CN107840847A

CN107840847A - Deuterated 3 (4,5 substituted-amino pyrimidine) phenyl derivatives and its application

Info

Publication number: CN107840847A
Application number: CN201610833361.3A
Authority: CN
Inventors: 朱永强; 刘兆刚; 冯超; 陈浩; 白恩赫; 王佳; 石晶淼
Original assignee: Jiangsu Chia Tai Fenghai Pharmaceutical Co Ltd
Current assignee: Jiangsu Chia Tai Fenghai Pharmaceutical Co Ltd
Priority date: 2016-09-19
Filing date: 2016-09-19
Publication date: 2018-03-27

Abstract

The invention discloses one kind deuterated 3 (4,5 substituted-amino pyrimidines) phenyl derivatives and its application, it is compound or its pharmaceutically acceptable salt with formula (I) structure, these compound or its salts can be used for the treatment or prevention of disease or the patient's condition by the EGF-R ELISA of some mutant forms, the growth of kinds of tumor cells can effectively be suppressed, and inhibitory action is produced to EGFR, Her family other protease, available for preparing antineoplastic.

Description

Deuterated 3- (4, 5-substituted aminopyrimidine) phenyl derivative and application thereof

Technical Field

The invention belongs to the technical field of antitumor drugs, and particularly relates to a deuterated 3- (4, 5-substituted aminopyrimidine) phenyl derivative and application thereof in preparation of an antitumor drug.

Background

Chemotherapy is the primary treatment in the traditional cancer treatment; chemotherapy drugs nonspecifically block cell division to cause cell death, and when they kill tumor cells, they also greatly destroy the growth of normal cells of human body, causing many adverse reactions. Many people worry about serious side effects of chemotherapy and are in a pessimistic state even abandon treatment, and due to the drug resistance of the chemotherapy drugs, the chemotherapy of the non-small cell lung cancer (NSCLC) is not optimistic, and the prolonging of the chemotherapy period only increases toxic and side effects and does not increase the curative effect. Meanwhile, cancer cells of the non-small cell lung cancer are insensitive to chemotherapy and conventional chemotherapy, and the total remission rate is only about 25%; due to these limitations, the five-year survival rate of non-small cell lung cancer patients is less than 20%.

In 50% -80% of NSCLC patients, their Epidermal Growth Factor Receptor (EGFR) is overexpressed, causing canceration. There are two main classes of EGFR-targeting drugs: one class is small molecule Tyrosine Kinase Inhibitors (TKIs) that act on the intracellular domain of the receptor; another class is monoclonal antibodies (MAbs) that act on the extracellular domain of the receptor. The first generation EGFR inhibitors which have been applied to clinical treatment, such as Iressa, erlotinib, lapatinib and the like, have achieved great success in the treatment of NSCLC lung cancer, and improve the five-year survival rate of non-small cell lung cancer patients. Meanwhile, compared with chemotherapy, the traditional Chinese medicine composition has the advantages that side effects such as bone marrow suppression, nausea and neurotoxicity are avoided; however, the traditional Chinese medicine composition has low efficacy when being used alone, has very obvious side effects of rash, diarrhea and the like, and can cause drug resistance of patients after being used for one year. The research considers that the mutation of the T790M site of the EGFR gene is the main cause of drug resistance of the drugs, and clinical case data show that about 50 percent of patients have acquired drug resistance caused by the mutation of the T790M site. Further studies have shown that steric hindrance caused by the mutation of the EGFR gene T790M, i.e., the conversion of the encoded threonine to methionine, prevents the binding of the inhibitor to the ATP binding domain, which ultimately results in loss of inhibitor activity. It has also been shown that the mutation at the T790M site does not directly affect the affinity of the inhibitor for EGFR, but rather the mutation results in a large increase in the affinity of EGFR for ATP, resulting in a relatively large decrease in the affinity of the inhibitor for EGFR (competitive binding of the inhibitor to ATP). Second generation inhibitors such as afatinib, Dacomitinib, which are better than the first generation inhibitors characterized by increased recognition of EGFR, allow discrimination between tumor cells and normal cells, and thus have reduced side effects. However, these molecules have poor selectivity for the T790M mutant of EGFR, resulting in a clinically tolerated dose of the drug which, at its Maximum Tolerated Dose (MTD), fails to reach its effective concentration in vivo and is ineffective in most drug resistant patients.

In conclusion, the current EGFR-TKI still cannot solve the clinical requirement caused by drug resistance, and most of the existing drugs are EGFR reversible or irreversible inhibitors taking quinazoline or quinoline amine as a basic parent nucleus, and toxic and side effects caused by poor selectivity on wild cells are inevitable. Therefore, a new class of compounds, especially compounds with novel frameworks, is urgently needed clinically to solve the problems of drug resistance, poor selectivity and the like.

Disclosure of Invention

The invention aims to provide a deuterated 3- (4, 5-substituted aminopyrimidine) phenyl derivative.

The object of the invention can be achieved by the following measures:

the deuterated 3- (4, 5-substituted aminopyrimidine) phenyl derivative is a compound with a structure shown in a formula (I) or a pharmaceutically acceptable salt thereof,

wherein R is₁,R₂,R₃And R₄Is independently selected from-CH₃or-CD₃And R is₁,R₂,R₃And R₄At least one is-CD₃。

Further, the compound with the structure of the formula (I), preferably R₁is-CH₃。

The compounds of the invention, or pharmaceutically acceptable salts thereof, wherein some specific compounds are selected from:

the preparation route of the compound having the general formula (I) is shown below:

the preparation route comprises the following specific steps:

step 1: dimethyl ether (DME) is used as a solvent, and the compound II and 2, 4-dichloropyrimidine are subjected to nucleophilic substitution reaction in the presence of Lewis acid such as aluminum trichloride to obtain III.

Step 2: and (3) obtaining IV by the intermediate III and 4-fluoro-2-methoxyl-5-nitroaniline under the action of p-toluenesulfonic acid.

And step 3: and (3) taking 1, 4-dioxane as a solvent, and reacting the intermediate IV with deuterated organic amine under the action of DIPEA to obtain an intermediate V.

And 4, step 4: and reducing the intermediate V into an intermediate VI by using methanol or ethanol as a solvent and Pd/C as a reducing agent.

And 5: in THF/H₂And O is used as a solvent, the intermediate VI reacts with chloropropionyl chloride to obtain an intermediate compound, and the intermediate compound is directly added with sodium hydroxide to continuously react without separation to obtain the final product, namely the compound shown in the formula I.

The salts which the compounds of the invention may form are also within the scope of the invention. Unless otherwise indicated, the compounds of the present invention are understood to include salts thereof. For example, the compounds of formula (I) may be prepared by reacting a suitable amount, e.g. an equivalent amount, of an acid or base, salting out in a medium, or lyophilizing in an aqueous solution. The compounds of the invention may contain basic moieties, including but not limited to amine or pyridine or imidazole rings, which may form salts with organic or inorganic acids. Typical acids that may be salified include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, p-toluenesulfonate, bisulfate, borate, butyrate, citrate, camphor, camphorsulfonate, cyclopentanepropionate, diglycolate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate.

The compounds of the present invention, obtained by preparing, isolating and purifying the compound in sequence, have a weight content of 90% or more, for example, 95% or more, 99% or more ("very pure" compounds), as set forth in the text. Such "very pure" compounds of the invention are also part of the invention herein.

The invention also discloses application of the compound of the general formula (I) or pharmaceutically acceptable salt thereof in preparing or preventing tumor medicaments.

The tumor is selected from non-small cell lung cancer, pancreatic cancer, breast cancer, prostatic cancer, hepatocarcinoma, skin cancer, epithelial cell cancer, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, and nasopharyngeal carcinoma.

One aspect of the invention provides a compound of formula (I) for use in the treatment or prevention of a disease, disorder or condition mediated by EGFR or mediated by an activating mutant or resistant mutant form of EGFR.

The disease, disorder or condition mediated by EGFR or mediated by EGFR in the form of an activating mutant or a resistant mutant is selected from non-small cell lung cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma or nasopharyngeal cancer.

The EGFR in the form of an activating mutant or a resistance mutant is selected from the group consisting of an L858R activating mutant, an Exon19 deletion activating mutant, and a T790M resistance mutant.

The compounds of general formula (I) of the invention may be used in combination with other drugs known to treat or ameliorate similar conditions. When administered in combination, the mode of administration and dosage of the original drug is maintained, while the compound of formula (I) is administered simultaneously or subsequently. When the compound of formula (I) is administered simultaneously with one or more other drugs, it is preferable to use a pharmaceutical composition containing both one or more known drugs and the compound of formula (I). The pharmaceutical combination also comprises the administration of a compound of formula (I) in an overlapping time period with one or more other known drugs. When a compound of formula (I) is administered in a pharmaceutical combination with one or more other drugs, the dose of the compound of formula (I) or the known drug may be lower than the dose when they are administered alone. Drugs or active ingredients that may be used in pharmaceutical combination with the compounds of formula (I) include, but are not limited to, the following:

estrogen receptor modulators, androgen receptor modulators, retinal receptor modulators, cytotoxins/cytostatics, antiproliferatives, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine/threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, inhibitors of apoptosis, pharmaceutical preparations containing these compounds, and methods of using them, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, ATK inhibitors, integrin blockers, interferons, interleukin-12, COX-2 inhibitors, P53, P53 activators, VEGF antibodies, EGF antibodies, and the like.

In one embodiment, the pharmaceutical or active ingredient which can be used in combination with the compound of formula (I) includes, but is not limited to, a pharmaceutical preparation containing aldesleukin, alendronate, interferon, atranoxine, allopurinol sodium, palonosetron hydrochloride, altretamine, aminoglutethimide, amphetamine, amrubicin, amrinine, valprozin, valtretinomycin, valtretin, doxepirubicin, doxepirubicine, doxine hydrochloride, doxepirubicine, doxine hydrochloride, doxine, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride, doxycycline hydrochloride.

The invention also provides a pharmaceutical composition which comprises the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable auxiliary material or carrier. The phrase "pharmaceutically acceptable adjuvant or carrier" as used herein refers to a pharmaceutically acceptable material, ingredient or vehicle, such as a liquid or solid filler, diluent, adjuvant, solvent or encapsulating material, which includes carrying or transporting a primary pharmaceutical agent from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" and compatible with the other forms of the pharmaceutical composition without causing harm to the patient. Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as wheat starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, powdered tragacanth, malt, gelatin, talc; adjuvants, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butanediol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; physiological saline; ringer's solution; ethanol; phosphate buffer, and other compatible substances that are non-toxic for use in pharmaceutical formulations.

When the compounds of the present invention are administered as medicaments for the treatment of humans and animals, they may be administered per se or as pharmaceutical compositions. For example, it comprises 0.1% to 99.5% (preferably 0.5% to 90%) of the active ingredient and a pharmaceutically acceptable carrier.

The compounds of the present invention may be used to treat diseases by intravenous, intramuscular, intraperitoneal, subcutaneous, topical, oral administration, or other acceptable methods.

The invention also provides a pharmaceutical pack or kit comprising one or more packages containing a pharmaceutical combination of one or more of the ingredients of the invention. Such packaging is optionally produced by a governmental agency regulating the production of bulletins, the use or sale of pharmaceuticals or biologicals by public methods permitted in the manufacturing regulations, and the use or sale of therapeutic agents to humans.

Compared with the prior art, the invention has the beneficial effects that:

the deuterated compound has the biological activity of enzyme and cell levels similar to those of AZD9291 and has lower cardiotoxicity. The deuterated compound provides more choices for novel antitumor drugs and has good drug application prospects.

Detailed Description

The following representative examples are intended to aid in the illustration of the present invention and are not intended to, and should not be construed to, limit the scope of the present invention. Indeed, the entire contents of the documents in this invention, including examples in accordance with the scientific literature and patents cited herein, as well as various modifications and numerous further variations thereof, will be readily apparent to those skilled in the art, except to those shown and described herein. It should also be understood that the citation of these references is helpful in setting forth the disclosure herein. The following examples contain important supplementary information, examples and guidance, and are adaptable to various variations and the like in the present invention.

Example 1

The synthetic route is as follows:

compound 1

N-methylethanolamine (10g, 133.1mmol), TEA (26.9g, 266.3mmol) and acetonitrile (100mL) are added into a 250mL single-neck flask respectively, then benzyl chloride (23.9g, 139.8 mmol) is slowly dropped into the reaction liquid at 0 ℃, stirring is continued for 1h at room temperature, TLC is used for monitoring that no raw material is left, the solvent is removed by reduced pressure distillation, and column chromatography purification is carried out to obtain 21g of colorless liquid, namely compound 1, with the yield of 95.5%.

Compound 2

A250 mL eggplant-shaped bottle was taken, and Compound 1(21g, 127.1mmol), TEA (25.7g, 254.2mmol) and DCM (100mL) were added, followed by dropwise addition of MsCl (14.6g, 127.1mmol) at 0 ℃. The reaction solution was stirred at room temperature for 3h, TLC monitored that no starting material remained, the solvent was removed by distillation under reduced pressure, and column chromatography purification yielded 20g of pale yellow liquid, compound 2, in 85.6% yield.

Compound 3

And taking 500mL of a sealed tube, adding the compound 2(20g, 108.9mmol) and 215mL of ammonia water, stirring the mixed solution at 40 ℃ overnight, monitoring by TLC that no raw material remains, and purifying by column chromatography to obtain 15g of colorless liquid, namely the compound 3, wherein the yield is 84%.

Compound 4

A500 mL eggplant-shaped bottle was charged with Compound 3(15g, 91.3mmol) and DCM (200mL), and Boc was slowly dropped at room temperature₂O (19.9g, 91.3mmol), after the addition was complete, the mixture was stirred at room temperature for 3h, TLC monitored for no starting material remaining, the solvent was distilled off under reduced pressure and column chromatography purification afforded 21g of compound 4 as a white solid in 87% yield.

Compound 5

A100 mL eggplant-shaped bottle was taken, and compound 4(10g, 37.8mmol) and DMF (40mL) were added, followed by addition of NaH (2.3g, 56.7mmol) in portions,stirring for 30mins, adding TsOCD₃(7.9g, 41.6mmol) in DMF (10mL) and then stirred at room temperature for 3H, TLC monitored for no starting material remaining, 150mL H was added₂Quenching with O, extracting with EA (50 mL. times.3), combining the organic phases, washing with brine, and removing anhydrous Na₂SO₄Drying, distilling under reduced pressure to remove solvent, and purifying by column chromatography to obtain 8.5g of compound 5 as white solid with yield of 80%.

Compound 6

A250 mL eggplant-shaped bottle was charged with Compound 5(8.5g, 30.18mmol) and THF (80mL), and LiAlH was added in portions in ice bath₄(3.4g, 90.59mmol), then heated to 60 ℃ overnight, monitored by TLC for no starting material remaining, Na was added slowly₂SO₄·10H₂Quenching with O, filtering to remove solid, collecting filtrate, distilling under reduced pressure to remove solvent, and purifying by column chromatography to obtain 4.5g colorless liquid, i.e. compound 6, with yield of 76.3%.

Compound 7

A100 mL eggplant-shaped bottle was charged with Compound 6(4.5g, 23mmol), MeOH (50mL), Pd (OH)₂C (200mg), changing hydrogen by evacuation for 3 times, stirring overnight at room temperature, monitoring by TLC that no starting material remains, filtering to remove Pd (OH)₂Then, the reaction solution was adjusted to acidic pH, and the solvent was distilled off under reduced pressure to obtain 2.8g of a white solid, i.e., Compound 7, in a yield of 85.9%.

Compound 8

Adding 250mL eggplant-shaped bottles respectively2, 4-dichloropyrimidine (11.37g, 76.33mmol), AlCl₃(10.18g, 76.33mmol) and 100mL of DMDMME, and stirred at room temperature for 20 minutes. Then, compound 8-01(10.00g, 63.61mmol) was added in portions, and the reaction was raised to 80 ℃ for 6 h. The reaction is stopped and cooled to room temperature, 100ml of water is added, the mixture is stirred for 2 hours, filtered, the solid is washed by ethanol and dried in vacuum, 15.46 g of red crude product, namely the compound 8-02 is obtained, and the yield is 90.1%.

300mL of 1, 4-dioxane was placed in a 500mL eggplant-shaped flask, and compound 8-02(20.00g, 82.07mmol), compound 8-03(16.80g, 90.28mmol) and p-toluenesulfonic acid (17.17g, 90.28mmol) were added, respectively. And (3) raising the temperature to 85 ℃ for reaction for 8h, cooling to room temperature, adding water, stirring, dropwise adding a 40% sodium hydroxide solution until the pH value is 9, filtering, washing the solid with ethanol, and drying in vacuum to obtain 30.00g of yellow solid, namely the compound 8, wherein the yield is 92.9%.

Compound 9

A120 mL sealed tube was taken, and compound 8(2g, 4.77mmol), 7(810mg, 5.72mmol), DIPEA (1.23g, 9.54mmol) and DMA (10mL) were added. And then sealing the tube at 140 ℃ for reaction for 6h, monitoring by TLC (thin layer chromatography) that no raw material remains, cooling the reaction solution to room temperature, adding 20mL of water, precipitating a solid, filtering, adding a filter cake, adding 2mL of methanol, pulping, washing, filtering, and drying to obtain 1.7g of red solid, namely the compound 9, wherein the yield is 70.6%.

Compound 10

Adding compound 9(1.7g, 3.37mmol), Pd/C (200mg) and MeOH (100mL) into a 250mL single-neck flask, vacuumizing and changing hydrogen for 3 times, stirring overnight at room temperature, monitoring by TLC that no raw material remains, filtering to remove Pd/C, distilling off the solvent under reduced pressure to obtain a yellow-green solid, purifying by column chromatography, and eluting with an eluent (DCM: MeOH: NH)₃H₂O ═ 20:1:0.1) to give 1.2g of compound 10 as a yellowish green solid in 75% yield.

Compound 11

Taking a 20mL single-neck flask, adding the compound 10(500mg, 1.05mmol) and DCM (30mL), then adding 3-chloropropionyl chloride (133.7mg, 1.05mmol) into the reaction solution slowly and dropwise at 0 ℃, continuing to stir at room temperature for 30min, monitoring by TLC that no raw material remains, then adding NaOH (168mg, 4.2mmol), heating to 65 ℃, stirring overnight, monitoring by HPLC that no raw material remains, distilling under reduced pressure to remove the solvent, purifying by column chromatography, eluting with eluent (DCM: MeOH ═ 10:1) to obtain 280mg of light yellow solid, namely the compound 11, with the yield of 50.4%.

¹H NMR(400MHz,CDCl3)δ10.19(s,1H),9.88(s,1H),9.12(s,1H),8.39(d,J＝5.3Hz,1H),7.85(d,J＝8.0Hz,1H),7.74(s,1H),7.26–7.14(m,2H),7.00(d,J＝7.0Hz,1H),6.82(s,1H),6.49(dd,J＝16.9,2.2Hz,1H),6.39(dd,J＝16.9,9.8Hz,1H),5.73(dd,J＝9.8,2.2Hz,1H),4.49–4.32(m,2H),3.91(s,3H),3.05(t,J＝6.0Hz,2H),2.95–2.87(m,2H),2.73(s,3H),2.39–2.23(m,7H),1.82(s,3H).LC-MS[M+H]⁺528.7

Example 2

The synthetic route is as follows:

compound 12

120mL of the tube was sealed, and Compound 8(500mg, 1.19mmol), N-methylethanolamine (107.26mg, 1.42mmol), DIPEA (307.59mg, 2.38mmol) and DMA (5mL) were added. Then the tube is sealed and the reaction is carried out for 6h at 140 ℃, TLC monitoring shows that no raw material remains, the reaction liquid is cooled to room temperature, 20mL of water is added, solid is separated out and filtered, then 2mL of methanol is added into the filter cake, pulping and washing are carried out, filtering and drying are carried out, 480mg of red brown solid, namely the compound 12 is obtained, and the yield is 85%.

Compound 13

A20 mL eggplant-shaped bottle was taken and Compound 12(450mg, 1.05mmol), TEA (159.39mg, 1.57mmol) and DCM (5mL) were added. MsCl (120.28mg,1.05mmol) was slowly added dropwise at 0 deg.C, after the addition was complete, stirring was carried out at that temperature, 1h later TLC was monitored that no starting material remained, the solvent was removed by distillation under reduced pressure, and column chromatography purification gave 320mg of compound 13 as a red solid in 55.15% yield.

Compound 14

Adding compound 13(220mg, 0.40mmol), deuterated dimethylamine (175.16mmg,2mmol), DIPEA (103.39mg,0.8mmol) and THF (2mL) into a 5mL sealed tube, stirring at 80 deg.C overnight, monitoring by TLC until no raw material remains, distilling off the solvent under reduced pressure to obtain a red solid, purifying by column chromatography, and eluting with an eluent (DCM: MeOH: NH)₃H₂O ═ 40:1:0.1) to give compound 14 as a red solid, 90mg, yield 44.32%.

Compound 15

Adding compound 14(90mg, 0.18mmol), Pd/C (30mg) and MeOH (10mL) into a 250mL single-neck flask, changing hydrogen for 3 times by vacuumizing, stirring overnight at room temperature, monitoring by TLC that no raw material remains, filtering to remove Pd/C, distilling off the solvent under reduced pressure to obtain a yellow-green solid, purifying by column chromatography, and eluting with an eluent (DCM: MeOH: NH)₃H₂O ═ 20:1:0.1) to give 40mg of compound 15 as a yellowish green solid in 46.53% yield.

Compound 16

Adding compound 15(40mg, 0.08mmol) and DCM (4mL) into a 10mL single-neck flask, then slowly dropping 3-chloropropionyl chloride (10.63mg, 0.08mmol) into the reaction solution at 0 deg.C, stirring at room temperature for 30min, monitoring by TLC that no raw material remains, then adding NaOH (16mg, 0.4mmol), heating to 65 deg.C, stirring overnight, monitoring by HPLC that no raw material remains, distilling under reduced pressure to remove solvent, purifying by column chromatography, and eluting with eluent (DCM: MeOH: NH)₃H₂O ═ 40:1:0.1) to give compound 16 as a pale yellow solid, 38mg, in 89.34% yield.

¹H NMR(400MHz,CDCl3)δ10.12(s,1H),9.88(s,1H),9.10(s,1H),8.39(d,J＝5.2Hz,1H),7.86(d,J＝8.0Hz,1H),7.74(s,1H),7.19(dd,J＝17.1,6.5Hz,2H),7.00(d,J＝6.9Hz,1H),6.81(s,1H),6.46(d,J＝8.4Hz,2H),5.73(dd,J＝8.6,3.1Hz,1H),4.51–4.28(m,2H),3.90(s,3H),3.05(t,J＝5.6Hz,2H),2.98–2.87(m,2H),2.72(s,3H),2.31(dd,J＝16.4,11.2Hz,4H).LC-MS[M+H]⁺531.7

Example 3

The synthetic route is as follows:

compound 17

Taking 2L three-necked bottle, and adding CD respectively₃OH (15g, 415.8mmol), THF (600 mL). n-BuLi (174.6mL, 436.6mmol) was then slowly added dropwise at-40 ℃. After stirring for 1h, a solution of TsCl (79.3g, 415.8mmol) in tetrahydrofuran was added dropwise, stirring was continued for 3h after the addition was complete, and no starting material was left by TLC. Adding 600mLH₂Quenching with O, EA extraction (200 mL. multidot.3), brine washing, anhydrous Na₂SO₄Drying, combining organic phases, distilling under reduced pressure to remove the solvent to obtain a crude compound, and purifying by column chromatography to obtain 73 g of a white solid, namely the compound 17, with the yield of 92.7%.

Compound 18

A250 mL eggplant-shaped bottle was taken and added with Compound 17(8g, 42.3mmol), N-benzylethanolamine (5.3g, 35.2mmol), triethylamine (9.8mL, 70.4mmol) and tetrahydrofuran (100mL), respectively. The mixture was reacted under reflux for 8h, TLC monitored no starting material remained, the solvent was distilled off under reduced pressure and purified by column chromatography to give 5g of a colorless viscous liquid, compound 18, in 84.4% yield.

Compound 19

Adding compound 18(3g, 15.87mmol), triethylamine (3.2g,31.74mmol) and dichloromethane (20mL) into a 50mL eggplant-shaped bottle, slowly adding methanesulfonyl chloride (2.18g, 19.05mmol) dropwise at 0 ℃, stirring the reaction solution for 3h at room temperature, monitoring by TLC to remove the raw material, distilling under reduced pressure to remove the solvent to obtain a light yellow viscous liquid, and purifying by column chromatography to obtain 2.8g of light yellow liquid, namely compound 19, with the yield of 71.6%.

Compound 20

After a 10mL sealed tube was taken, Compound 19(2.8g, 11.37mmol), dimethylamine solution (1.5mL), and THF (3mL) were added, respectively. The mixture was stirred at 80 ℃ for 5h and no starting material was left by TLC. Diluting with 5mL of water, adding ethyl acetate (5 mL. times.3), combining the organic phases, washing with saturated brine, and removing anhydrous Na₂SO₄Drying, distilling under reduced pressure to remove solvent, and purifying under medium pressure to obtain 2g colorless liquid, compound 20, with yield of 75%.

Compound 21

Compound 20(2g, 10.2mmol), 10% palladium on carbon (500mg), methanol (30mL) were added to 100mL eggplant-shaped bottles, respectively, stirred overnight at 35 ℃ under hydrogen, TLC (thin layer chromatography) monitored that no raw material remained, filtered off solids, hcl (in ea) was added dropwise to the filtrate until pH was acidic, and the solvent was distilled off under reduced pressure to obtain 900mg of white solid, compound 21, with a yield of 62.3%.

Compound 22

120mL of the tube was sealed, and compound 8(965mg, 2.3mmol), 21(467mg, 2.76mmol), diisopropylethylamine (1.18g, 9.2mmol) and DMA (5mL) were added thereto. Then the tube is sealed and the reaction is carried out for 6h at 140 ℃, TLC monitoring shows that no raw material remains, the reaction liquid is cooled to room temperature, 20mL of water is added, solid is separated out and filtered, then 2mL of methanol is added into the filter cake, pulping and washing are carried out, filtering and drying are carried out, 900mg of red solid, namely the compound 22 is obtained, and the yield is 77.5%.

Compound 23

Adding compound 22(900mg, 1.78mmol), Pd/C (300mg) and MeOH (100mL) into a 250mL single-neck flask, changing hydrogen for 3 times by vacuumizing, stirring overnight at room temperature, monitoring by TLC that no raw material remains, filtering to remove Pd/C, distilling off the solvent under reduced pressure to obtain a yellow-green solid, purifying by column chromatography, and eluting with an eluent (DCM: MeOH: NH)₃H₂O ═ 20:1:0.1) to give 600mg of compound 23 as a yellowish green solid in 71% yield.

Compound 24

A20 mL single-neck flask was charged with compound 23(300mg, 0.63mmol), THF (6mL), and H₂O (1mL), then 3-chloropropionyl chloride (80.36mg, 0.63mmol) is slowly added dropwise to the reaction solution at 0 ℃ and stirred for 30min at room temperature, TLC monitors no raw material remaining, then NaOH (100.8mg, 2.52mmol) is added, the temperature is raised to 65 ℃ and stirred overnight, HPLC monitors no raw material remaining, the solvent is removed by distillation under reduced pressure, column chromatography purification is performed, and eluent (DCM: MeOH ═ 10:1) is eluted to give 170mg of a pale yellow solid, i.e. compound 24, yield 51%.

¹H NMR(400MHz,CDCl3)δ10.07(s,1H),9.87(s,1H),9.10(s,1H),8.39(d,J＝5.3Hz,1H),7.85(d,J＝8.0Hz,1H),7.75(s,1H),7.25–7.14(m,2H),7.00(d,J＝7.0Hz,1H),6.80(s,1H),6.57–6.38(m,2H),5.74(dd,J＝8.2,3.8Hz,1H),4.49–4.30(m,2H),3.91(s,3H),3.05(t,J＝6.0Hz,2H),2.98–2.89(m,2H),2.42–2.21(m,13H).LC-MS[M+H]⁺528.7

Example 4: biological Activity testing of the prepared Compounds

The kinase activity IC of the compounds on EGFR wild type, EGFR (T790M, L858R) double mutant and EGFR (L858R) single mutant₅₀And (6) testing. The above kinase was purchased from Yinxie funding (Shanghai) trade company Limited.

A homogeneous phase time-resolved fluorescence (HTRF) method is adopted to establish a kinase activity detection method of EGFR wild type, EGFR (T790M, L858R) double mutant and EGFR (L858R) single mutant, and the inhibitory activity of the compound is measured. 8uL of reaction solution was prepared, including 1 Xenzymatic buffer (Cisbio, HTRF KinEASE)^TM-TK)，5mM MgCl₂，1mM MnCl₂1mM DTT, 0.5. mu.M TK substrate-biotin (Cisbio, HTRF KinEASETM-TK) 10. mu.M ATP, graded concentrations of compound and either 0.04 ng/. mu.L EGFR or 0.025 ng/. mu.L EGFR (T790M, L858R) or EGFR (L858R). Compound response concentration is 1000nM from 3 times dilution of 9 concentrations. The DMSO concentration in the reaction system was 2%. The enzyme and compound were preincubated for 15 minutes, and then the reaction was started by adding ATP and substrate. All enzyme-catalyzed reactions were carried out at 25 ℃ for 60 minutes. After the completion of the enzyme-catalyzed reaction, 4. mu.L of TK antibody-cryptate and 4. mu.L of streptavidin-XL665 (reaction concentration: 62.5nM) were added to the reaction mixture, and incubation was continued at 25 ℃ for 60 minutes. HTRF fluorescence values were measured on a Clariostat (BMG LABTECH) after the end of incubation and IC was calculated using GraphPad Prism 5.0₅₀。

TABLE 1 in vitro enzymatic Activity test data (IC)₅₀，nM)

The deuterated compound has enzyme biological activity similar to AZD 9291.

EGFR wild type, EGFR Exon19 deletion (activating single mutant) and EGFR (T790M, L858R) double mutant cell phosphorylation assay

Test 1: EGFR wild type cell phosphorylation assay

The human skin squamous cell carcinoma cell line a431 expresses wild-type EGFR and was purchased from the cell bank of the chinese academy of sciences. A431 was maintained in EMEM medium containing 10% fetal bovine serum. Cells were maintained at 5% CO₂Grown at 37 ℃ in a humidified incubator. Endogenous p-EGFR was detected in cell lysates following the protocol described in the Phospho-EGFR HTRF kit (Cisbio, cat #64HR1 PEG). 100 μ L of cells were seeded in 96-well plates (50000 cells/well) at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator overnight. Compounds were added to the cells in 4-fold serial dilutions, with a maximum reaction concentration of 10 μ M. After further incubation for 2 hours, 100 ng/well of EGF was added for 10 minutes at 37 deg.C, the culture was discarded, and 25. mu.L/well of lysis solution was immediately added to lyse the cells at room temperature for 10 minutes, and then 12. mu.L/well was added to Greiner white low volume 384 well plates, and detection antibodies (Anti-phospho EGFR-d2 and Anti-EGFR-Tb) were added and incubated at 25 deg.C for 60 minutes. HTRF fluorescence values were measured on a Clariostat (BMG LABTECH) after the end of incubation and IC was calculated using GraphPad Prism 5.0₅₀。

Test 2: examination of Exon19 deletion of EGFR (activating Single mutant) cell phosphorylation

The human non-small cell lung cancer cell line HCC827(Exon19 lacks EGFR, activates single mutants) was purchased from cell banks of the chinese academy of sciences. HCC827 was maintained in RPMI1640 medium containing 10% fetal bovine serum. Cells were maintained at 5% CO₂Grown at 37 ℃ in a humidified incubator. Endogenous p-EGFR was detected in cell lysates following the protocol described in the Phospho-EGFR HTRF kit (Cisbio, cat #64HR1 PEG). 100 μ L of cells were seeded in 96-well plates (50000 cells/well) at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator overnight. Compounds were added to the cells in 4-fold serial dilutions, with a maximum reaction concentration of 10 μ M. After a further 2 hours of cultivation the culture medium is discarded and 25. mu.L of culture medium are added immediatelyThe cells were lysed in a well lysis solution at room temperature for 10 min, 12. mu.L/well was added to Greiner white low volume 384 well plates, and the detection antibodies (Anti-phosphoEGFR-d2 and Anti-EGFR-Tb) were added and incubated at 25 ℃ for 60 min. HTRF fluorescence values were measured on a Clariostat (BMG LABTECH) after the end of incubation and IC was calculated using GraphPad Prism 5.0₅₀。

Test 3: EGFR (T790M, L858R) double mutant cell phosphorylation assay

The human non-small cell lung cancer cell line NCI-H1975 expresses the EGFR (T790M, L858R) double mutant and was purchased from cell banks of the Chinese academy of sciences. NCI-H1975 was maintained in RPMI1640 medium containing 10% fetal bovine serum. Cells were maintained at 5% CO₂Grown at 37 ℃ in a humidified incubator. Endogenous p-EGFR was detected in cell lysates following the protocol described in the Phospho-EGFR HTRF kit (Cisbio, cat #64HR1 PEG). 100 μ L of cells were seeded in 96-well plates (50000 cells/well) at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator overnight. Compounds were added to the cells in 4-fold serial dilutions, with a maximum reaction concentration of 10 μ M. After further incubation for 2 hours the medium was discarded and immediately 25. mu.L/well of the lysate was added and the cells lysed at room temperature for 10 minutes, then 12. mu.L/well was added to Greiner white low volume 384 well plates, the detection antibodies (Anti-phosphoEGFR-d2 and Anti-EGFR-Tb) were added and incubated for 60 minutes at 25 ℃. HTRF fluorescence values were measured on a Clariostat (BMG LABTECH) after the end of incubation and IC was calculated using GraphPad Prism 5.0₅₀。

TABLE 2 cellular level EGFR wild and mutant phosphorylation assay (IC)₅₀，nM)

The deuterated compound has the biological activity similar to that of AZD9291 at a cellular level.

Example 9: test of the Effect of the prepared Compounds on the hERG Potassium ion channel

HEK293 cells stably expressing the hERG channel were cultured in 35mm dishes at 37 ℃/5% CO₂The incubator was left for at least 24 hours and then used for the experiment. The cell culture medium was DMEM containing 10% fetal bovine serum and 250. mu.g/mL G418.

The extracellular fluid used in the whole cell patch clamp experiment was composed of (mM): NaCl, 137; KCl, 4; CaCl₂,1.8；MgCl₂1, 1; HEPES, 10; glucose 10; pH 7.4(NaOH titration). All test and control compound solutions contained 0.3% DMSO. Intracellular fluid (mM) was: k asparate, 130; MgCl₂5, 5; EGTA 5; HEPES, 10; Tris-ATP 4; pH 7.2(KOH titration).

One culture dish was removed for each experiment, washed twice with extracellular fluid, and placed on the stage of an inverted microscope. The whole-cell patch clamp experiment is carried out at room temperature, and the tip resistance of the used borosilicate glass microelectrode is 3-5M omega. After the whole cell recording mode, the membrane potential is clamped at minus 80mV, the cell is given with plus 50mV depolarization voltage stimulation every 30s, after 2s, repolarization is carried out to minus 50mV, and after 3s, the hERG tail current can be led out. Depolarization voltage prior to stimulation, the cells were given a repolarization voltage of 50ms and-50 mV, with the current recorded at this voltage as a baseline for calculation of hERG tail current. Before the compound was added, the hERG tail current was stably recorded in the extracellular fluid for at least 3 minutes. Drug action is considered to reach steady state when hERG tail current amplitude changes by less than < 5% after perfusion administration. Data acquisition and analysis used the pCLAMP 10.1 software program. And selecting 4-5 sweep with steady-state current before adding the compound, and calculating the average value of peak values to be used as a reference current amplitude. And selecting 4-5 sweep with the current in a stable state after the compound is added, and calculating the average value of the peak values to be used as the residual amplitude after the current is inhibited. The inhibition rate of the test compound on hERG current was calculated according to the following equation:

% inhibition rate {1- (current residual amplitude)/(control current amplitude) } × 100

Obtaining a plurality of concentrations of the compound to be detected according to the calculation methodAfter measuring the inhibition ratio of hERG current (mean. + -. standard deviation), the data were fitted using logistic equation to obtain IC₅₀The value is obtained.

Table 3 compounds in cellular levels on hERG potassium channel inhibition of IC₅₀(μM)

Compound ID	IC₅₀
		AZD9291	0.37
Compound 11	1.85
		Compound 16	2.67
Compound 24	1.97

The deuterated compound has lower cardiac toxicity relative to AZD 9291.

Claims

1. A compound having the structure of formula (I) or a pharmaceutically acceptable salt thereof,

wherein,

R₁,R₂,R₃and R₄Is independently selected from-CH₃or-CD₃And R is₁,R₂,R₃And R₄At least one is-CD₃。

2. A compound having the structure of formula (I) according to claim 1, characterised in that R is₁is-CH₃。

3. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

4. a compound having the structure of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of claims 1-3, wherein the pharmaceutically acceptable salt is selected from acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, p-toluenesulfonate, bisulfate, borate, butyrate, citrate, camphor, camphorsulfonate, cyclopentanepropionate, diglycolate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oxalate, pectate, persulfate, phenylpropionate, phosphate, picrate, pivalate, salts, Propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate.

5. A pharmaceutical composition, which comprises a compound with a structure of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-4, and a pharmaceutically acceptable adjuvant.

6. Use of a compound having a structure of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 4 in the preparation of a medicament for treating or preventing a tumour.

7. The use of claim 6, wherein the tumor is selected from non-small cell lung cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, or nasopharyngeal carcinoma.

8. Use of a compound having the structure of formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-4, in the manufacture of a medicament for the treatment or prevention of a disease, disorder or condition mediated by EGFR or mediated by an activating mutant or resistant mutant form of EGFR.

9. The use of claim 8, wherein the disease, disorder, or condition mediated by EGFR or mediated by an activating mutant or a resistant mutant form of EGFR is selected from non-small cell lung cancer, pancreatic cancer, breast cancer, prostate cancer, liver cancer, skin cancer, epithelial cell cancer, gastrointestinal stromal tumor, leukemia, histiocytic lymphoma, or nasopharyngeal carcinoma.

10. The use of claim 8, wherein the EGFR that is an activating mutant or a resistant mutant form is selected from the group consisting of an L858R activating mutant, an Exon19 deletion activating mutant, and a T790M resistant mutant.