use of the pharmaceutical composition of claim 3 in the manufacture of a medicament for treating cancer. Use of the salt compound according to claim 1 or 2 for the preparation of a medicament for treating inflammation. Use of the pharmaceutical composition of claim 3 in the manufacture of a medicament for treating cancer. Use of the pharmaceutical composition of claim 3 in the manufacture of a medicament for the treatment of inflammation.

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