CN113582995B - 9-9H-purine compound containing acrylamide amino fragment at 9-position, and salt and application thereof - Google Patents
9-9H-purine compound containing acrylamide amino fragment at 9-position, and salt and application thereof Download PDFInfo
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Abstract
The invention discloses a 9-position-9H-purine compound containing an acrylamide fragment, and salt and application thereof, belonging to the technical field of anti-cancer drugs. The compounds have novel structures, have the activity of inhibiting Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, have obvious inhibitory activity on EGFR single-mutation cell HCC827 and EGFR double-mutation (L858R/T790M) cell H1975, can be used for treating cancers related to EGFR mutation, and have easily-obtained synthesis raw materials and easily-realized synthesis method.
Description
Technical Field
The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a 9-position acrylamide fragment-containing-9H-purine compound, and a salt and an application thereof.
Background
Cancer is one of the malignant diseases that seriously threatens human health. In recent 30 years, the incidence of cancer in our country is in a rapid rise. In recent years, the incidence rate of cancer is about 300/10 ten thousand, more than 400 ten thousand new cases per year, about 250 or more than ten thousand deaths, and more than 700 ten thousand deaths. The main treatment means of cancer at present are still surgical treatment, drug therapy and radiation therapy, wherein drug therapy plays a major role. Therefore, the research and development of new anti-cancer drugs are of great significance. The traditional antitumor drug has strong activity, but lacks selectivity and has high toxicity, and patients cannot take the drug for a long time. In recent years, with the progress of tumor molecular biology research, the pathogenesis of tumors is more understood, and a plurality of new targets of the action of anti-cancer drugs, such as Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, PI3Ks, BTK and the like, are found.
In non-small cell lung cancer, about 50% of patients exhibit mutations in EGFR. In response to this feature, first generation EGFR tyrosine kinase inhibitors such as gefitinib, erlotinib, second generation EGFR tyrosine kinase inhibitor afatinib, third generation EGFR tyrosine kinase inhibitor oxitinib have been developed for use as anti-cancer drugs. Therefore, the development of an EGFR single mutation or EGFR double mutation (L858R/T790M) inhibitor with novel structure and high activity for treating EGFR mutation cancer is of great significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a-9H-purine compound containing an acrylamide amino fragment at the 9-position, and salt and application thereof, wherein the compound has a novel structure, has remarkable activity of inhibiting single mutation and double mutation (L858R/T790M) of EGFR tyrosine kinase, has obvious external anti-tumor activity, can be applied to the preparation of anti-cancer pharmaceutical preparations, and has easily available synthetic raw materials and easily realized synthetic method.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a 9-position-9H-purine compound containing an acrylamide fragment, which has a structural formula as follows:
wherein ring A contains a nitrogen-containing heterocycle having 4 to 7 atoms;
R 1 is alkoxy or halogen substituted;
R 2 is alkoxy or substituted amino;
R 3 is hydrogen or fluorine;
x is CH or N;
y is NH or S.
Preferably, the a ring is an azetidine ring, a pyrrolidine ring, a piperidine ring or an azepane.
Preferably, said acrylamido fragment at the 9-position is N-acryloyl-3-azetidinyl, N-acryloyl-3-pyrrolidinyl, N-acryloyl-3-piperidinyl, N-acryloyl-4-piperidinyl, or N-acryloyl-3-azepanyl; wherein the chiral center in the N-acryloyl-3-pyrrolidinyl-, N-acryloyl-3-piperidinyl-, and N-acryloyl-3-azepanyl groups is of the S-type.
Preferably, R is 1 Is methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, fluoro or chloro.
Preferably, R 2 Is 2- (dimethylamino) ethoxy, 1-pyrrolidinyl, 1-piperidinyl, 4-dimethylamino-1-piperidinyl, 4-methyl-1-piperazinyl, 4-ethyl-1-piperazinyl, N-methyl-N- (2-dimethylaminoethyl) amino or 4-morpholinyl.
Preferably, R 3 Is hydrogen substitution, 2-fluoro substitution, 3-fluoro substitution, 4-fluoro substitution or 2, 4-difluoro substitution.
Preferably, the compound is one of the following compounds:
the invention also discloses a pharmaceutically acceptable salt of the-9H-purine compound containing the acrylamide amino fragment at the 9-position, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, nitrate, phosphate, sulfate, acetate, fumarate, malate, citrate, tartrate, maleate, lactate, citrate, camphorsulfonate, benzoate, gluconate, glutamate, isethionate, succinate or methanesulfonate.
The invention also discloses an application of the-9H-purine compound containing the acrylamide amino fragment at the 9-position or the pharmaceutically acceptable salt of the-9H-purine compound containing the acrylamide amino fragment at the 9-position in the preparation of an anti-cancer medicinal preparation.
Preferably, the anti-cancer pharmaceutical preparation is a tablet, a capsule or an injection, wherein each tablet, each grain or each preparation contains 10-300 mg of-9H-purine compounds containing acrylamide amino fragments at 9-positions or pharmaceutically acceptable salts thereof.
Compared with the prior art, the invention has the following beneficial effects:
the-9H-purine compound or salt compound thereof with 9-position containing the acrylamide amino segment provided by the invention has a novel structure and is easy to synthesize, the acrylamide amino segment introduced into the 9-position of the purine ring can generate Michael addition reaction with the sulfydryl of the side chain of the cysteine residue at the 797 position of EGFR to form a C-S bond, so that the covalent inhibition effect is generated, the activity is stronger, and the effect is durable. Therefore, the present inventionthe-9H-purine compound or salt compound thereof containing the acrylamide segment at the 9-position has the activity of inhibiting Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, has obvious inhibitory activity on single-mutation and double-mutation (L858R/T790M) EGFR, has stronger activity than that of marketed medicaments gefitinib and ocitinib, and can be used for treating cancers related to EGFR mutation. The compound provided by the invention has remarkable activity of human lung cancer cell proliferation such as HCC827, H1975 and the like. IC inhibition of human lung carcinoma cells HCC827 and H1975, for example, by Compound 3 50 Values of 0.33 and 12 nmol.L, respectively -1 . While the positive drug gemfibrozil inhibits the IC of HCC827 under the same conditions 50 A value of 10 nmol.L -1 (ii) a The positive drug Niocitinib (AZD9291) inhibits IC of HCC827 and H1975 50 Values of 1.47 and 17 nmol. L, respectively -1 。
The 9-position-9H-purine compound containing acrylamide fragments or salt compounds thereof provided by the invention have the activity of inhibiting Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, and have obvious inhibitory activity and IC (integrated Circuit) on single-mutation or double-mutation EGFR 50 The value is nanomolar, and can be used for treating cancers related to EGFR mutation.
The 9-position-9H-purine compound containing the acrylamide segment and the salt compound thereof provided by the invention can be applied to the preparation of anti-cancer pharmaceutical preparations, wherein each tablet or granule or branch of the pharmaceutical preparation contains 10-300 mg. When the active compound provided by the invention is used for preparing an anticancer medicine preparation, the medicine can be prepared into tablets, capsules or injections. The pharmaceutical preparations can be prepared according to the conventional preparation process of various preparations. The preferable content is 50-100 mg for a tablet or capsule. The oral preparation of the invention can contain pharmaceutic adjuvants including additives, stabilizers, solubilizers, lubricants, disintegrating agents and the like, such as starch, dextrin, glucose, lactose, cellulose, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, pectin, cyclodextrin, Tween-80, polyvinyl alcohol, magnesium stearate, talcum powder and the like.
Drawings
FIG. 1 shows a method for synthesizing a-9H-purine compound having an acrylamido moiety at the 9-position;
FIG. 2 shows the inhibitory activity of compounds 1 and 3 on phosphorylation of EGFR (L858R/T790M); wherein (A) is compound 1 at a concentration of 10 nmol.L -1 Almost completely inhibits the phosphorylation of EGFR (L858R/T790M); (B) is compound 3 at a concentration of 10 nmol.L -1 The phosphorylation of EGFR (L858R/T790M) was completely inhibited.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
referring to fig. 1, a method for synthesizing-9H-purine compounds and salts thereof containing an acrylamido fragment at the 9-position comprises the following steps:
the method comprises the following steps: in the presence of tertiary amine, stirring 2, 4-dichloro-5-nitropyrimidine and cyclic amine in a solvent to obtain 4-substituted-2-chloro-5-nitropyrimidine, namely an intermediate a;
step two: in the presence of alkali, the intermediate a reacts with arylamine or substituted arylamine to obtain an intermediate b;
step three: reducing the intermediate b by iron powder to obtain an intermediate c;
step four: in the presence of tertiary amine, reacting the intermediate c with phenyl isothiocyanate or substituted phenyl isothiocyanate and EDCI to form an intermediate d;
step five: removing tert-Butyloxycarbonyl (BOC) from the intermediate d under the action of acid to obtain an intermediate e;
step six: and reacting the intermediate e with acryloyl chloride to obtain a target product.
1. Specific examples of Synthesis of Compounds 1 to 21
The structural formula and the number of the representative compound are as follows:
examples of the synthesis of the above compounds, whose structures were characterized by MS, are given below.
Example 1: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (Structure 1):
the method comprises the following steps: synthesis of tert-butyl (S) -3- ((2-chloro-5-nitro-4-pyrimidinyl) amino) pyrrole-1-carboxylate (a1)
Reference eur.j.med.chem.2020,186,111888 makes a 1. The yield thereof was found to be 90%.
Step two: synthesis of (S) -3- (2- (4- (4-methyl-1-piperazinyl) phenylamino) -5-nitro-4-pyrimidinyl) amino) pyrrole-1-carboxylic acid tert-butyl ester (b1)
A1(0.50g,1.46mmol) was dissolved in absolute ethanol (5mL), and then a solution of 4- (4-methyl-1-piperazinyl) aniline (0.28g,1.46mmol) in ethanol (5mL) was added, and Na was added with stirring 2 CO 3 (0.23g,0.22mmol), stirring overnight at room temperature, evaporating the ethanol to dryness, and separating the residue by silica gel column chromatography (dichloromethane: methanol 50:1) to give compound b1 in 81% yield.
Step three: synthesis of tert-butyl (S) -3- ((2- (4- (4-methyl-1-piperazinyl) phenylamino) -5-amino-4-pyrimidinyl) amino) pyrrole-1-carboxylate (c1)
B1(0.50g, 1.00mmol), reduced iron powder (0.56g, 10.00mmol), ammonium chloride (0.54g, 10mmol) were added to a round bottom flask, then anhydrous ethanol (10mL) and water (2mL) were added, heated under reflux for 1h, slightly cooled, filtered with celite while hot, the filter cake was washed with hot ethanol, the filtrate was evaporated to dryness, water (10mL) and saturated sodium carbonate solution (20mL) were added to the residue, extracted with dichloromethane (6X 30mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to give 0.40g of a black solid in 85% yield.
Step four: synthesis of (S) -3- (2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9H-9-purinyl) pyrrolidine-1-carboxylic acid tert-butyl ester (d1)
C1(0.48g,1.02mmol), EDCI (0.30g, 1.54mmol) and phenylisothiocyanate (0.21g, 1.54mmol) were mixed in a flask, followed by the addition of DCE and DIPEA. The flask was placed in a 65 ℃ oil bath and stirred for 4h, the solvent was distilled off, the residue was dissolved in dichloromethane (80mL), washed with water (3X 20mL) and then with saturated brine (20mL), and the organic phase was dried over anhydrous sodium sulfate. Silica gel column chromatography (DCM: MeOH ═ 50:1-30:1) afforded d1 as a yellow solid in 52% yield.
Step five: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (3-pyrrolyl) -9H-purine (e1)
D1(0.25g, 0.44mmol) was dissolved in dichloromethane (4mL), trifluoroacetic acid (2mL) was added, stirring at room temperature for 2h, the solvent was evaporated, toluene was added to the residue, evaporation under reduced pressure was carried out, the residue was dissolved in a mixed solvent of dichloromethane and methanol, K was added 2 CO 3 The powder was dried until no more bubbles were formed and chromatographed on silica gel (DCM: MeOH ═ 15:1) to give e 1. The yield thereof was found to be 73%.
Step six: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (1):
e1(0.10g, 0.21mmol) was suspended in acetone (2mL), cooled in an ice bath and stirred for 5min, then a solution of acryloyl chloride (26. mu.L, 0.32mmol) in acetone (3mL) was added dropwise, after which the ice bath was removed and the mixture stirred at room temperature for 10min, then 10 drops of saturated sodium carbonate solution were added to quench the reaction. The solvent was evaporated to dryness, water (10mL) and saturated sodium carbonate solution (20mL) were added to the residue, dichloromethane was extracted (5 × 30mL), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness, and the residue was subjected to silica gel column chromatography (DCM: MeOH ═ 30:1 to 10:1) to obtain the product. Crystallization from ethyl acetate gave 1 in 46% yield, ms (esi): calculated values: 524.3[ M + H] + And (3) measuring the following values: 524.4.
example 2: synthesis of (S) -2- (4- (4-dimethylamino-1-piperidinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (structural formula 2):
methods are provided for the preparation of compound 1. Compound 2 can be synthesized by substituting 4- (4-dimethylamino-1-piperidinyl) aniline for 4- (4-methyl-1-piperazinyl) aniline. Ms (esi): calculated values: 552.3[ M + H] + And (3) measuring the following values: 552.4.
example 3: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine (Structure 3):
methods also include the preparation of compound 1. Compound 3 can be synthesized by replacing tert-butyl 3-aminopyrrole-1-carboxylate with tert-butyl (S) -3-aminopiperidine-1-carboxylate. Ms (esi): calculated values: 538.3[ M + H] + And (3) measuring the following values: 538.4.
example 4: synthesis of (S) -2- (4- (4-dimethylamino-1-piperidinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine (structural formula 4):
methods are provided for the preparation of compound 1. Ms (esi): calculated values: 566.3[ M + H] + And (3) measuring the following values: 566.4.
example 5: synthesis of (S) -2- (4- (N-methyl-N- (2-dimethylaminoethyl) amino) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (Structure 5):
methods also include the preparation of compound 1. Ms (esi): calculated values: 526.3[ M + H] + And (3) measuring the following values: 526.4.
example 6: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8- (4-fluorophenyl) -9- (N-acryloyl-3-pyrrolyl) -9H-purine (Structure 6):
methods are provided for the preparation of compound 1. Synthesis of MS (ESI) by substituting phenyl isothiocyanate with p-fluoroisothiocyanate: calculated values: 542.3[ M + H] + And (3) measuring the following values: 542.3.
example 7: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-4-piperidinyl) -9H-purine (Structure 7):
methods are provided for the preparation of compound 1. Ms (esi): calculated values: 538.3[ M + H] + And (3) measuring the following values: 538.3.
example 8: synthesis of 2- (6- (4-methyl-1-piperazinyl) -3-pyridylamino) -8-phenylamino-9- (N-acryloyl-4-piperidinyl) -9H-purine (Structure 8):
methods are provided for the preparation of compound 1. Ms (esi): calculated values: 539.3[ M + H] + And (3) measuring the following values: 539.3.
example 9: synthesis of (S) -2- (4- (4-morpholinyl)) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine (Structure 9):
methods are provided for the preparation of compound 1. Ms (esi): calculated values are: 525.3[ M + H] + Measurement value: 525.4.
example 10: synthesis of (R) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine (Structure 3):
methods are provided for the preparation of compound 1. Compound 10 can be synthesized by substituting (R) -3-aminopiperidine-1-carboxylic acid tert-butyl ester for (S) -3-aminopyrrole-1-carboxylic acid tert-butyl ester. 538.3[ M + H] + And (3) measuring the following values: 538.4.
example 11: synthesis of (S) -2- (4- (2-dimethylaminoethoxy) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine (Structure 11):
methods are provided for the preparation of compound 1. Ms (esi): calculated values: 527.3[ M + H] + And (3) measuring the following values: 527.4.
example 12: synthesis of (S) -2- (4- (1-piperidinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (structural formula 12):
methods are provided for the preparation of compound 1. Ms (esi): calculated values: 509.3[ M + H] + And (3) measuring the following values: 509.4.
example 13: synthesis of (S) -2- (4- (1-pyrrolyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (Structure 13):
methods also include the preparation of compound 1. Ms (esi): calculated values: 495.3[ M + H] + And (3) measuring the following values: 495.4.
example 14: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-azetidinyl) -9H-purine (Structure 14):
method of using Compound 1And (4) preparation. Ms (esi): calculated values: 510.3[ M + H] + And (3) measuring the following values: 510.4.
example 15: synthesis of (S) -2- (2-methoxy-4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (formula 15)
Methods are provided for the preparation of compound 1. Ms (esi): calculated values: 572.3[ M + H] + And (3) measuring the following values: 572.4.
example 16: synthesis of 2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-azepanyl) -9H-purine (Structure 16):
methods also include the preparation of compound 1. Ms (esi): calculated values are: 552.3[ M + H] + And (3) measuring the following values: 552.4.
example 17: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine hydrochloride (Structure 17):
compound 1(0.30g, 0.57mmol) was dissolved in acetone (10mL), concentrated hydrochloric acid (1mL) was added dropwise to precipitate a solid, which was filtered off, washed with acetone and dried to give compound 17 in 79% yield. Ms (esi): calculated values: 524.3[ M + H] + And (3) measuring the following values: 524.4.
example 18: synthesis of (S) -2- (4- (4-dimethylamino-1-piperidinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine methanesulfonate (Structure 18):
dissolving the compound 4 in dichloromethane, adding methanesulfonic acid with the molar weight of 1.2 times, stirring the mixture at room temperature overnight, performing suction filtration, and drying to obtain a compound 18. Ms (esi): calculated values: 566.3[ M + H] + And (3) measuring the following values: 566.4.
example 19: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-thiophenyl-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (Structure 19):
to a 100mL round-bottomed flask was added EtOH (30mL), H 2 O (3mL), intermediate c1(350mg), CS 2 (85. mu.L) and KOH (50mg) were refluxed for 4 hours and cooled to room temperature. The solvent was evaporated under reduced pressure and the residue was separated by silica gel column chromatography (eluent: dichloromethane/methanol-20/1) and purified to give a pale yellow solid in 80% yield.
To a round bottom flask was added c1(0.24g), CuI (8.2mg), 1, 10-phenanthroline (16.2mg), K 2 CO 3 (160mg) and DMSO (5 mL). The mixture was stirred at 140 ℃ for 24h under nitrogen. After cooling to room temperature, dichloromethane (50mL) was added, the organic phase was washed with water 3 times, dried over anhydrous sodium sulfate, and the solvent was removed, the residue was subjected to silica gel column chromatography (dichloromethane/methanol ═ 10: 1). The yield thereof was found to be 66%.
The last two reaction steps of the synthesis of compound 19 are analogous to the preparation of compound 1. Ms (esi): calculated values: 541.2[ M + H] + And (3) measuring the following values: 541.3.
example 20: synthesis of (S) -2- (4- (4-dimethylamino-1-piperidinyl) phenylamino) -8-thiophenyl-9- (N-acryloyl-3-pyrrolidinyl) -9H-purine (Structure 20):
preparation method and compound 19. Ms (esi): calculated values: 569.3[ M + H] + And (3) measuring the following values: 569.4.
example 21: synthesis of (S) -2- (4- (4-methyl-1-piperazinyl) phenylamino) -8-phenylamino-9- (N-acryloyl-3-piperidinyl) -9H-purine (Structure 3):
methods are described for the preparation of compound 19. Compound 21 can be synthesized by substituting tert-butyl (S) -3-aminopiperidine-1-carboxylate for tert-butyl 3-aminopyrrolidine-1-carboxylate. Ms (esi): calculated values are: 554.3[ M + H] + And (3) measuring the following values: 554.4.
2. assays for inhibiting EGFR-tyrosine kinase Activity
The method comprises the following steps: respectively dissolving a compound to be measured by DMSO to prepare a mother solution of 10mmol/L, diluting step by step to obtain a solution with the concentration of 0.1 mu mol/L, and respectively adding 5mL of different drugs or DMSO to be measured into an enzyme reaction system. The composition of the enzyme reaction system is as follows: 40mmol/L Tris, pH 7.4, 10mmol/LMgCl 2 0.1mg/mL BSA, 1mmol/L DTT, 10. mu. mol/L ATP, 25mg/L EGFR (L858R), 0.2mg/mL Poly (Glu, Tyr) as substrate, 50. mu.L of the final volume of the reaction system, and 10nmol/L of the drug. After the reaction system is placed at 30 ℃ for reaction for 40min, stop solution is added to stop the reaction, the ATP content in the system is detected by a luciferase method, a chemiluminescence signal is detected on an MD-SpectraMax M5 multifunctional microplate reader, and the intensity of the chemiluminescence signal is inversely proportional to the activity of enzyme in the reaction system. Substituting the detected chemiluminescent signal value into the formula:
inhibition rate ═ 1- ((Lu) Enzyme -Lu Background of the invention )/Lu Medicine -Lu Background of the invention )]×100％
In the formula: lu (Lu) Medicine Denotes the administration group, Lu Background of the invention Represents blank group (without enzyme or medicine), Lu Enzyme The solvent control group is shown.
The results of the experimental determination are shown in table 1.
TABLE 1 Compound at a concentration of 10 nmol.L -1 Inhibition ratio (%) of EGFR (L858R) Activity at that time
Similarly, the inhibitory activity of the compounds on EGFR double mutant kinase could be determined by substituting EGFR (L858R/T790M) for EGFR (L858R), and the results are shown in Table 2.
TABLE 2 Compound concentration of 10 nmol.L -1 Inhibition ratio (%) of EGFR (L858R/T790M) Activity
Wherein the compound 1-4,19-20 has strong inhibitory activity on EGFR double mutant with the concentration of 10 nmol.L -1 The inhibition rate of the activity of the compound on EGFR (L858R/T790M) is more than 93 percent.
3. Western Blotting analysis
To further verify the activity of the synthesized-9H-purine compounds containing an acrylamido fragment at the 9-position of the present invention to inhibit EGFR-TK, we used Western Blotting to determine the effect of compounds 1 and 3 on EGFR (L858R/T790M) phosphorylation.
The method comprises the following steps: h1975 cells were arranged at 8X 10 5 One well per well was inoculated in six well plates at 37 ℃ with 5% CO 2 Culturing in an incubator for 24h, adding 2 μ L of DMSO solutions of compounds 1 or 3 with different concentration gradients, continuously culturing for 24h, lysing cells by using lysis solution containing protease inhibitor and phosphatase inhibitor, centrifuging at 12000rpm for 20min, taking supernatant, adding loading buffer, uniformly mixing, heating in a water bath kettle at 95 ℃ for 10min for denaturation, separating proteins by using 8% SDS-PAGE according to the loading amount of 25 μ g per hole, transferring to a PVDF membrane, sealing the PVDF membrane in 5% skimmed milk for 1h, incubating in primary antibody solution at 4 ℃ overnight, incubating in secondary antibody solution at room temperature for 1h, and finally exposing and photographing, wherein the result is shown in figure 2.
4. Verification of in vitro anti-cancer Activity
In order to verify the anticancer activity of the synthesized 9-position-9H-purine compound containing acrylamide fragments and salts thereof, gefitinib and oxitinib (AZD9291) are used as positive control drugs, and an in vitro MTT method is adopted to determine the compound 1-18 to human lung cancer cell HCC827 (EGFR) Del19 )，H1975(EGFR L858R/T790M ) And human epidermal carcinoma cell A431 (EGFR) WT ) The growth inhibitory effect of (1).
The verification method comprises the following steps: tumor cell HCC827 is cultured in RPMI1640 medium containing 10% fetal bovine serum and containing penicillin 100 U.mL -1 Streptomycin 100. mu.g.mL -1 At 37 ℃ and 5% CO 2 Subculturing in an incubator. Taking 0.25% pancreatin to digest adherent tumor cells, preparing cell suspension by RPMI1640 culture solution containing 10% fetal bovine serumLiquid with a concentration of 2.5X 10 4 Individual cells/ml. mu.L (containing about 5000 tumor cells) per well of 96-well culture plate was inoculated and cultured at 37 ℃ for 24 hours. The administration groups were added with drugs at different concentrations, each set at 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10. mu. mol. L -1 10 concentration gradients, each set with 3 parallel wells. Adding solvent with the same volume as the medicine into the control group, placing at 37 deg.C and 5% CO 2 Culturing for 72h in incubator, discarding culture solution, adding 20 μ L of 5 mg/mL -1 After incubation for 4h, the supernatant was discarded, 200. mu.L of DMSO was added to each well, and the Optical Density (OD) was measured at 490nm using a microplate reader after gentle shaking. And (4) calculating a result:
taking tumor cells treated by solvent control as a control group, and calculating the inhibition rate of the drug on the tumor cells according to the following formula:
and further using linear regression to determine the median Inhibitory Concentration (IC) 50 )。
The results are shown in Table 3.
TABLE 3 antiproliferative Inhibition (IC) of the Compounds on three human cancer cells 50 ，nmol·L -1 )
"ND": and (4) not measuring.
The results in Table 3 demonstrate that the compounds 3, 4, 6 and 18, 19-21 provided by the present invention inhibit H1975 cells more strongly than the positive drug AZD 9291; the activity of compounds 3 and 20 in inhibiting HCC827 cells is 4.45 times stronger than that of oxitinib and 31.82 times stronger than that of gefitinib. And the activity of the compound 3 on A431 cells is weak, which indicates that the compound 3 has weak effect on wild type EGFR and has small side effect after being applied to clinic.
The most active compound reported in document j.med.chem.2012,55,10685-10699 is 9 e. However, the CLogP of 9e is 6.40, and the water solubility and potency are poor. 9e belongs to reversible EGFR-TKI, and has strong activity on HCC827 cells and weak activity in inhibiting H1975 cells. The compound D9(Eur.J.Med.chem.2020,186,111888) reported by this group, also belonging to reversible EGFR-TKI, has strong activity against HCC827 cells and weak activity against H1975 cells (IC) 50 A value of 200 nmol.L -1 ). The compound C-ZLF002 disclosed in Chinese patent ZL201811205782.7 belongs to irreversible EGFR-TKI and is similar to the structure of the compound synthesized by people. We have synthesized the compound C-ZLF002, and determined its activity in inhibiting HCC827 and H1975 cells under the same conditions, and its IC 50 Respectively having values of 0.38 and 41 nmol.L -1 . The compound 3 provided by the invention inhibits the IC of H1975 cells 50 A value of 12 nmol.L -1 . The activity of compound 3 was increased 3.4-fold relative to C-ZLF 002. The activity of the compound provided by the invention is remarkably improved. In addition, the ClogP value of the compound 9e and C-ZLF002 is larger than 6, and the drug property is poor.
The invention relates to application of a composition of-9H-purine compounds containing acrylamide amino fragments at 9-position and salts thereof in preparing anticancer drugs, and the application can be capsules, oral liquid or granules or injections. The preparation can be prepared according to conventional preparation process of various preparations, wherein the content of effective components is 1-300mg, preferably 30-100 mg.
The oral preparation of the invention can contain pharmaceutic adjuvants, including a stabilizer, a solubilizer, a lubricant and the like, such as glucose, lactose, cellulose, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, starch, pectin, cyclodextrin, Tween-80, polyvinyl alcohol, magnesium stearate, talcum powder and the like.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (4)
1. A-9H-purine compound containing an acrylamido fragment at the 9-position, wherein the compound is one of the following compounds:
2. a pharmaceutically acceptable salt of a-9H-purine compound having an acrylamido moiety in the 9-position as claimed in claim 1 wherein the pharmaceutically acceptable salt is the hydrochloride, hydrobromide, nitrate, phosphate, sulphate, acetate, fumarate, malate, citrate, tartrate, maleate, lactate, citrate, camphorsulfonate, benzoate, gluconate, glutamate, isethionate, succinate or mesylate salt.
3. Use of a pharmaceutically acceptable salt of an-9H-purine compound having an acrylamido moiety in the 9-position as claimed in claim 1 or of a-9H-purine compound having an acrylamido moiety in the 9-position as claimed in claim 2 in the preparation of an anti-cancer pharmaceutical formulation.
4. The use according to claim 3, wherein the anticancer pharmaceutical preparation is a tablet, capsule or injection, wherein each tablet, granule or preparation contains 10-300 mg of-9H-purine compounds or their pharmaceutically acceptable salts, each of which contains an acrylamido segment at the 9-position.
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