Application of substituted crotonamide

Abstract

An application of a substituted crotonamide, in particular being an application of (E)-N-(3-cyano-7-ethoxyl-4-(3-ethynylphenylamino)quinoline-6-yl)-4-(dimethylamino)but-2-enamide and a pharmaceutically acceptable salt and solvate thereof in the preparation of a drug for treating cancer mediated by a rare EGFR mutation.

Claims

1. An application of (E)-N-(3-cyano-7-ethoxyl-4-(3-ethynylphenylamino)quinoline-6-yl)-4-(dimethylamino)but-2-enamide, or a pharmaceutically acceptable salt, or solvate thereof in the preparation of a drug for treating cancer mediated by a rare EGFR mutation.

2. The application according to claim 1, wherein the pharmaceutically acceptable salt is hydrochloride salt, benzenesulfonate salt, methanesulfonate salt, or maleate salt.

3. The application according to claim 1, wherein the pharmaceutically acceptable salt is a hydrate, preferably a hemihydrate or a monohydrate.

4. The application according to claim 1, wherein the rare EGFR mutations include but are not limited to any one or a combination of EGFR mutants: L861Q, G719X, and/or S768I.

5. The application according to claim 1, wherein the cancer is non-small cell lung cancer.

6. A method for treating rare EGFR mutation-mediated cancer, which is characterized by administering a therapeutically effective amount of (E)-N-(3-cyano-7-ethoxyl-4-(3-ethynylphenylamino)quinoline-6-yl)-4-(dimethylamino)but-2-enamide, or a pharmaceutically acceptable salt, or solvent thereof.

7. The method according to claim 6, wherein the therapeutically effective amount is from 50 to 250 mg per day, preferably 100 mg a day.

8. The method according to claim 6, wherein the therapeutically effective amount is daily dose/body weight from 0.01 to 500 mg/kg, preferably from 0.01 to 50 mg/kg, more preferably from 0.01 to 30 mg/kg, further from 0.1 to 10 mg/kg or from 0.5 to 3mg/kg.

9. The method according to claim 6, wherein the rare EGFR mutations include but are not limited to EGFR mutants: L861Q, G719X, and/or S768I.

10. The method according to claim 6, wherein the pharmaceutically acceptable salt is hydrochloride salt, benzenesulfonate salt, methanesulfonate salt, maleate salt, and more preferably hemihydrate or monohydrate of the pharmaceutically acceptable salt.

Description

DETAILED DESCRIPTION

[0033] The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.

[0034] (E)-N-(3-cyano-7-ethoxy-4-(3-ethynylphenylamino)quinolin-6-yl)-4-(dimethylamino) involved in the examples of the application) But-2-enamide was prepared according to the method described in WO2010151710, (E)-N-(3-cyano-7-ethoxy-4-(3-ethynylphenylamino)quinolin-6-yl)-4-(dimethylamino)but-2-enamide maleate monohydrate was prepared according to the method described in CN104513200A, and it was also named (E)-N-{4-[(3-ethynyl Anilino)-3-cyano-7-ethoxy-6-quinolinyl]}-4-(dimethylamino)-2-butenamide maleate.

Example 1 IC50 Determination of Protein Kinase

[0035] The compound tested in this example is (E)-N-(3-cyano-7-ethoxy-4-(3-ethynylphenylamino)quinolin-6-yl)-4-(dimethyl Amino)but-2-enamide.

[0036] Part 1. Experimental Design

[0037] The IC50 of the test compound against protein kinase, the test substance is set at 10 semi-logarithmic concentrations (from 1×10.sup.−06 M to 3×10.sup.−11 M), and the IC50 is determined in a single well.

[0038] Part 2. Test Substance

[0039] The test substance is two tubes of solid, and ProQinase is transferred to −20° C. for subsequent use.

[0040] Before the test, a storage solution of 1×10.sup.−03 M test substance was prepared with 100% DMSO and further diluted to 1×10.sup.−04 M/100% DMSO.

[0041] The test substance was diluted semi-logarithmically into a 96-well plate with 100% DMSO and prepared from 1×10.sup.−04 M to 3×10.sup.−09 M. Before use, the test substance is diluted 1:10 with water to obtain 1×10.sup.−05 M to 3×10.sup.−10 M test substance samples containing 10% DMSO.

[0042] Add 5 μl of each concentration to the test (see Part 3 Protein Kinase Test), the final volume of the test is 50 μl. The test substance is measured at a concentration of 1×10.sup.−06 M to 3×10.sup.−11 M. The final concentration of DMSO in all reaction wells is 1%.

[0043] Part 3. Protein Kinase Test

[0044] Protein kinase test by radiometry (.sup.33PanQinase® activity determination method) is used to determine the activity of 8 protein kinases. All experiments were performed in a 96-well plate of Perkin Elmer (Boston, Mass., USA) with a reaction volume of 50 μl. Reagents and samples are added in 4 steps:

[0045] 10 μl non-radioactive ATP aqueous solution;

[0046] 25 μl buffer/[γ.sup.−33 P]-ATP mixture;

[0047] 5 μl test substance containing 10% DMSO;

[0048] 10 μl enzyme/substrate mix.

[0049] The substrate is Poly(Glu, Tyr) 4:1, code name SIG_20K5903.

[0050] All experiments include 70 mM HEPES-NaOH buffer at pH7.5, 3 mM MgCl.sub.2, 3 mM MnC, 3 μM sodium orthovanadate, 1.2 mM DTT, ATP (concentration varied, concentration related to the apparent ATP-Km of each enzyme, see Table 1), [γ.sup.−33P]-ATP (approximately 8×1005 cpm per well), protein kinase (concentration varied, see Table 1), and substrate (concentration varied, see Table 1).

[0051] The concentrations of enzymes and substrates used in the experiment are shown in Table 1:

TABLE-US-00001 TABLE 1 Concentrations of enzymes and substrates used kinase [kinase] [kinase] [ATP] [substrate] No. name ng/50 μL nM* μM ng/50 μL 1 EGF-R G719C 20 4.5 1.0 0.125 2 EGF-R G719S 10 2.2 0.3 0.125 3 EGF-R L861Q 25 5.6 0.3 0.125 4 EGF-R 790M 10 2.2 0.3 0.125 5 EGF-R 10 2.2 0.3 0.125 T790M/L858R 6 EGF-R wt 5 1.1 0.3 0.125 7 ERBB2 100 21.3 1.0 0.125 8 ERBB4 5 1.0 0.3 0.125

[0052] The reaction system was incubated at 39° C. for 60 minutes. The reaction was terminated with 50 μl 2% (v/v) H.sub.3PO.sub.4, and the plate was washed twice with 200 μl 0.9% (w/v) NaCl. Microplate scintillation counter (Microbeta, Wallac) measures the enzyme activity-dependent transfer of .sup.33Pi (in “cpm”).

[0053] All experiments were done with Beckman Coulter Biomek 2000/SL robot.

[0054] Part 4. Data Analysis

[0055] The blank control places 3 reaction wells without adding enzyme, and taking the median of the cpm value of the 3 reaction wells as the reading for the blank. This value reflects the non-specific binding of radioactivity in the absence of protein kinase or substrate. In addition, set 3 reaction wells without adding test substance, and take the median of the cpm value of 3 reaction wells as a negative control, that is, the enzyme activity without any inhibitor. The difference between the negative control and the blank is the 100% activity of each enzyme.

[0056] As with the negative control, the blank value should be subtracted from the value of the corresponding 10 test substances. The residual enzyme activity of each well is calculated according to the following formula: residual enzyme activity (%)=100×[(test substance cpm value-blank control)/(negative control-blank control)].

[0057] Each test substance is set to 10 concentrations to act on each enzyme, so the data analysis is based on these 10 residual enzyme activity values. Use Prism5.04 (Graphpad, San Diego, Calif., USA www.graphpad.com), fix the maximum value of 100%, the minimum value of 0%, and perform S-curve fitting to calculate IC50.

[0058] Part 5. Results

[0059] Table 2 lists the IC50 values of the tested compounds acting on 8 kinases.

TABLE-US-00002 TABLE 2 IC50 values of the tested compounds acting on 8 kinases No. Kinase IC50 (M) 1 EGF-R G719C 1.40 × 10.sup.−09 2 EGF-R G719S 1.50 × 10.sup.−09 3 EGF-R L861Q 1.90 × 10.sup.−09 4 EGF-R 790M 1.20 × 10.sup.−07 5 EGF-R 8.80 × 10.sup.−08 T790M/L858R 6 EGF-R wt 1.30 × 10.sup.−09 7 ERBB2 2.70 × 10.sup.−08 8 ERBB4 1.00 × 10.sup.−08

[0060] The above data shows that the inhibitory effect of the tested compound on rare mutations is better than the general non-rare mutation inhibitory effect.

Example 2 An Exploratory Clinical Research Trial for the Treatment of Locally Advanced or Metastatic Non-Small Cell Lung Cancer

[0061] (I) Subject selection criteria

[0062] 1. Inclusion criteria:

[0063] (1) Age from 18 to 75 (including 18 and 75) years old, regardless of gender.

[0064] (2) Patients with locally advanced or metastatic NSCLC confirmed by histopathology and/or cytology.

[0065] (3) The patient's EGFR has one or more of L861Q, G719X, and S768I mutations, and does not have T790M mutation, exon 19 deletion mutation, exon 20 insertion mutation, or L858R mutation.

[0066] (4) The ECOG physical strength score is 0, 1, or 2.

[0067] (5) Expected survival time>3 months.

[0068] (6) According to Version 1.1 of the Curative Effect Evaluation Criteria for Solid Tumors (RECIST), there is at least one evaluable tumor lesion.

[0069] (7) Sufficient blood system function, liver function, kidney function and blood coagulation function: Absolute neutrophil count≥1.5×109/L, platelet count≥90×109/L, hemoglobin≥90g/L; Total bilirubin≤1.5×upper limit of normal (ULN), alanine aminotransferase (ALT)≤2.5×ULN, aspartate aminotransferase (AST)≤2.5×ULN (total bilirubin in patients with liver metastasis≤3.0×ULN , ALT≤5.0×ULN, AST≤5.0×ULN); Creatinine≤1.0×ULN, or creatinine clearance≥60mL/min (using Cockcroft-Gault method); International normalized ratio (INR)≤1.5.

[0070] (8) Eligible patients (male and female) with fertility must agree to use reliable contraceptive methods (hormonal or barrier method or abstinence) during the trial period and at least 90 days after the last medication; female patients of childbearing age within 7 days before enrollment The blood human chorionic gonadotropin (HCG) pregnancy test must be negative; male patients cannot do sperm donation within 90 days after the first dose to the last dose.

[0071] (9) All patients must be informed of this study before beginning to accept any inspections prescribed by this trial, and voluntarily sign a written informed consent (ICF) approved by the ethics committee.

[0072] 2. Exclusion criteria:

[0073] (1) Have received any epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) anti-tumor therapy before enrollment.

[0074] (2) Patients who have received chemotherapy, anti-angiogenesis and other systemic anti-tumor treatments in the past require a washout period of 4 weeks or more; those who have received anti-PD-1/PD-L1 immunotherapy in the past and have not experienced immune pneumonia For patients, a washout period of 4 weeks or more is required; for patients who have previously received radiotherapy or anti-tumor Chinese patent medicine for non-target lesions for the purpose of relieving symptoms, a washout period of 2 weeks or more is required.

[0075] (3) Major organ surgery (excluding needle biopsy) or significant trauma occurred within 4 weeks before enrollment.

[0076] (4) At the time of screening, the adverse reaction (ADR) of any previous treatment has not yet recovered to the Common Terminology Criteria for Adverse Events (CTCAE) 5.0 grade evaluation≤grade 1 (except for hair loss).

[0077] (5) Inability to take drugs orally, severe chronic gastrointestinal dysfunction (CTCAE 5.0 rating≥3), malabsorption syndrome or any other conditions that affect the absorption of the gastrointestinal tract (for example: peptic ulcer, intestinal tract) Obstruction, irritable bowel syndrome, Crohn's disease, gastroesophageal reflux disease, etc.).

[0078] (6) Have immunodeficiency, including but not limited to human immunodeficiency virus (HIV) antibody test (enzyme-linked immunoassay or Western spot method) positive, and active rheumatic immune disease.

[0079] (7) There is unstable central nervous system metastasis or meningeal metastasis with clinical symptoms, or there is other evidence that the patient's central nervous system metastasis or meningeal metastasis has not been controlled, and the investigator judges that it is not suitable for inclusion; clinical symptoms suspect that there is brain Or patients with leptomeningeal disease need to be excluded by computed tomography/magnetic resonance imaging (CT/MRI).

[0080] (8) A history of severe bullous or exfoliative skin diseases (CTCAE 5.0 grade evaluation≥grade 3).

[0081] (9) During screening, there are uncontrolled active infections (such as syphilis, HIV, hepatitis B virus (HBsAg positive, HBV-DNA>1000 cps/ml and AST/ALT>2.0 xULN) or hepatitis C virus (HCV) infection.

[0082] (10) A history of severe cardiovascular disease [New York College of Cardiology (NYHA) Heart Function Grade III or IV], including but not limited to ventricular arrhythmia requiring clinical intervention; within 6 months before enrollment Acute coronary syndrome, congestive heart failure, stroke, or other cardiovascular events of grade III and above; at the time of screening, NYHA cardiac function grade≥grade II or left ventricular ejection fraction (LVEF)<50%.

[0083] (11) There is a history of other serious systemic diseases (CTCAE 5.0 grade evaluation≥grade 3), and the investigator judges it is not suitable for participating in clinical trials.

[0084] (12) Participated in other clinical trials within 4 weeks before enrollment.

[0085] (13) Known alcohol or drug dependence.

[0086] (14) Patients with mental disorders or researchers who believe that poor compliance are not suitable for participating in the study.

[0087] (15) Women during pregnancy or lactation.

[0088] (16) Known to be allergic to the active ingredients or excipients of the test drug.

[0089] (17) Long-term treatment with adrenal steroids is required (equivalent to the daily dose of prednisolone≥20 mg).

[0090] (18) Suffered from other malignant tumors in the past 5 years or at the same time (except for cured skin basal cell carcinoma and cervical carcinoma in situ; except for those without recurrence>3 years after radical mastectomy).

[0091] (II). Administration method and dosage

[0092] (E)-N-(3-cyano-7-ethoxy-4-(3-ethynylphenylamino)quinolin-6-yl)-4-(dimethylamino)but-2-ene Amide maleate monohydrate capsules (refer to Chinese patent application 201911180660.1), taken orally at least 1 hour before meals or 2 hours after meals; 100 mg once a day for 28 days. Every four weeks since the start of administration is a cycle.

[0093] (III). Therapeutic effect

TABLE-US-00003 TABLE 3 The sum of the longest diameters of the target lesions at the end of the treatment cycle Sum of the longest Patient Mutation Diameters of all lesions No. Gender Type Selection cycle 2.sup.nd cycle 4.sup.th cycle Changes % 001 Female L861Q 65.8 mm   35 mm 30.1 mm −54% 002 Female L861Q 58.6 mm 40.4 mm 36.7 mm −37% 003 Female G719X 20.8 mm   16 mm 13.6 mm −35% 004 Female G719X, S768I 105.0 mm  48.0 mm 43.0 mm −59% 005 Male S768I 62.8 mm 38.7 mm 39.6 mm −37% 006 Male G719X 81.0 mm 31.0 mm 34.8 mm −57% 007 Male S768I 89.1 mm 54.8 mm 57.3 mm −38% 008 Female G719X 23.9 mm 14.7 mm 18.2 mm −38% 009 Female L861Q, G719X 88.4 mm 51.6 mm 47.9 mm −46% 010 Female L861Q 18.0 mm  8.9 mm     mm −51%

TABLE-US-00004 TABLE 4 The sum of the shortest diameters of the target lesions in the treatment cycle Sum of the longest Sum of shortest Patient Mutation Diameters of all Diameters of all PFS No. Gender Type lesions lesions Evaluation (month) 001 Female L861Q 65.8 mm 29.9 mm PR 8 002 Female L861Q 58.6 mm 36.7 mm PR 14 003 Female G719X 20.8 mm 13.6 mm PR 14 004 Female G719X, S768I 105.0 mm  41.0 mm PR 13 005 Male S768I 62.8 mm 38.7 mm PR 10 006 Male G719X 81.0 mm 27.0 mm PR 12 007 Male S768I 89.1 mm 54.8 mm PR 12 008 Female G719X 23.9 mm 13.5 mm PR 11 009 Female L861Q, G719X 88.4 mm 47.3 mm PR 9

[0094] PFS: progression-free survival

[0095] The above treatment results show that cancer patients with rare EGFR mutation-mediated cancers have shown good therapeutic effects after treatment.