CN108530450B

CN108530450B - Compound with EGFR (epidermal growth factor receptor) inhibitory activity, preparation method and application of compound in disease treatment

Info

Publication number: CN108530450B
Application number: CN201810416627.3A
Authority: CN
Inventors: 赖建智
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-05-03
Filing date: 2018-05-03
Publication date: 2021-03-30
Anticipated expiration: 2038-05-03
Also published as: CN108530450A

Abstract

The invention relates to an anti-tumor compound, a preparation method and application thereof in preparing a medicament for treating EGFR drug-resistant mutant mediated diseases, and the compound has good selectivity on EGFR T790M mutant tumors.

Description

Compound with EGFR (epidermal growth factor receptor) inhibitory activity, preparation method and application of compound in disease treatment

Technical Field

The invention belongs to the field of medicines, and particularly relates to a compound with EGFR (epidermal growth factor receptor) inhibitory activity, a preparation method and application thereof in disease treatment.

Background

Egfr (epidemal Growth Factor receptor) is a member of the erbB receptor family of transmembrane protein tyrosine kinases. First generation small molecule EGFR inhibitors, including gefitinib and erlotinib, showed superior efficacy in the treatment of lung cancer. However, almost all NSCLC patients develop resistance to small molecule inhibitors of this type after treatment with first generation small molecule EGFR inhibitors. The drug resistance mechanism comprises EGFR secondary mutation, bypass activation and the like. In half of the patients, the drug resistance is caused by the secondary mutation of T790M, thereby reducing the affinity of the drug and the target point to generate drug resistance, and causing the recurrence of the tumor or the disease progression.

Second generation small molecule EGFR inhibitors, such as natantib, etc., but have not shown therapeutic efficacy due to severe skin and gastrointestinal toxicity resulting from stronger inhibition of wild type EGFR by natantib, limiting the dose administered.

Therefore, there is a need for the development of third generation small molecule EGFR inhibitors that inhibit the EGFR T790M mutant with high selectivity and no or low activity against wild-type EGFR. Multiple classes of third-generation small molecule EGFR inhibitors are currently reported, as disclosed in CN201180030338.6, and in patent documents CN201180060966.9, CN201280033773.9, etc., wherein AZD-9291 has obtained FDA approval for marketing.

Disclosure of Invention

Through research, the inventors found a series of compounds with completely novel structures and the activities of inhibiting an L858R EGFR mutant, a T790M EGFR mutant and an exon 19 deletion activation mutant. Has good application prospect in preventing or treating various cancers, such as non-small cell lung cancer.

In one aspect, the invention provides a compound of formula I

Wherein R is₁And R₂Independently selected from hydrogen, alkyl, halogen;

R₃optionally selected from alkyl, alkoxy, haloalkoxy;

R₄and R₅Independently selected from hydrogen, alkyl, substituted alkyl, - (CH)₂)n-NR₆R₇、R₆R₇N-(CH₂) n-; or R₄、R₅Form a ring, which may be substituted by one or two N atoms;

R₆、R₇h, methyl, ethyl;

n is an integer of 0 to 4.

In a further aspect of the invention there is provided a compound of formula I

Wherein R is₁And R₂Independently selected from hydrogen, methyl, fluorine, chlorine;

R₃is C₁-C₄Alkyl radical, C₁-C₄Alkoxy, perhalogenated C₁-C₄Alkoxy or partially halogenated C₁-C₄An alkoxy group; in a preferred embodiment of the invention, R₃Is methyl, methoxy, trifluoromethoxy or trifluoroethoxy;

R₄、R₅independently selected from hydrogen, C₁-C₆Alkyl, - (CH)₂)n-NR₆R₇、R₆R₇N-(CH₂) n-; or R₄、R₅Form a ring, which may be substituted by one N atom; in a preferred embodiment of the invention, R₄R₅N-is piperidinyl or 4-methylpiperazinyl; r₆、R₇Is independently selected fromHydrogen, methyl;

n is an integer of 0 to 2.

In a preferred embodiment of the invention, R₃Is methyl, methoxy or trifluoromethoxy.

In a preferred embodiment of the invention, R₄R₅N-is 4-methylpiperazinyl.

In a preferred embodiment of the invention, n is 1.

Preferred compounds of the invention include:

in yet another aspect, the invention provides a method for synthesizing a compound of formula I,

wherein X is halogen, hydroxyl, alkoxy; r₁～R₅As defined for formula I; the method is prepared by reacting the formula II with the formula III under the condition of a condensation reagent, wherein the condensation reagent is a condensation reagent which is conventional in condensation reaction, such as dicyclohexylcarbodiimide.

In yet another aspect of the invention, there is provided a process for the preparation of formula II,

the preparation of formula II can be described by reference to the method disclosed in Tetrahedron letters.53,2012, Page 1720-1724, to Hiroki Wada et al, wherein R₁～R₅As defined for formula I; y is halogen, preferably fluorine, chlorine, bromine or iodine.

In another aspect, the present invention provides a pharmaceutical preparation containing the compound of formula I, which can be solid dosage forms for oral administration, including capsules, tablets, granules, powders, etc., and the corresponding dosage forms can be quick release, sustained release, enteric coated, etc. In solid dosage forms, the compounds of formula I of the present invention are mixed with one or more excipients including, but not limited to, solubilizers, binders, disintegrants, surfactants, lubricants, and the like. The solubilizer is selected from the components commonly used in the preparation, such as starch, sucrose, mannitol and the like; binders such as polyvinylpyrrolidone, hydroxymethyl cellulose, and the like; disintegrants such as sodium carboxymethyl starch, low substituted celluloses, crospovidone, etc.; surfactants such as polyhydric alcohols, polysorbates, sodium alkylsulfonates, and the like; lubricants such as talc, magnesium stearate, calcium stearate, and the like.

In a further aspect, the invention provides the use of a compound comprising formula I for the preparation of a medicament for the treatment of diseases caused by EGFR, non-limiting cancers such as non-small cell lung cancer, breast cancer, ovarian cancer, adenocarcinoma and the like, preferably having a L858R mutation or a T790M mutation or a L858R mutation or a T790M mutation.

Detailed Description

The structure of the compounds of the invention was confirmed by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated methanol (CD)₃OD) and deuterated chloroform (CDCl)₃) Internal standard is Tetramethylsilane (TMS).

All reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere without specific indication, the solvent is a dry solvent, and the reaction is carried out at room temperature of 20-25 ℃. Unless otherwise specified, the reagents used in the preparation process of the present invention are commercially available and used directly, and the starting materials are synthesized by reference to the methods of the prior art.

EXAMPLE 1 preparation of intermediate A4

Step 1

In a reaction flask, compound a1(2g,8.0mmol) was dissolved in acetonitrile (100mL), triethylamine (0.45g,8.8mmol) was added dropwise at room temperature, the reaction was refluxed for 5h, diluted hydrochloric acid aqueous solution was added, ethyl acetate was extracted 3 times (300mL), sodium bicarbonate solution was washed, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried by spinning to give compound a2(1.6g, 87.9% yield).

Step 2

In a reaction flask, compound A2(1.6g,7.04mmol) is dissolved in tetrahydrofuran (100mL), cooled to-78 ℃, n-hexane solution of n-butyllithium (14.1mmol,2.0eq) is added dropwise, after stirring reaction for 0.5h, tetrahydrofuran solution (50mL) of p-toluenesulfonylazide (2.08g,10.56mmol) is added dropwise, after stirring reaction for 0.5h, the temperature is raised to room temperature, washing with ammonium chloride solution, extraction with diethyl ether (400mL) is carried out for 3 times, and the organic phase is dried with anhydrous sodium sulfate and then carried on to the next reaction. Pd/C (0.2eq) was added to the reaction solution, and reduction was performed by hydrogen gas, followed by reflux reaction for 3 hours, filtration, washing of the organic phase with a saturated sodium chloride solution, drying with anhydrous sodium sulfate, and spin-drying under reduced pressure to obtain compound a3(0.8g, yield 82.2%).

Step 3

Compound A3(0.8g, 5.79mmol) was dissolved in ethanol solution (100mL) in a reaction flask, CsCO was added₃(1.68g,8.68mmol), ethyl acrylate (5.91mmol,1.02eq) was added, the reaction was heated to reflux until no starting compound was present, cooled to room temperature, filtered, extracted with dichloromethane solution (400mL), and the combined organic phases were dried over anhydrous sodium sulfate to afford intermediate compound a4(0.8g, 71.9% yield). MS M/z (ESI) 193.1[ M +1]]

EXAMPLE 2 preparation of Compound 1

Step 1

Adding 50mL of 2-methoxy-4- (4-methyl-1-piperazinyl) -aniline (1.0g, 4.52mmol), adding 50mL of tetrahydrofuran, adding triethylamine (0.91g,9.04mmol), placing in a 150mL round-bottom flask, adding ethyl 4- (2-chloro-5-fluoro-pyrimidin-4-yl) formate (0.934g,4.56mmol) while stirring, refluxing for 6h, evaporating to remove the solvent after the reaction is finished, adding water for dilution, extracting with ethyl acetate (150mL), washing the organic phase with a saturated ammonium chloride solution and a saturated sodium chloride solution, adding anhydrous sodium sulfate for drying, and purifying by silica gel column chromatography to obtain 0.81g of a solid ethyl 2- [ 2-methoxy-4- (4-methyl-1-piperazinyl) -anilino ] -5-fluoro-pyrimidin-4-yl formate, the yield was 46%.

Step 2

The compound ethyl 2- [ 2-methoxy-4- (4-methyl-1-piperazinyl) -anilino ] -5-fluoro-pyrimidin-4-yl formate (0.81g,2.08mmol) obtained in step 1 was placed in a 150mL reaction flask, 50mL of xylene was added, triethylamine (0.42g,4.16mmol) was added, compound A4(0.4g,2.08mmol) was added, the mixture was reacted at 80-100 ℃ for 10 hours, the temperature was reduced to room temperature, the mixture was diluted with water and extracted with ethyl acetate (250mL), the organic phase was washed with a saturated ammonium chloride solution and a saturated sodium chloride solution, dried with anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain 0.72g of the title compound 1 as a solid with a yield of 64.6%. MS M/z (ESI) 535.3[ M +1]

1H NMR(400MHz，DMSO-d₆)：10.10(s,1H),9.31(d,J＝9Hz,1H),7.42(s,1H),7.18(s,1H),6.49(dd,J＝16.7,10.1Hz,1H),6.37(m,1H),6.24(m,1H),,6.05(m,1H),6.0(br,1H),5.65(dd,J＝10.0Hz，2.0Hz,1H),4.35(s,2H),4.27(m,2H),3.89(m,2H),3.65(s,3H),3.35(br,4H),2.35(br,4H),2.23(s,3H)

EXAMPLE 3 preparation of Compound 2

Referring to the procedure for the preparation of compound 1 of example 2, using ethyl 2- [ 2-methoxy-4- (dimethylaminoethyl) (methyl) amino-anilino ] -5-fluoro-pyrimidin-4-ylcarboxylate (1g, mmol) to react with compound A4, the title compound 2 was prepared as a solid 0.7g in 62.6% yield. MS M/z (ESI) 538.3[ M +1]

1H NMR(400MHz，DMSO-d₆)：10.07(s,1H),9.31(d,J＝9Hz,1H),7.42(s,1H),7.18(s,1H),6.49(dd,J＝16.7,10.1Hz,1H),6.37(m,1H),6.24(m,1H),6.05(m,1H),6.0(br,1H),5.65(dd,J＝10.0Hz，2.0Hz,1H),4.35(s,2H),4.27(m,2H),3.89(m,2H),3.65(s,3H),3.45(br,2H),2.72((s,3H),2.45(br,2H),2.23(s,6H)

EXAMPLE 4 preparation of Compound 3

Referring to the procedure for preparing compound 1 in example 2, using ethyl 2- [ 2-methoxy-4- (4-methyl-1-piperazinyl) -anilino ] -6-chloro-pyrimidin-4-ylcarboxylate (1g, mmol) to react with compound A4, the title compound 3 was prepared as a solid in 0.5g, 45.4% yield. MS M/z (ESI) 552.2[ M +1]

1H NMR(400MHz，DMSO-d₆)：10.10(s,1H),7.94(s,1H),7.42(s,1H),7.18(s,1H),6.49(dd,J＝16.7,10.1Hz,1H),6.37(m,1H),6.24(m,1H),6.05(m,1H),6.0(br,1H),5.65(dd,J＝10.0Hz，2.0Hz,1H),4.35(s,2H),4.27(m,2H),3.89(m,2H),3.65(s,3H),3.35(br,4H),2.35(br,4H),2.23(s,3H)

EXAMPLE 5 preparation of Compound 4

Referring to the procedure for the preparation of compound 1 of example 2, using ethyl 2- [ 3-trifluoromethoxy-4- (4-methyl-1-piperazinyl) -anilino ] -5-chloro-pyrimidin-4-ylcarboxylate (1g, mmol) to react with compound A4, the title compound 4 was prepared as a solid 0.55g in 41.7% yield. MS M/z (ESI) 606.2[ M +1]

1H NMR(400MHz，DMSO-d₆)：10.10(s,1H),8.54(s,1H),7.42(s,1H),7.18(s,1H),6.49(dd,J＝16.7,10.1Hz,1H),6.37(m,1H),6.24(m,1H),6.05(m,1H),6.0(br,1H),5.65(dd,J＝10.0Hz，2.0Hz,1H),4.35(s,2H),4.27(m,2H),3.89(m,2H),3.35(br,4H),2.35(br,4H),2.23(s,3H)

With reference to the above preparation methods, the present invention further includes, without limitation, compounds of the following structures:

example 10 biological assay

EGFR T790M mutant enzymology experiment

In the experiment, the inhibition effect of the compound on the exon 20T790M mutant EGFR enzyme is tested by adopting a fluorescence resonance energy transfer (TR-FRET) method, and the half inhibition concentration IC of the compound on the enzyme activity is obtained₅₀。

1) Adding 1-5 ul of EGFR T790M enzyme solution into a 96-well plate, wherein the final enzyme concentration is 0.1-1 nM.

2) And adding 1-5 ul of the gradient diluted solution of the compound to be detected, and incubating for 10 minutes at room temperature.

3) Adding 1-5 ul of substrate mixed solution containing 5-50 nM of substrate polypeptide and 1-10 uM of ATP final concentration, and incubating at room temperature for 0.5-2 hours.

4) 5ul of EDTA stop solution was added to stop the reaction for 5 minutes.

5) 5ul of the detection solution containing the labeled antibody was added and incubated at room temperature for 1 hour.

6) The microplate reader measures the 665nm fluorescence signal value of each plate.

7) The inhibition rate was calculated from the fluorescence signal value.

8) Obtaining the IC of the compound by curve fitting according to the inhibition rates of different concentrations₅₀。

EGFR Wild Type (WT) enzymology experiment

In the experiment, the inhibition effect of the compound on the wild type EGFR enzyme is tested by adopting a fluorescence resonance energy transfer (TR-FRET) method, and the half inhibition concentration IC of the compound on the activity of the enzyme is obtained₅₀。

1) Adding 1-5 ul of EGFR wild type enzyme solution into a 96-well plate, wherein the concentration is 0.1-1 nM.

2) Adding 1-5 ul of the compound solution which is diluted in a gradient manner.

3) Incubate for 10 minutes at room temperature.

4) Adding 1-5 ul of substrate mixed solution containing 5-50 nM of substrate polypeptide and 0.1-5 uM of ATP final concentration.

5) And incubating for 0.5-2 hours at room temperature.

6) 5ul of EDTA stop solution was added to stop the reaction for 5 minutes.

7) 5ul of the detection solution containing the labeled antibody was added and incubated at room temperature for 1 hour.

8) The microplate reader measures the 665nm fluorescence signal value of each plate.

9) The inhibition rate was calculated from the fluorescence signal value.

10) Obtaining the IC of the compound by curve fitting according to the inhibition rates of different concentrations₅₀。

The IC of the compounds of the invention was determined according to the above method₅₀As in the following table:

the embodiment of the invention has strong inhibitory activity to EGFR mutant kinase, and simultaneously has weak inhibitory activity to wild type kinase, thereby showing excellent selectivity.

Claims

1. A compound which is:

2. a pharmaceutical composition comprising the compound (1) as set forth in claim 1 and a pharmaceutically acceptable carrier.

3. Use of a compound of claim 1 for the manufacture of a medicament for treating a disease mediated by an EGFR-resistant mutant.

4. The use according to claim 3, wherein the EGFR-resistant mutant is an EGFR T790M mutant.