CN108658874B - Thiopyrimidine heterocyclic antitumor compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to a thiopyrimidine heterocyclic antitumor compound, a preparation method and application thereof, wherein the antitumor compound is specifically a structure shown in a formula (I). The invention also relates to the use of the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition containing the compound for treating tumor diseases by inhibiting wild type EGFR and mutant EGFR-T790M epidermal factor receptor protein tyrosine kinase, in particular for treating small cell lung cancer, non-small cell lung cancer and EGFR-T790M mutant non-small cell lung cancer;

Description

Thiopyrimidine heterocyclic antitumor compound and preparation method and application thereof

Technical Field

The invention relates to an anti-tumor compound and a preparation method and application thereof, in particular to the anti-tumor compound which is a thiopyrimidine heterocyclic compound, belonging to the technical field of medicines.

Background

The targeted therapy of malignant tumor greatly prolongs the life cycle of tumor patients and obviously improves the life quality of the patients. At the heart of "molecular targeting" therapy is the targeting of precise molecular "targets" followed by the application of small molecule drugs that are capable of binding specifically to these targets. Protein Tyrosine Kinases (PTKs) are important signal donors for cell life activities, can catalyze the transfer of gamma-phosphate groups at the ends of ATP to substrates, can catalyze the phosphorylation of tyrosine residues of various substrate proteins, thereby transmitting signals, and have important roles in cell proliferation, survival, apoptosis, metabolism, transcription and differentiation. PTKs are currently the most ideal "target" for anti-tumor targeted drug development, and over 50% of both proto-and oncogene products are tyrosine kinases. Epidermal growth factor receptor tyrosine kinase (EGFR) is one of the earliest discovered protein tyrosine kinases, the intracellular domain of EGFR has an ATP binding site, and an EGFR inhibitor can competitively bind with the ATP binding site, so as to inhibit the phosphorylation process of EGFR, block the conduction of downstream signals, and further inhibit the growth, differentiation and metastasis of tumor cells (Yun, et al. cancer Cell2007,11, 217-227). The biochemical process of EGFR as an anti-tumor target is elucidated, the crystal structure and the active site are relatively clear, and medicaments such as gefitinib, erlotinib and afatinib which take the EGFR as the target are applied to clinic. However, these drugs inevitably have a problem of poor resistance to drugs. The research shows that: threonine to methionine mutation at the site of EGFR kinase protein 790 (EGFR-T790M) is a major cause of resistance in such drugs. Clinical case data show that approximately 60% of patient acquired resistance results from mutations at the T790M site. Therefore, the development of novel EGFR inhibitors with stronger drug resistance, less toxicity and stronger activity has very important practical value.

Ostinib is orally irreversible, T790M mutation selective EGFR inhibitor, EGFR that is deleted at exon 19 in LoVo cells^L858R/T790MAnd EGFR^WTIC of₅₀11.44 and 493.8nM, respectively, were administered in the United states 12 months in 2015FDA approved for marketing (WO2013014448) for treating EGFR-T790M drug-resistant lung cancer patients. Rosentinib is another irreversible, mutation-selective EGFR-T790M inhibitor, acting on EGFR-T790M and EGFR in cell-free assays^WTIC of₅₀21.5nM and 303.3nM, respectively, are currently in phase III clinical study (WO 2012061299). Other examples of EGFR-T790M inhibitors (Ma et al, J.Med.chem.,2016,59, 6580-Asca 6594) such as HM61713, EGF816, PF-06747775, Avertinib, ASP8273 are also in clinical research: US20120157426, US8563568B2, CN102740847, CN 102083800. The discovery of the medicines meets the medication requirements of EGFR-T790M drug-resistant patients to a certain extent, but the medicines all belong to pyrimidine derivatives, have similar drug effects and pharmacological mechanisms, have certain toxic and side effects and are limited in application range.

In view of the urgent need for the treatment of cancer, especially drug-resistant cancer, there is a need in the art to develop new molecular scaffolds, mechanisms of action, and better antitumor drugs.

Disclosure of Invention

One of the purposes of the invention is to provide a novel antitumor compound or a pharmaceutically acceptable salt thereof, wherein the antitumor compound is a thiopyrimidine heterocyclic compound, and the compound has good antitumor activity.

Another object of the present invention is to provide a process for the preparation of the aforementioned antitumor compounds.

The invention also aims to provide a pharmaceutical composition containing the thiopyrimidine heterocyclic compound or the pharmaceutically acceptable salt thereof.

The invention further aims to provide the thiopyrimidine heterocyclic compound or the pharmaceutically acceptable salt thereof, or the application of the composition.

In one aspect, the present invention provides a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by general formula (I) has the following structure:

wherein the content of the first and second substances,

x is selected from O, NH;

m is selected from S (O)_n,n＝0,1；

R is selected from

Preferably, the compound represented by the general formula (I) has structures represented by (I-1) to (I-6):

preferably, the compound shown in the general formula (I) is I-1, I-2, I-4, I-5.

The structural compound is a thiopyrimidine heterocyclic compound, and the screening of the anti-tumor activity shows that the compound has the unexpected proliferation capacity of resisting lung cancer cells (A431 phosphorus cells, HCC827 adenocarcinoma cells and A549 adenocarcinoma cells) and EGFR T790M drug-resistant lung cancer cells (H1975 cells); some of the compounds also showed superior anti-EGFR-T790M kinase activity than was unexpected for the reference drugs gefitinib and rostinib. As a molecule with novel structure, the compound has the potential of being developed into a novel high-efficiency EGFR-T790M mutant kinase inhibitor, and has great application value in treating related tumor diseases, particularly small cell lung cancer, non-small cell lung cancer and EGFR-T790M drug-resistant non-small cell lung cancer.

The structures (I-1) to (I-6) have the following names, respectively:

(I-1) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) propylthio) aniline ] -4-pyrimidine ] amino ] phenyl ] -2-propenamide;

(I-2) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) propylthio) aniline ] -4-pyrimidine ] oxy ] phenyl ] -2-propenamide;

(I-3) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) propylsulfinyl) aniline ] -4-pyrimidine ] amino ] phenyl ] -2-propenamide;

(I-4) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) acetylthio) aniline ] -4-pyrimidine ] amino ] phenyl ] -2-propenamide;

(I-5) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) acetylthio) aniline ] -4-pyrimidine ] oxy ] phenyl ] -2-propenamide;

(I-6) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) acetylsulfinyl) aniline ] -4-pyrimidine ] amino ] phenyl ] -2-propenamide.

In another aspect, the present invention provides a method for preparing the above anti-tumor compound, wherein the anti-tumor compound is prepared according to the following route:

in another aspect, the present invention provides a pharmaceutical composition, which comprises an effective amount of the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The compounds of the invention are bases, wherein the desired salt form may be prepared by suitable methods known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, α -hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, and the like.

The pharmaceutical compositions of the invention will generally contain one compound of the invention. However, in some embodiments, the pharmaceutical compositions of the invention contain more than one compound of the invention. In addition, the pharmaceutical compositions of the present invention may optionally further comprise one or more other pharmaceutically active compounds.

The invention also provides the thiopyrimidine heterocyclic compound or the pharmaceutically acceptable salt thereof, and the pharmaceutical composition can inhibit the tumor proliferation by inhibiting EGFR-T790M kinase. Specifically, the application is mainly used for preparing the medicine for treating small cell lung cancer, non-small cell lung cancer and EGFR-T790M drug-resistant non-small cell lung cancer.

The invention provides an application of a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition in preparation of an EGFR-T790M kinase inhibitor.

The invention provides a compound shown in the general formula (I) or a pharmaceutically acceptable salt thereof, or application of a pharmaceutical composition in preparing a medicament for treating tumors. Preferably, the tumor is selected from one or more of small cell lung cancer, non-small cell lung cancer and EGFR-T790M drug-resistant non-small cell lung cancer, and further preferably EGFR-T790M drug-resistant non-small cell lung cancer. More preferably, the use is primarily through inhibition of EGFR-T790M kinase.

Drawings

FIG. 1 shows the inhibition of H1975 cells by different treatment groups of compound I-1 as a function of time.

FIG. 2 is a graph showing the results of the effect of Compound I-1 on apoptosis in EGFR-T790M mutant H1975 cells.

Detailed Description

The present invention is further described and explained below in conjunction with specific examples, which are not intended to limit the scope of the present invention.

The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

EXAMPLE 1 preparation of target molecules

The thin layer chromatography silica gel plate is prepared from natural sea GF254 or Qingdao GF254 silica gel plate, the silica-amine plate for Thin Layer Chromatography (TLC) is 0.15-0.2 mm, and the thin layer chromatography separation and purification product is 0.4-0.5 mm.

The raw materials used in the present invention are mainly purchased from chemical reagents of national medicine group, Beijing coupled technology, Inc., Aladdin chemical reagents, Inc., Darriy Chemicals, etc.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The technical scheme adopted by the invention is as follows:

the synthesis route, reagent and condition of the compounds (I-1), (I-2) and (I-3) are (a) acryloyl chloride, NaHCO₃，CH₃CN, room temperature, 0.5 hour, 60-80%; (b) when X is OH, K₂CO₃，DMF，60℃；X＝NH₂DIPEA, 1, 4-dioxane, room temperature, 2 hours, 91%; (c) 1-chloro-3-bromopropane, K₂CO₃，CH₃CN, 70 ℃, 12 hours, 95%; (d) morpholine, K₂CO₃KI, DMF, 80 ℃, 12 hours, 81%; (e) Fe-NH₄Cl，MeOH-H₂O, 2 hours, 60 ℃, 72%; (f) TFA, 2-BuOH, 100 ℃, 12 hours, 22-37%; (g) m-CPBA, CH₂Cl₂Room temperature, 31%.

Synthetic route of compounds (I-4), (I-5) and (I-6), reagents and conditions (a) ethyl 2-mercaptoacetate, K₂CO₃KI, DMF, 100 ℃, 12 hours, 95%; (b) KOH, H₂O, 50 ℃, 24 hours, 76%; (c) SOCl₂At 80 ℃ for 2 hours; (d) morpholine, NaHCO₃，CH₃CN, room temperature, 0.5 hour, 95%; (e) Fe-NH₄Cl，MeOH-H₂O, 2 hours, 60 ℃, 72%; (f) pyrimidine 3a-b, TFA, 2-BuOH, 100 ℃, 12h, 16-19%; (g) m-CPBA, CH₂Cl₂Room temperature, 25%.

The target molecule was synthesized according to the above method, and the physicochemical data of the synthesized target molecule were as follows:

(I-1) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) propylthio) aniline]-4-pyrimidines]Amino group]Phenyl radical]36.52% yield, yellow solid.¹H NMR(400MHz,DMSO-d₆):δ1.60-1.63(m,2H),2.28-2.32(m,6H),2.82(t,J＝8.0Hz,2H),3.53(t,J＝8.0Hz,4H),5.76(dd,J＝4.0Hz,8.0Hz,1H),6.26(dd,J＝4.0Hz,16.0Hz,1H),6.44(dd,J＝8.0Hz,16.0Hz,1H),7.11(d,J＝8.0Hz,2H),7.18-7.35(m,2H),7.52-7.57(m,3H),7.89(s,1H),8.15(s,1H),9.00(s,1H),9.41(s,1H),10.22(s,1H)；¹³C NMR(100MHz,DMSO-d₆):δ26.37,32.42,53.98(2C),57.39,66.86(2C),104.60,116.17,116.24,120.03(2C),120.14,127.35,127.64,129.32,131.03(2C),132.53,139.46,139.76,139.83,155.48,156.96,158.18,163.84；HRMS(ESI)，C₂₆H₂₉ClN₆O₄S,m/z，[M+H]⁺And (4) theoretically calculating: 525.1834, found 525.1843.

(I-2) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) propylthio) aniline]-4-pyrimidines]Oxy radical]Phenyl radical]2-acrylamide, yield 22.14%; a yellow solid;¹H NMR(400MHz,DMSO-d₆):δ1.64-1.69(m,2H),2.32-2.36(m,6H),2.87(t,J＝8.0Hz,2H),3.58(t,J＝8.0Hz,4H),5.83(dd,J＝4.0Hz,8.0Hz,1H),6.32(dd,J＝4.0Hz,16.0Hz,1H),6.49(dd,J＝12.0Hz,16.0Hz,1H),7.06-7.10(m,3H),7.40(d,J＝8.0Hz,2H),7.52(d,J＝8.0Hz,2H),7.76(s,1H),8.54(s,1H),9.89(s,1H),10.46(s,1H)；¹³C NMR(100MHz,DMSO-d₆):δ26.01,31.83,53.67(2C),57.04,66.56(2C),105.09,113.30,117.02,117.39,119.75(2C),127.79,128.01,130.31(2C),130.39,131.97,138.56,140.78,152.66,157.75,158.51,163.73,164.26；HRMS(ESI),C₂₆H₂₈ClN₅O₃S,m/z,[M+H]⁺the theoretical calculation is 526.1674, and the actual measurement is 526.1678.

(I-3) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) propylsulfinyl) aniline]-4-pyrimidines]Amino group]Phenyl radical]Yield of 31.0 percent of 2-acrylamide; a brown solid;¹H NMR(400MHz,DMSO-d₆):δ1.56-1.68(m,2H),2.26-2.32(m,6H),2.74-2.86(m,2H),3.52(t,J＝8.0Hz,4H),5.76(dd,J＝4.0Hz,8.0Hz,1H),6.27(dd,J＝4.0Hz,16.0Hz,1H),6.48(dd,J＝12.0Hz,16.0Hz,1H),7.30-7.40(m,5H),7.53-7.58(m,1H),7.83(d,J＝8.0Hz,2H),8.22(s,1H),9.10(s,1H),9.73(s,1H),10.28(s,1H)；¹³C NMR(100MHz,DMSO-d₆):δ19.14,19.47,53.68(2C),57.13,66.78(2C),105.28,114.31,116.17,116.35,119.33(2C),125.25(2C),126.64,127.63,129.53,132.56,135.60,139.91,143.62,155.47,157.03,158.01,163.86；HRMS(ESI),C₂₆H₂₉ClN₆O₃S,m/z,[M+H]⁺the theoretical calculation is 541.1783, and the actual measurement is 541.1788.

(I-4) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) acetylthio) aniline]-4-pyrimidines]Amino group]Phenyl radical]2-acrylamide, yield 16.04%; a white solid;¹H NMR(400MHz,DMSO-d₆):δ3.41-3.54(m,8H),3.80(s,2H),5.76-5.79(m,1H),6.28(dd,J＝4.0Hz,16.0Hz,1H),6.48(dd,J＝8.0Hz,16.0Hz,1H),7.20(d,J＝8.0Hz,2H),7.36(d,J＝8.0Hz,2H),7.45-57(m,3H),7.91(s,1H),8.18(s,1H),9.03(s,1H),9.46(s,1H),10.24(s,1H)；¹³C NMR(100MHz,DMSO-d₆):δ37.70,42.74,47.14,67.04(2C),105.06,116.42,116.59,120.18(2C),120.48,126.44,127.95,129.62,132.30(2C),132.84,139.75,140.12,140.71,155.75,157.25,158.43,164.15,167.63；HRMS(ESI),C₂₅H₂₅ClN₆O₃S,m/z,[M+H]⁺the theoretical calculation is 525.1470, and the actual measurement is 525.1484.

(I-5) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) acetylthio) aniline]-4-pyrimidines]Oxy radical]Phenyl radical]19.01% of yield; a white solid;¹H NMR(400MHz,DMSO-d6):δ3.39(t,J＝4.0Hz,4H),3.50(t,J＝4.0Hz,4H),3.77(s,2H),5.77(dd,J＝4.0Hz,8.0Hz,1H),6.26(dd,J＝4.0Hz,16.0Hz,1H),6.45(dd,J＝8.0Hz,16.0Hz,1H),7.00-7.03(m,1H),7.11(d,J＝8.0Hz,2H),7.35(d,J＝8.0Hz,2H),7.46(d,J＝8.0Hz,1H),7.57(d,J＝8.0Hz,1H),7.70(s,1H),8.48(s,1H),9.85(s,1H),10.39(s,1H)；¹³C NMR(100MHz,DMSO-d₆):δ36.89,42.14,46.51,66.43(2C),105.30,113.26,117.09,117.43,119.60(2C),126.84,127.81,130.40,131.36(2C),131.98,139.24,140.78,152.67,157.74,158.50,163.77,164.28,166.95；HRMS(ESI),C₂₅H₂₄ClN₅O₄S,m/z,[M+H]⁺and (4) theoretically calculating: 526.1310, found 526.1323.

(I-6) N- [3- [ [ 5-chloro-2- [4- ((1-morpholino) acetylsulfinyl) aniline]-4-pyrimidines]Amino group]Phenyl radical]25.0 percent of yield; a brown solid;¹H NMR(400MHz,DMSO-d₆):δ3.37-3.52(m,8H),3.93(d,J＝12.0Hz,1H),4.11(d,J＝12.0Hz,1H),5.77(dd,J＝4.0Hz,8.0Hz,1H),6.27(dd,J＝4.0Hz,16.0Hz,1H),6.47(dd,J＝8.0Hz,16.0Hz,1H),7.37(d,J＝8.0Hz,2H),7.49(d,J＝8.0Hz,2H),7.82-7.94(m,4H),8.22(s,1H),9.11(s,1H),9.72(s,1H),10.25(s,1H)；¹³CNMR(100MHz,DMSO-d₆):δ42.39,46.71,61.09,66.61,66.78,105.36,116.23,116.43,119.23(2C),120.43,125.78(2C),127.71,129.36,132.53,135.41,139.40,139.91,143.96,155.48,157.09,158.01,163.89,163.91；HRMS(ESI),C₂₅H₂₅ClN₆O₄S,m/z,[M+H]⁺the theoretical calculation is 541.1419, and the actual measurement is 541.1441.

Method for salifying target molecule

The preparation method of the inorganic acid salt comprises the following steps: dissolving a target molecule (1mmol) in 10mL of anhydrous methanol, slowly dropwise adding a 5mL of anhydrous methanol solution of inorganic acid (1mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the methanol at normal temperature to obtain the inorganic acid salt of the target molecule. By the method, hydrochloride (I-1-1), hydrobromide (I-1-2), sulfate (I-1-3) and phosphate (I-1-4) of the compound I-1 are prepared;

the preparation method of the organic acid salt comprises the following steps: dissolving a target molecule (1mmol) in 10mL of anhydrous methanol, slowly dropwise adding 5mL of dry ether of organic acid (1mmol) in ice bath, stirring for 30 minutes at the temperature after dropwise adding, and then evaporating the solvent at normal temperature to obtain the organic acid salt of the target molecule. By this method, maleate salt (I-1-5), succinate salt (I-1-6) and fumarate salt (I-1-7) of Compound I-1 were prepared.

Example 2 evaluation of biological Activity of target molecule

1. Method for testing in vitro inhibitory activity of receptor tyrosine kinase

Preparation of kinase assay buffer

① the kinase assay buffer was thawed at room temperature and observed for precipitation.

② if precipitation occurs, the kinase assay buffer is incubated at 37 ℃ for 15 minutes with constant shaking to dissolve the precipitate or the supernatant is carefully aspirated off to remove the precipitate.

Preparation of kinase assay reagent

① the kinase assay buffer and the kinase assay substrate were equilibrated at room temperature prior to use.

② pouring the kinase detection buffer solution into a brown bottle filled with a kinase detection substrate to dissolve the freeze-dried powder substrate, thus preparing the kinase detection reagent.

③ mix well by gentle shaking, vortexing or inversion to form a homogeneous solution, and the substrate should be dissolved within 1 minute.

④ the kinase detection reagent should be used immediately after preparation, or be packaged at-20 deg.C, and the prepared reagent has no loss of cyclic signal activity after several times of freeze thawing.

Standard Curve for conversion of ATP to ADP was generated

① ultrapure ATP and ADP provided by the kit was diluted with 1 Xkinase reaction buffer to make 900. mu.L of 50. mu.M ATP and 500. mu.L of 50. mu.M ADP.

② the 50. mu.M ATP and 50. mu.M ADP solutions prepared in the previous step were mixed in a 384-well plate A1-A12 as shown in Table 1, simulating the concentration of ATP and ADP for each conversion percentage, and mixed well.

TABLE 1 preparation of 50. mu.M series of ATP + ADP standards

③ mu.L of ADP-Glo was added per well^TMReagents to terminate the kinase reaction. Incubate at room temperature for 40 minutes.

④ mu.L of kinase detection reagent per well was added to convert ADP to ATP and luciferase and luciferin were introduced to detect ATP.

⑤ were incubated at room temperature for 30-60 minutes, fluorescence was measured with a multifunctional microplate reader and fluorescence values were recorded.

⑥ Standard curves for conversion of ATP to ADP were plotted.

Determination of IC50 values for kinase inhibitors

① 1 Xkinase reaction buffer, 2.5X 50 ng/. mu.L kinase and 2.5X 0.5. mu.g/. mu.L substrate and 125. mu.M ATP were prepared according to the Promega kit instructions.

② mu.L of 1 Xkinase reaction buffer, 2. mu.L of 2.5 X0.5. mu.g/. mu.L substrate and 125. mu.M ATP are added to the enzyme-free control wells 1. mu.L of 1 Xkinase reaction buffer, 2. mu.L of 2.5 X50 ng/. mu.L kinase, 2. mu.L of 2.5 X0.5. mu.g/. mu.L substrate and 125. mu.M ATP are added to the negative control wells 1. mu.L of 5 Xtest drug, 2. mu.L of 2.5 X50 ng/. mu.L kinase, 2. mu.L of 2.5 X0.5. mu.g/. mu.L substrate and 125. mu.M ATP are added to the test wells.

③ the plates were mixed and incubated for 60 minutes.

④ mu.L of ADP-Glo was added per well^TMReagents to terminate the kinase reaction. Incubate at room temperature for 40 minutes.

⑤ mu.L of kinase assay reagent was added per well to convert ADP to ATP and luciferase and luciferin were introduced to detect ATP, incubated at room temperature for 30-60 minutes, fluorescence was measured using a multifunctional microplate reader and the fluorescence value recorded.

⑥ results analysis, the results are shown in Table 2.

2. Cell growth experiments (MTT assay)

Cell inoculation: cells in the logarithmic growth phase were collected and the cell suspension concentration was adjusted to 7 × 10 per well³Each cell, 100 μ L per well volume, was seeded into 96-well plates, each set of 4 replicates (marginal wells filled with sterile PBS);

cell culture: after cell attachment, 0% FBS RPMI-1640 was starved for 8h, and the control group was cultured with 10% FBS RPMI-1640 at 37 ℃ and 5% CO₂Continuously culturing in an incubator (culturing for different times according to experimental requirements);

color generation: adding 10 μ L MTT solution (5mg/mL) into three groups of cells after culturing for 72h, terminating the culture after 4h, adding 100 μ L triple solution into each well, and oscillating on a shaking table at low speed for 10min to fully dissolve crystals;

color comparison: the absorbance value (OD value) of each well is measured on an enzyme linked immunosorbent assay detector, the wavelength of 570nm is selected, the blank well of cell-free RPMl-1640 culture solution is adjusted to zero, and the absorbance value of each well is measured. The experiment was repeated three times

The results are recorded: cell growth inhibition rate (control absorbance value-experimental absorbance value)/control absorbance value x 100%, cell proliferation rate (experimental absorbance value/control absorbance value) x 100;

drawing a cell growth curve: the cell growth curve was plotted with time as abscissa and inhibition/proliferation rate as ordinate.

Inhibitor concentrations were plotted in GraphPad Prism mapping software in GraphPad software to determine log [ inhibitor [ ]]Variable slope model estimation of IC versus response₅₀。

The test results are shown in Table 2, Table 3 and Table 4, Table 2 shows that the compounds inhibit the EGFR and EGFR-T790M kinase activities, Table 3 shows the proliferation activities of the compounds against tumor cells and normal human cells, and FIG. 1 shows the time-varying inhibitory effects of different treatment groups of compound I-1 on H1975 cells.

TABLE 2

Using ADP-Glo^TMThe kinase assay system evaluated the inhibitory activity of such molecules against wild-type EGFR and mutant EGFR-T790M/L858R kinase, with representative EGFR inhibitor drugs gefitinib and rosatinib as positive controls. The test results given in table 2 clearly indicate that such compounds haveProminent EGFR-T790M/L858R kinase inhibitory activity, with the strongest inhibitory activity of compounds I-1 and I-5, and IC thereof₅₀Values were 27.5 and 9.1nM, respectively. With oxygen-containing compounds I-7 (IC)₅₀100.5nM) activity, sulfur-containing compound I-1 (IC)₅₀27.5nM) increased by a factor of approximately 10; compared with the reference drug, the activity is more advantageous. Of particular concern are compounds that are not effective against wild-type EGFR (IC)₅₀>980nM), indicating that they have highly selective inhibitory activity against mutant EGFR-T790M. Especially inhibitor I-1 (SI)>36.4) and I-5 (SI)>110.2) have a much higher selectivity than rosteinib (SI ═ 25) and are also indicative of lower toxic side effects.

TABLE 3

^aFour typical NSCLC cell lines: a431^EGFR-WTCells, H1975^EGFR-T790MMutant cell line, A549^{EGFR -WT and k-ras mutations}And HCC827^{EGFR del E746_ A750 mutation}Cell lines and two normal cell lines (human bronchial epithelial cells (HBE)) and hepatocytes (LO-2)

In addition, all compounds were evaluated for anti-proliferative activity against NSCLC lung cancer cell lines using the MTT assay. As shown in Table 3, compounds I-1 to I-5 had significantly higher anti-H1975 cell proliferation activity than gefitinib at concentrations ranging from 0.074 to 1.050. mu.M; its potency on a431 cells carrying wild-type EGFR is comparable to rostinib and gefitinib. The selection indexes of the compounds I-1 to I-5 of the invention are all higher than that of gefitinib, the selection indexes of the compounds I-1, I-2 and I-5 are higher than that of Rostinib, and particularly the compound I-1 has strong efficacy on H1975 cells at the concentration of 0.074 mu M. Compound I-1 also has moderate activity (IC) in inhibiting A431 cell proliferation₅₀3.893 μ M), indicating its high selectivity for mutant H1975 cells (SI 52.6). Furthermore, all of these compounds were also very effective against EGFR del E746-A750 mutation, which is highly sensitive to cells carrying HCC827, IC₅₀Values below 0.308. mu.M. The compound I-4 most strongly inhibits HCC827 cellsActive, IC₅₀The value was 0.050. mu.M. Of further interest is the non-toxicity of most of these compounds against normal cell lines HBE and LO-2, especially the inhibitors I-1, I-4 (IC)₅₀Values all above 40 μ M) showed a rather low cytotoxicity.

Figure 1 shows the effect of the strongest inhibitor I-1 to significantly block the proliferation of H1975 cells with increasing time (24, 48 and 72 hours) and drug concentration (0.2,0.5 and 1.0 μ M). When I-1 was used at a concentration of 1.0 μ M, cell viability (26.8%) was about 2-fold lower than that of rosatinib (44.8%) 72 hours after administration, indicating enhanced resistance to gefitinib. These biological evaluations indicate that compound I-1 may potentially be useful as an inhibitor of EGFR-T790M resistance to NSCLC. The present invention also investigated the effect of the most potent inhibitor I-1 on apoptosis in EGFR-T790M mutant H1975 cells. The results obtained are shown in FIG. 2, and Compound I-1 induces apoptosis in H1975 cells in a dose-and time-dependent manner. The apoptosis rate of H1975 cells treated with compound I-1(0.2, 0.5 and 1.0. mu.M) for 72 hours increased significantly from 45.6% to 96.8%.

The biological activity results show that the molecular anti-tumor effect activity effect is remarkable, the toxic and side effect is low, and the molecular anti-tumor effect activity is mainly shown in the following aspects that 1) the compounds I-1 and I-5 show very prominent anti-EGFR-T790M/L858R activity and IC thereof₅₀Values of 27.5 and 9.1nM, respectively, are stronger than those unexpected for the reference drug gefitinib; 2) the selective inhibition index SI of the compounds I-1 and I-5 to EGFR-T790M is also obviously superior to that of two reference drugs, and the molecules are indicated to have unexpected lower toxic and side effects; 3) the inhibitory activity of the compound I-1 on drug-resistant lung cancer cells H1975 is less than 100nM, which is obviously superior to gefitinib and reaches the Rosentinib level; 4) the inhibitory effect of the I-4 and I-6 compounds on lung cancer cells HCC827 is less than 100nM, and the anti-NSCLC cell proliferation effect is very prominent; 4) most compounds also have very strong inhibitory activity on NSCLC squamous carcinoma cells A431 and adenocarcinoma cells A549, and have more obvious progress and advantages than the EGFR inhibitor drugs on the market; 5) most compounds have no obvious interference and inhibition effect on human upper respiratory tract normal cells HBE and human liver normal cells LO-2, and the molecular has unexpected effectLow cytotoxicity.

As a class of molecules with novel structures, the research compound has the potential of being developed into a novel high-efficiency EGFR-T790M kinase inhibitor, and has great application value in treating related tumor diseases, particularly small cell lung cancer, non-small cell lung cancer and EGFR-T790M drug-resistant non-small cell lung cancer.

Claims (5)

1. An anti-neoplastic compound of formula (I-1) or formula (I-5), or a pharmaceutically acceptable salt thereof, wherein the compound has the structure shown below:

2. a pharmaceutical composition comprising an effective amount of the anti-neoplastic compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. Use of the anti-neoplastic compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the preparation of an EGFR-T790M mutant epidermal factor receptor protein tyrosine kinase inhibitor.

4. Use of the anti-neoplastic compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating EGFR-T790M mutant non-small cell lung cancer.

5. The use of claim 4, wherein the use is primarily through inhibition of EGFR T790M mutant epidermal factor receptor protein tyrosine kinase.

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