WO2019001425A1 - Deuterated osimertinib derivative and application thereof - Google Patents

Abstract

The present invention discloses a deuterated osimertinib derivative of formula I, and a pharmaceutically acceptable salt, a stereoisomer, a solvate, or a prodrug thereof, wherein the symbols in the formula are as defined in the claims. The deuterated osimertinib derivative of the present invention can inhibit activation or resistance mutations of one or more EGFRs, can be used for the treatment of EGFR-sensitive cancer mutations, and is an ideal therapeutic drug for diseases caused by EGFR mutations.

Description

A Deuterated Ostinid Derivative and Its Application Technical field

The present invention relates to the field of medical technology, and in particular to a deuterated Osimintinib derivative for use as an EGFR inhibitor and its preparation for regulating EGFR tyrosine kinase activity or treating EGFR related diseases, especially non-small cell lung cancer. The application of the drug.

Background technique

Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein tyrosine kinase of the erbB receptor family that binds to growth factor ligands (eg, epidermal growth factor (EGF)). Homogeneous dimerization can occur with additional EGFR molecules or with another family member (eg, erbB2 (HER2), erbB3 (HER3), or erbB4 (HER4)). Homologous dimerization and/or heterodimerization of the erbB receptor results in phosphorylation of key tyrosine residues in the intracellular domain and results in stimulation of many intracellular signaling pathways involved in cell proliferation and survival. Deregulation of erbB family signaling promotes proliferation, invasion, metastasis, angiogenesis, and tumor cell survival, and has been described in many human cancers, including lung cancer, head and neck cancer, and breast cancer.

Lung cancer is the world's highest incidence of cancer. It ranks first among all cancers in China, and it is also the cancer with the highest morbidity and mortality in China. About 30% of lung cancer patients in China have EGFR mutations, of which L858R and exon 19 deletion mutations account for more than 90%. These patients are more sensitive to EGFR inhibitors. The existing first-generation EGFR inhibitors such as erlotinib and gefitinib have good curative effect on such patients, which can reduce tumors in more than 60% of patients and significantly prolong the progression-free survival of patients. . However, the vast majority of patients will acquire resistance within 6-12 months. This resistance pattern is a further mutation of EGFR, which reduces its sensitivity to first-generation EGFR inhibitors. The most common of these mutations is the so-called "gatekeeper" mutation T790M (Science, 2004, Vol. 304, 1497-1500; New England Journal of Medicine 2004, 350, 2129-2139), from the original L- at this position. Threonine (T) is replaced by L-methionine (M), and the mutated EGF tyrosine kinase R no longer binds to gefitinib or erlotinib, thus making the first generation of EGFR inhibitors No longer effective, resulting in such patients currently in a state of no drug availability. Clinically, 50% of patients who developed resistance to first-generation EGFR inhibitors had EGFR T790M mutations. In the T790M mutant cell line H1975, first-generation EGFR inhibitors, such as gefitinib and erlotinib, were all greater than 3 [mu]M and were essentially inactive.

In order to increase the inhibitory activity against mutations such as the resistant EGFR T790M, WO2013014448 discloses a pyrimidine derivative which can be used as an EGFR inhibitor and its use for treating cancer, the structure is as shown in the formula A, wherein G is selected from 4, 5, 6, 7-tetrahydropyrazolo[1,5-a]pyridin-3-yl, 1H-indol-3-yl, 1-methyl-1H-indol-3-yl or pyrazolo[1,5 -a]pyridin-3-yl, R ² is methyl or methoxy;

The structural formula of the compound (AZD9291) which has been successfully marketed in WO2013014448 is:

On November 13, 2015, it was approved by the US FDA (trade name TagAsso, AZD9291, Osimertinib) for the treatment of advanced non-small cell lung cancer patients with positive EGFR T790M mutation. However, Osimertinib (AZD9291, TagAsso) has a short half-life, a high dose, and some metabolites cause adverse reactions, the most common adverse reactions in the clinical application process due to excessive inhibition or activation of wild-type EGFR (≧25). %) is toxic to diarrhea, rash, dry skin and nails. This also limits the therapeutic window for the application of this drug, and for some less sensitive patients, the treatment effect is poor due to the limitations of the treatment window. At the same time, for some patients who are too sensitive, due to excessive side effects, this drug cannot be used. Therefore, it is very necessary to optimize the structure of the drug, increase the half-life, reduce the toxicity of the drug, and increase the treatment window, so that more patients benefit.

To date, CN104140418B discloses some methyl deuterated azinib (AZD9291) compounds having the structural formula shown in formula B, wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are methyl or deuterated. methyl,

Although these compounds disclosed in CN104140418B increase the half-life of Ostiminib, it has been reported in the literature and our study that the binding mode of Osimertinib to the target EGFR kinase, the pyrimidine ring moiety and the EGFR kinase Hinge-binding, once the pyrimidine ring moiety is metabolized to other forms, the binding of Osimertinib to EGFR kinase becomes weak or unable to bind and loses activity. CN105153122 reports AZD9291 compounds of methyl deuterated and anthracycline full deuterated. Although these compounds can increase the half-life of Osimertinib, due to too many sites in the deuteration, the production price is inevitably expensive and increased. Production and development costs, resulting in increased patient spending. CN105237515 discloses an Osimertinib compound which only has a pyrimidine ring, and our study found that when the methyl group on the oxime is metabolized, a compound of the formula C is formed.

Although this compound still inhibits EGFR activity, it does not pass through the blood-brain barrier and enters the brain, thereby losing its effect on tumor patients with brain metastasis.

Therefore, it is necessary to develop drugs that can increase the half-life of Osimertinib, reduce toxic side effects well, and enter the brain through the blood-brain barrier, which is effective for patients with brain tumor metastasis.

Summary of the invention

The invention provides a compound of formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

among them:

R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from methyl or deuterated methyl;

R _{6 is} selected from H or hydrazine.

Preferably, the deuterated methyl group -CD _3. The R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each independently selected from methyl or -CD ₃ .

At least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ is -CD ₃ .

One to three of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are -CD ₃ .

a compound of the formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein the compound is selected from the group consisting of the compounds represented by the following structural formula:

Wherein the pharmaceutically acceptable salt is a mineral acid salt or an organic acid salt selected from the group consisting of a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a sulfate salt, a hydrogen sulfate salt, and a nitrate salt. , phosphate, acid phosphate; the organic acid salt is selected from the group consisting of formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, propylene Acid salt, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, besylate, salicylate, picric acid Salt, glutamate, ascorbate, camphorate, camphor sulfonate.

Preferably, the inorganic acid salt is a hydrochloride or a sulfate; the organic acid salt is a methanesulfonate.

Another technical solution provided by the present invention is: a pharmaceutical composition comprising the compound of the above formula I and a pharmaceutically acceptable carrier.

The deuterated Ostinidil derivative provided by the present invention is a compound of Formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, which inhibits one or more EGFR activation or resistance mutations, such as L858R activating mutant, Exon19 deletion EGFR activating mutant, T790M resistant mutant; this compound increases the inhibitory activity against mutations such as resistant EGFR T790M, and simultaneously reduces wild-type EGFR The inhibitory activity can be used to develop third-generation EGFR mutant selective inhibitors with higher activity, better selectivity, and lower toxicity.

The solvate referred to in the present invention means a complex of the compound of the present invention and a solvent. They either react in a solvent or precipitate out of the solvent or crystallize out. For example, a complex formed with water is referred to as a hydrate; others include an alcoholate, a ketone compound, and the like. The solvates of the present invention include the compounds of the formula I of the present invention and salts thereof, and solvates of stereoisomers.

A stereoisomer as referred to in the present invention means that the compound of formula I in the present invention may contain one or more chiral centers and exist in different optically active forms. When the compound contains a chiral center, the compound contains the enantiomer. The invention includes mixtures of the two isomers and isomers, such as racemic mixtures. Enantiomers can be resolved by methods known in the art, such as crystallization and chiral chromatography. When a compound of formula I contains more than one chiral center, diastereomers may be present. Stereoisomers of the invention include resolved optically pure specific isomers as well as mixtures of diastereomers. Diastereomers can be resolved by methods known in the art, such as crystallization and preparative chromatography.

A prodrug as referred to in the present invention refers to a parent compound which includes a known amino protecting group and a carboxy protecting group, which are hydrolyzed under physiological conditions or released by an enzymatic reaction. Specific prodrug preparation methods can be referred to the prior art (Saulnier, MG; Frennesson, DB; Deshpande, MS; Hansel, SB and Vysa, DM Bioorg. Med. ChemLett. 1994, 4, 1985-1990; and Greenwald, RB; Choe, YH; Conover, CD; Shum, K.; Wu, D.; Royzen, MJ Med. Chem. 2000, 43, 475.).

In a practical application, a compound of the formula I according to the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, may be administered in a suitable dosage form with one or more pharmaceutical carriers. These dosage forms include those suitable for oral, rectal, topical, intraoral, and other parenteral administration (e.g., subcutaneous, intramuscular, intravenous, etc.).

The pharmaceutical compositions of this invention may be formulated, quantified, and administered in a manner consistent with medical practice. The "effective amount" of a compound administered is determined by the particular condition being treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.

The deuterated Ostinidil derivative provided by the invention can be used for preparing drugs for regulating EGFR tyrosine kinase activity or treating EGFR-related diseases, such as cancer, diabetes, immune system diseases, neurodegenerative diseases or cardiovascular diseases, etc. It is especially useful for the treatment of non-small cell lung cancer caused by EGFR mutations, including sensitive mutations (such as L858R mutation or deletion of exon 19) and drug resistance mutations (such as EGFR T790M mutation).

Accordingly, in another aspect, the invention provides a compound of formula I of the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, for use in the modulation of EGFR tyrosine kinase activity or in the treatment of EGFR The application of drugs for related diseases.

In one embodiment, the modulating EGFR tyrosine kinase activity or treating an EGFR-related disease refers to treating cancer, diabetes, an immune system disease, a neurodegenerative disease, or a cardiovascular disease.

In another embodiment, the invention provides the use of a compound of formula I according to the invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, for the manufacture of a medicament for the treatment of non-small cell lung cancer. The deuterated Ostinidyl derivatives of the present invention are particularly useful for the preparation of a medicament for the treatment of cancer, such as non-small cell lung cancer.

The compound of the formula I of the present invention, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, can be used as a single therapeutic drug in anticancer therapy, or can be used in addition to conventional Surgical or radiation therapy or a combination of chemotherapy or immunotherapy. The above therapy and the EGFR inhibitor of the present invention can be administered in parallel, simultaneously, sequentially, or separately.

The medicament for regulating EGFR tyrosine kinase activity or treating EGFR-related diseases of the present invention may further comprise any one or more of the following drugs in addition to the EGFR inhibitor of the present invention: gefitinib, erg Lotitinib, ectatinib, lapatinib, XL647, NVP-AEE-788, ARRY-334543, vandetanib, PF00299804, cetuximab, panituzumab, pertuzumab , zarumimumab, nimotuzumab, MDX-214, CDX-110, IMC-11F8, CNF2024, tancomycin, aspironmycin, IPI-504, NVP-AUY922.

Detailed ways

The technical solution of the present invention will be further described below in conjunction with specific embodiments.

Example 1

Target compound 1, the structural formula is:

The synthetic route is:

The specific synthesis steps are as follows:

1) Synthesis of compound TRN158-a: Under a nitrogen atmosphere, 18 g of 5-bromo-2,4-dichloropyrimidine and 180 ml of anhydrous tetrahydrofuran were sequentially added to a 1-liter three-necked flask, and the temperature was dropped to -60 ° C. 393 ml of isopropylmagnesium chloride in tetrahydrofuran solution for 40 minutes; then, the temperature was raised to -30 ° C for 1 hour, and 20 ml of hydrophobic water was added dropwise at -30 ° C for 20 minutes, then slowly raised to room temperature for 1 hour; After completion, 1 L of ethyl acetate was added to the reaction mixture, and the mixture was stirred for 10 minutes, and filtered, and the filtrate was evaporated to dryness.

2) Synthesis of compound TRN158-b: Under a nitrogen atmosphere, 4 g of sodium hydrogen and 600 ml of anhydrous tetrahydrofuran were sequentially added to a 1-liter three-necked flask, and the temperature was lowered to 0 ° C, and 9.75 g of hydrazine tetrahydrofuran was added dropwise to the reaction liquid. 60 ml of the solution, after the completion of the dropwise addition, gradually warmed to room temperature, and stirred for 1 hour; the temperature was lowered to 0 ° C, and 14.5 g of deuterated methyl iodide was added dropwise to the reaction solution. After the completion of the dropwise addition, the temperature was gradually raised to room temperature and stirred overnight; After disappearing, ice water and EA were added to the reaction solution, and the aqueous layer was washed twice with EA, then the EA layer was washed twice with saturated brine, and then dried and dried to dryness. The analysis data is as follows:

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 7.63 (d, J = 8.64, 1H); 7.30 (dd, J = 0.76, J = 9.0, 1H); 7.21. (t, J = 16.24, 1H); (t, J = 15.88, 1H); 7.00 (d, 3.08, 1H); 6.46 (dd, J = 0.76, J = 3.84, 1H); MS+1: 135.

3) Synthesis of compound TRN15801-1: Under a nitrogen atmosphere, 3.76 g of TRN158-a, 40 ml of DME, 4.06 g of anhydrous ferric chloride and 2.8 g of TRN158-b were sequentially added to a 100 ml single-mouth bottle at 60 ° C. The reaction was continued overnight; TLC detected the disappearance of TRN158-b, the reaction solution was cooled to room temperature, poured into a mixture of ice water and EA, and the aqueous layer was washed twice with EA, then the EA layer was washed twice with saturated brine, then Dry and spin dry the organic layer; pass the column to obtain 0.746 g of a yellow solid; the analytical data of the compound is as follows:

^{1 H-NMR (400MHz, CDCl} 3): δ: 8.42 (s, 1H); 8.28-8.34 (m, J = 22.76,1H); 7.92 (s, 1H); 7.30-7.40 (m, J = 41.32, 3H); MS+1: 248.

4) Synthesis of target compound 1: Under a nitrogen atmosphere, 540 mg of TRN15801-1, 574 mg of TRN158-c, 30 ml of DCE, 20 ml of 2-pentanol and 374 mg of p-toluenesulfonic acid were sequentially added to a 100 ml single-mouth bottle. The oil bath was heated to 80 ° C for overnight reaction; TLC detection TRN158-c disappeared, the reaction solution was cooled to room temperature, poured into a mixture of ice water and DCM, and the aqueous layer was washed twice with DCM, then the DCM layer was combined with saturated brine. After washing twice, then drying and drying the organic layer; passing the column to obtain 200 mg of a brown solid, the analytical data of the compound is as follows:

^{1 H-NMR (400MHz, CDCl} 3): δ: 10.18 (br, 1H); 9.85 (s, 1H); 9.12 (br, 1H); 8.38 (s, 1H); 8.06 (dd, J = 2.92, J =8.64,1H);7.72(s,1H);7.39(dd,J=1.84,J=9.24,1H);7.23-7.30(m,2H);6.79(s,1H);6.33-6.48(m, , 2H); 5.71 (dd, J=2.20, J=11.92, 1H); 3.88 (s, 3H); 2.89 (t, J = 11.2, 2H); 2.70 (s, 3H); 2.26-2.29 (m, 8H); MS+1: 504, which is the target compound 1.

Example 2

Target compound 2, the structural formula is as follows:

The synthetic route is as follows:

Synthesis of compound TRN15802-2:

The compound TRN15801-1 (500 mg, 2.02 mmol) was added to a reaction flask, then 10 ml of isopropanol was added, followed by TRN158-d (416 mg, 2.2 mmol) and p-toluenesulfonic acid monohydrate (475 mg, 2.5 mmol). Then, under reflux with argon, a reflux reaction was added overnight. The reaction solution was cooled to room temperature, and then a part of the solvent was concentrated, then placed in an ice bath, and then allowed to stand for crystallized, filtered, and the filter cake was washed twice with acetonitrile and dried to give 524 mg of product. MS+1: 401.3.

Synthesis of compound TRN15802-3:

Compound TRN15802-2 (520 mg, 1.3 mmol), N,N,N'-trimethylethylenediamine (265 mg, 2.6 mmol) and potassium carbonate (359 mg, 2.6 mmol) were added to the reaction flask, followed by 10 ML N-methylpyrrolidone, reacted at 120 °C for 5 hours, the reaction solution was brought to room temperature, then poured into ice water, stirred for 30 minutes, then filtered, and the filter cake was collected, washed twice with methyl t-butyl ether, and dried. Get 500mg of product. MS+1: 483.3.

Synthesis of compound TRN15802-4:

Compound TRN15802-3 (500 mg, 1.03 mmol) was added to a three-necked flask, then 10 ml of ethanol and 10 ml of a saturated aqueous solution of ammonium chloride were added, followed by the addition of iron powder (560 mg, 10 mmol). The mixture was heated to reflux for 8 hours, then the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated to dryness, and then 50 ml of dichloromethane was added, and then washed with water, and washed once with brine, dried organic layer and concentrated to give 420 mg of product . MS+1: 453.3.

Synthesis of compound TRN15802:

The compound TRN15802 (400 mg, 0.88 mmol) was added to a three-necked flask, and then 20 ml of anhydrous dichloromethane and diisopropylethylamine (206 mg, 1.6 mmol) were added, and then cooled to 0 degree under argon atmosphere. Then, 5 ml of acryloyl chloride (90 mg, 1 mmol) in anhydrous dichloromethane was added dropwise, and the addition was completed in about 30 minutes. After the addition, the temperature was automatically raised to room temperature overnight. The reaction solution was poured into ice water, and then extracted with dichloromethane (3 ml of dichloromethane), and the organic phase was combined, and the organic phase was washed with brine, and the organic phase was dried and concentrated to give 245 mg of product. The analytical data for this compound are as follows:

^{1 H-NMR (400MHz, CDCl} 3): δ: 10.18 (br, 1H); 9.85 (s, 1H); 9.12 (br, 1H); 8.38 (s, 1H); 8.06 (dd, J = 2.92, J =8.64,1H);7.72(s,1H);7.39(dd,J=1.84,J=9.24,1H);7.23-7.30(m,2H);6.79(s,1H);6.33-6.48(m, , 2H); 5.71 (dd, J=2.20, J=11.92, 1H); 2.89 (t, J=11.2, 2H); 2.70 (s, 3H); 2.26-2.29 (m, 8H); MS+1: 507.3.

The synthesis of the compound TRN158-d was synthesized by referring to the preparation method of the patent document CN105237515B.

Example 3

Target compound 3, the structural formula is as follows:

The synthetic route is as follows:

The intermediate TRN158-e was purchased from the BEHRINGER Reagent Company.

The synthetic route of the intermediate TRN158-f is as follows:

Synthesis of Compound 3:

Compound 1 (5 g, 71 mmol) was added to a three-necked flask, then 100 ml of ethanol, triethylamine (7.2 g, 71 mmol), benzaldehyde (7.5 g, 71 mmol) was added at 0 °. Tetraisopropyl titanate (21.6 g, 76 mmol) was added dropwise at 0 ° C. After the addition was completed, the mixture was slowly warmed to room temperature overnight. On the next day, the reaction solution was brought to 0 °C, and sodium borohydride (2.8 g, 74 mmol) was added in portions over 2 hours. After the addition, the reaction was continued at 0 °C for 4 hours, and then saturated with a saturated aqueous solution of ammonium chloride. The reaction was quenched, filtered, and the filtrate was concentrated and then then th MS+1: 125.1.

Synthesis of Compound 5:

Compound 3 (4.6 g, 36.8 mmol) and Compound 4 (5.3 g, 36.8 mmol) were added to the reaction flask, and then 30 ml of ethanol was added thereto, and 30 ml of an aqueous sodium hydroxide solution (1.5 g) was added dropwise at 0 °C. 36.9 mmol), after the addition was completed, the reaction solution was slowly heated to reflux for 3 hours. The reaction was allowed to come to room temperature, then the mixture was evaporated, evaporated, mjjjjjjj MS+1: 196.2.

Synthesis of compound TRN158-f:

Compound 5 (3.1 g, 15.8 mmol) was added to a reaction flask, then 100 mg of wet palladium carbon was added, and the reaction was allowed to stand overnight under a hydrogen atmosphere. MS+1: 106.1.

The synthesis procedures of the compounds TRN15803-2, TRN15803-3, TRN15803-4 and TRN15803 were carried out in accordance with the corresponding steps in Example 2. The analytical data of the synthesized target compound TRN15803 is as follows:

^{1 H-NMR (400MHz, CDCl} 3): δ: 10.18 (br, 1H); 9.85 (s, 1H); 9.12 (br, 1H); 8.38 (s, 1H); 8.06 (dd, J = 2.92, J =8.64,1H);7.72(s,1H);7.39(dd,J=1.84,J=9.24,1H);7.23-7.30(m,2H);6.79(s,1H);6.33-6.48(m, , 2H); 5.71 (dd, J=2.20, J=11.92, 1H); 3.88 (s, 3H); 2.89 (t, J = 11.2, 2H); 2.26-2.29 (m, 8H); MS+1: 507.3.

Example 4

Target compound 4, the structural formula is:

The synthetic route is:

The synthesis of the intermediate TRN158-g was carried out in accordance with the preparation method of the patent CN105237515B.

The synthesis of the compounds TRN15804-2, TRN15804-3, TRN15804-4 and TRN15804 was carried out in accordance with the corresponding steps in Example 2. The analytical data of the synthesized target compound TRN15804 is as follows:

^{1 H-NMR (400MHz, CDCl} 3): δ: 10.18 (br, 1H); 9.85 (s, 1H); 9.12 (br, 1H); 8.38 (s, 1H); 8.06 (dd, J = 2.92, J =8.64,1H);7.72(s,1H);7.39(dd,J=1.84,J=9.24,1H);7.23-7.30(m,2H);6.79(s,1H);6.33-6.48(m, , 2H); 5.71 (dd, J=2.20, J=11.92, 1H); 3.88 (s, 3H); 2.89 (t, J = 11.2, 2H); 2.70 (s, 3H); 2.26-2.29 (m, 2H); MS+1: 510.3.

Referring to the synthesis method of the above examples, a series of specific compounds were separately prepared. See Table 1.

Example 5 - Example 12

The target compounds synthesized in Examples 5-12 and their characterization data are shown in Table 1.

Table 1

Example 13

The methanesulfonate salt of the compound TRN15801 was synthesized, and the synthesis route was as follows:

Specific procedure: Compound TRN15801 (100 mg, 0.2 mmol) was added to the reaction flask, then 10 ml of acetonitrile was added, then methanesulfonic acid (20 mg) was added, followed by heating under reflux for 3 hours, then cooled to room temperature, and the product was filtered. 96 mg.

Effect experiment

Experimental example 1

Inhibition of the activity of wild-type EGFR and mutant EGFR kinase was examined using the compounds provided in the above examples.

The inhibitory effect of the test substance on the activity of double mutant EGFR kinase (EGFR T790M/L858R kinase) and wild type EGFR kinase (EGFR WT) was determined. Both wild-type EGFR and mutant EGFR (T790M/L858R) kinases used in the assay were purchased from Carna Bioscience.

The experimental process is as follows:

First, the preparation of the test compound

1. The compound to be tested was separately prepared into a 10 mM (mmol/L) DMSO solution, and the control compound AZD9291 was formulated into a 1 mM (mmol/L) DMSO solution;

2. Serially dilute the test compound solution to 12 concentrations (or other desired test concentration) on a 384-well plate of TECAN EVO200 by 3-fold dilution;

3. Transfer 20 nL of the test compound solution to a 384-well plate (Coring 3570) using Echo550. DMSO was used as a blank control.

Second, carry out enzyme testing

1. Prepare a 1.3X enzyme solution containing enzyme, matrix and cofactor, as shown in Table 2 below;

2, at room temperature, 15 μL of 1.3X enzyme solution was added to the wells of each well for 30 minutes;

3. Start the test by adding 5 μL of 4X ATP solution. The volume of the solution in each well should be 20 μL, and the composition is as shown in Table 2 below;

4. The well plate was incubated at room temperature for 90 minutes, and then the test reaction was terminated by adding 40 μL of stop buffer (containing 0.5 M EDTA);

5. Analyze the experimental data of each well using EZ detection.

Table 2 Enzyme solution parameter table in enzyme test

Third, data analysis

1. Using the read conversion ratio (CR), calculate the inhibition ratio according to the following formula:

2. Calculate the IC50 and Ki values using XLFit (equation 201) according to the following formula.

The test results of some of the compounds are shown in Table 3 below.

Table 3 Activity inhibition test results of wild-type EGFR and mutant EGFR kinase

The respective test compounds in Table 3 are as follows.

The structural formula of the compound of Example 1 of the present invention is:

Control compound 2 is a compound disclosed in the authorization publication number CN105237515B, and its structural formula is:

Control compound 3 is a compound disclosed in the authorization publication number CN104140418B, and its structural formula is:

The structure of Control Compound 1 (AZD9291, trade name: Ostinib) is as follows:

As can be seen from the test data of Table 3, the compound of Example 1 of the present invention had better kinase activity than Comparative Compound 1, Control Compound 2 and Control Compound 3.

Experimental example 2

Compounds tested for cell growth inhibitory activity. Test methods and procedures are carried out using methods well known to those skilled in the art, and the reagents used in the methods are commercially available.

Test Methods:

2.1 Experimental steps:

(1) Using Echo (Non-Contact Nano-Upgrade Acoustic Pipetting System), take 40 nL of the test compound solution to the test plate.

(2) The cells were formulated into a 25000 cell/mL solution, and then 40 μL was taken to the designated 384-well test plate.

(3) The plate was incubated at 37 ° C, 5% carbon dioxide, 95% humidity for 72 hours.

试剂。 (4) Add 40 μL to each well

Reagents.

(5) The test plate was incubated at room temperature for 30 minutes to stabilize the luminescence signal.

(6) Seal the test plate to remove air bubbles at a centrifugal speed of 1000 rpm.

(7) Shake the test plate on a shaker for 1 minute.

(8) Read the test board data.

2.2 Data Processing

(1) Calculate the residual rate using the following formula:

S: test sample reading;

V: blank sample reading;

M: AZD9291 test sample (1 μM for testing PC-9 and H1975, 30 μM for A431 test) reading;

The IC50 was calculated using XLFIT (V5.3.1.3) software.

The test results are shown in Table 4:

Table 4 Compound cytostatic activity

The structural formulas of Comparative Compound 1, Control Compound 2, and Control Compound 3 in Table 4 were the same as the corresponding compounds in Table 3.

As can be seen from Table 4, the compound of Example 1 of the present invention exhibited strong inhibitory activity against EGFR mutant cells (H1975, PC-9), compared with the control compound 1 and the control compound 2 and the control compound 3. The compounds of the invention have a higher inhibitory activity on the growth of EGFR mutant cells.

Experimental example 3

Experimental studies on the in vitro metabolic stability of compounds, methods and procedures are carried out using methods well known to those skilled in the art, and the reagents used in the methods are commercially available.

Test Methods:

3.1 Experimental design:

(1) The main solution of this experiment was configured according to the following table.

table 5

Reagent Spare concentration volume Final concentration Phosphate buffer 200mM 200μL 100mM

Ultra-pure water - 106μL - Magnesium chloride solution 50mM 40μL 5mM

(2) Perform two test experiments as follows:

a) Reducing Coenzyme II (NADPH) is required: 10 μl of 20 mg of liver microparticles per ml is mixed with 10 mmol of reduced coenzyme II (NADPH) to incubate. Finally, the concentrations of liver microparticles and reduced coenzyme II (NADPH) were set at 0.5 mg per ml and 1 mmol, respectively.

b) Reducing Coenzyme II (NADPH) is not required: 10 μl of 20 mg of liver microparticles per ml and 40 μl of ultrapure water are added to incubate. The final concentration of liver particles is 0.5 mg per ml.

(3) Start the test by adding 4 μl of 200 μmol of the internal control compound or the compound to be tested. In this study, Verapamil was used as a positive control sample. The concentration of the compound or internal control compound to be tested is finally 2 micromolar.

(4) At 0, 15, 30, 45 and 60 minutes, a small amount of incubation solution was taken for testing. A small amount of the incubation solution was diluted with 4 volumes of cold acetonitrile with IS (100 nanomoles of alprazolam, 200 nanomoles of labetalol, 200 nanomolar caffeine and 2 micromoles of ketoprofen) stopped the reaction. The sample was centrifuged for 40 minutes, and 100 μl of a small amount of filtrate and 100 μl of ultrapure water were mixed and analyzed by liquid chromatography/mass spectrometry.

(5) Data analysis

All recorded data is recorded in the Microsoft excel table.

The peak area was determined from the extracted ion chromatogram. The slope value K is determined by a linear regression of the natural logarithm of the percentage of parent drug residue versus the incubation time curve.

The in vitro half-life (in vitro t _1/2 ) is calculated by the following formula:

In vitro half-life = - (0.693 / k).

In vitro t _1/2 (min) conversion to intrinsic clearance in vitro (CL _int (in vitro CL _int ), μL/min/mg protein) was performed using the following equation (average of repeated assays):

The in vitro t _1/2 (min) was converted to an amplified intrinsic clearance (magnification CL _int at mL/min/kg) using the following equation (average of repeated determinations):

Table 6 Conversion factors for prediction of clearance of liver microsomes in vivo

a. Iwatsubo et al., Davies and Morris, 1993, 10(7) pp 1093-1095

b. Barter et al., 2007, Curr Drug Metab, 8(1), pp 33-45; Iwatsubo et al., 1997, JPET, 283 pp 462-469.

3.2 Experimental results

The results of in vitro liver particle metabolism stability experiments are shown in Table 7 below:

Table 7 Stability of in vitro liver particle metabolism of compounds

The control compound 2 and the control compound 3 in Table 7 have the same structural formula as in Table 3.

As is apparent from the experimental data of Table 7, the compound of Example 1 of the present invention has better metabolic stability in human liver microparticles than AZD9291 and Comparative Compound 2 and Control Compound 3. This indicates that the compounds of the present invention have better pharmacokinetic stability and in vivo activity in humans, and are very suitable for subsequent drug development.

The compounds provided in the above examples of the invention can be used to prepare EGFR inhibitors.

The compounds provided in the examples of the present invention can be used for the preparation of a medicament for regulating EGFR tyrosine kinase activity or treating EGFR-related diseases. Among them, the EGFR-related diseases are selected from the group consisting of cancer, diabetes, immune system diseases, neurodegenerative diseases, and cardiovascular diseases.

The compounds provided in the examples of the present invention can be used for the preparation of a medicament for treating non-small cell lung cancer.

The compounds provided in the examples of the present invention may be mixed with a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.

The above is only the embodiment of the present invention, and is not intended to limit the scope of the invention, and equivalent transformations made by the contents of the specification of the present invention, or directly or indirectly applied to other related technical fields, are included in the present invention. Within the scope of patent protection.

Claims (12)

a compound of formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

among them: R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently selected from methyl or deuterated methyl; R _{6 is} selected from H or hydrazine. The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein each of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ Independently selected from methyl or -CD ₃ . The compound according to claim 2, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein said R ₁ , R ₂ , R ₃ , R ₄ , R ₅ At least one is -CD ₃ . The compound according to claim 3, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein said R ₁ , R ₂ , R ₃ , R ₄ , R ₅ There are 1 to 3 of -CD ₃ . The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, wherein the compound is selected from the group consisting of the compounds represented by the following structural formula:

The compound according to any one of claims 1 to 5, wherein the pharmaceutically acceptable salt is a mineral acid salt or an organic acid salt selected from the group consisting of a hydrochloride salt, a hydrobromide salt, and a hydrogen salt. Iodate, sulphate, hydrogen sulphate, nitrate, phosphate, acid phosphate; the organic acid salt is selected from the group consisting of formate, acetate, trifluoroacetate, propionate, pyruvate , glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate , benzenesulfonate, salicylate, picrate, glutamate, ascorbate, camphorate, camphorsulfonate. The compound according to claim 6, wherein the inorganic acid salt is a hydrochloride or a sulfate; and the organic acid salt is a methanesulfonate. A pharmaceutical composition comprising the compound of any one of claims 1-7 and a pharmaceutically acceptable carrier. Use of a compound according to any one of claims 1 to 7 for the preparation of an EGFR inhibitor. Use of a compound according to any one of claims 1 to 7 for the manufacture of a medicament for the modulation of EGFR tyrosine kinase activity or for the treatment of EGFR related diseases. The use according to claim 10, wherein the disease is selected from the group consisting of cancer, diabetes, immune system diseases, neurodegenerative diseases, and cardiovascular diseases. Use of a compound according to any one of claims 1 to 7 for the manufacture of a medicament for the treatment of non-small cell lung cancer.