CN108250187B - Indole-1-carbonate compound, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of drug synthesis, and particularly relates to indole-1-carbonate compounds shown as a general formula (I), pharmaceutically acceptable salts, deuterated compounds or solvates thereof, a preparation method thereof and application thereof in preparing drugs for selectively inhibiting active EGFR resistant mutation T790M and activating mutation.

Description

Indole-1-carbonate compound, preparation method and application thereof

Technical Field

The invention belongs to the field of drug synthesis, and particularly relates to indole-1-carbonate compounds, a preparation method thereof, and application thereof in preparation of antitumor drugs.

Background

An Epidermal Growth Factor Receptor (EGFR), also called ErbB1 or HER1, is an important member of the ErbB receptor family, and is homodimerized or heterodimerized with other members of the ErbB family (such as HER2, HER3, and HER4) after binding with an endogenous ligand such as Epidermal Growth Factor (EGF), and through phosphorylation of key tyrosine residues at the intracellular end, downstream pathways are activated, and cell proliferation and survival are regulated.

Epidemiologically, it has been found that overexpression or overactivation of EGFR is closely related to the development of many tumors, such as 40-80% of non-small cell lung cancers (NSCLC) with EGFR overexpression (Fujino, S.; Enokibiori, T.; Tezuka, N.; Asada, Y.; Inoue, S.; Kato, H.; Mori, A.A, complex of epidemic growth factor receptors and other pathological parametersin non-small cell lung cancer. Eur. J. cancer1996,32, 2070-2074.); 3-5 fold amplification of the EGFR gene occurred in 50% of rectal cancers (Cappouzzo, F.; Finococchiaro, G.; Rossi, E.;

P.A.; carnaghi, c.; calandri, c.; benciadino, k.; ligorio, c.; ciardiello, f.; presssiani, t.; EGFR FISH assay precursors for response to therapy in chemotherapy recovery chromatography, Ann. Oncol.2008,19,717, 723.); it also has significant overexpression or activation phenomena in glial cell line carcinoma and head and neck cancer. EGFR is therefore one of the important tumor therapeutic targets. Lung cancer death is the first killer of malignant tumors, the incidence of lung cancer is increased year by year in China, the number of new diseases is up to 70 ten thousand every year, the proportion of EGFR activating mutation is up to 40-60%, and China has a great demand for EGFR third-generation inhibitors. Therefore, the development of third-generation EGFR inhibitors with high activity, high selectivity and safety is of great significance.

Currently, the drugs approved to be marketed for selectively targeting EGFR include Gefitinib (Gefitinib), Erlotinib (Erlotinib), Afatinib (Afatinib), Dacomitinib (Dacomitinib) and oxitinib (osiertinib), wherein Gefitinib and Erlotinib are first-generation EGFR inhibitors and are non-covalent binding inhibitors; afatinib and Dacomitinib are second generation EGFR covalent binding inhibitors; while the activity of oxitinib on EGFR resistance mutation T790M and activating mutation (L858R, delE746-A750 or Exon19 are deleted) is higher, and is obviously superior to the activity of EGFR wild type (EGFRWT), so that the skin and gastrointestinal toxicity caused by strong inhibition of EGFRWT by first-generation and second-generation inhibitors is reduced, and the oxitinib is a third-generation inhibitor. The Oxitinib has great clinical success, the objective remission rate is 61%, and the tumor control rate after administration is up to 91%. The important metabolite AZ5104 has better activity (almost 10-fold difference), has more than 10-fold significant selectivity difference on EGFRWT and mutation, and has poor oral exposure.

In order to better improve the druggability of the third-generation EGFR inhibitor and possibly achieve better treatment effect in human bodies, the patent designs and synthesizes AZD9291 and related compounds, carbonate structures are innovatively introduced into indole nitrogen, and the research on the drugs is very rare (only 2 compounds containing indole-1-carbonate are obtained by ThOMSON REUTERS CORTELLISS (TM) search for treating advanced melanoma, and the search time is 11 months in 2017).

Disclosure of Invention

The invention aims to provide indole-1-carbonate compounds, pharmaceutically acceptable salts, deuterated compounds or solvates thereof.

Another object of the present invention is to provide a method for preparing the indole-1-carbonate compound, a pharmaceutically acceptable salt, a deuterated compound or a solvate thereof,

the invention also aims to provide a pharmaceutical composition containing the indole-1-carbonate compound, the pharmaceutically acceptable salt, the deuterated compound or the solvate thereof.

Another object of the present invention is to provide the indole-1-carbonate compound, a pharmaceutically acceptable salt, a deuterated compound or a solvate thereof; or the application of the composition containing the indole-1-carbonate compound, the pharmaceutically acceptable salt, the deuterated compound or the solvate thereof in preparing the antitumor drugs.

The invention provides an indole-1-carbonate compound shown in a general formula (I), and pharmaceutically acceptable salts, deuterated compounds or solvates thereof,

wherein the content of the first and second substances,

r is selected from substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₃-C₆Cycloalkyl, substituted or unsubstituted C₆-C₁₂Aryl, substituted or unsubstituted benzyl; the substituted substituent is selected from halogen, nitro, cyano, C₁-C₆Alkyl radical, C₁-C₆Acyloxy, C₁-C₆Alkoxy or C₂-C₆An alkenyl group;

r' is selected from C₁-C₆Alkyl or C₁-C₆A deuterated alkyl group; preferably methyl or deuterated methyl.

In a preferred embodiment of the invention, the compound of the general formula (I) is selected from indole-1-carbonate compounds shown in the following general formula (I-A), pharmaceutically acceptable salts, deuterated compounds or solvates thereof,

wherein R is as defined above.

Preferably, the first and second electrodes are formed of a metal,

r is selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, phenyl, benzyl, methylbenzyl, acetoxybenzyl or allyl; further preferably methyl, ethyl, propyl, isopropyl, cyclopropyl, phenyl, benzyl, o-methylbenzyl, o-acetoxybenzyl or 2-allyl;

r' is methyl or deuterated methyl.

More preferably, the indole-1-carbonate compound shown in the formula (I), the deuterated compound, the pharmaceutically acceptable salt or the solvate thereof is selected from compounds shown in the following formula:

“C₁-C₆alkyl "refers to straight or branched chain saturated having 1 to 6 carbon atoms in the chainAnd hydrocarbyl groups including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like.

“C₁-C₆Deuterated alkyl "refers to C wherein one or more hydrogen atoms are replaced on the alkyl with deuterium atoms₁-C₆An alkyl group.

“C₁-C₆Acyloxy "refers to straight or branched chain alkanoyloxy having 1 to 6 carbon atoms in the alkyl moiety, for example, methacryloyloxy, ethylacryloxy, n-propylacyloxy, isopropylacyloxy, n-butylacyloxy, isobutylacyloxy or tert-butylacyloxy.

“C₁-C₆Alkoxy "refers to straight or branched chain O-alkyl groups containing from 1 to 6 carbon atoms in the alkyl moiety, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy.

“C₃-C₆Cycloalkyl "refers to a group containing one or more saturated and/or partially saturated rings, all ring-forming atoms being carbon atoms, which includes from 3 to 6 carbon atoms; for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl and the like.

“C₆-C₁₂Aryl "refers to an aromatic ring group containing 6 to 12 ring atoms, but no hetero atom in the ring atoms, preferably a 6-to 10-membered aryl group (i.e., an aryl group having 6 to 10 carbon atoms), such as phenyl or naphthyl.

"halogen" refers to fluorine, chlorine, bromine and iodine.

In a second aspect of the present invention, a method for preparing indole-1-carbonate compounds represented by general formula (I) is provided, where the method includes a step of subjecting a compound represented by formula (II) to an acylation reaction with an acylating agent to obtain a compound represented by general formula (I), and the reaction is represented by the following reaction formula:

wherein R, R' is as defined in formula (I);

the acylating agent is selected from haloformate XCOOR and dicarbonate

Or carbonates

Wherein X is halogen;

preferably, the haloformate XCOOR is selected from chloroformates or bromoformates.

In a preferred embodiment, the method for preparing the indole-1-carbonate compound shown in formula (i) comprises one of the following methods:

the method comprises the following steps:

wherein R, R' is as defined in formula (I);

reacting the compound shown in the formula II with chloroformic ester ClCOOR under an alkaline condition to obtain a compound shown in a general formula (I);

the alkali is selected from one or more of sodium hydride, potassium tert-butoxide, potassium carbonate and triethylamine, and is preferably sodium hydride;

the solvent used in the reaction is one or more selected from tetrahydrofuran, dichloromethane, N-dimethylformamide, acetonitrile, dioxane and toluene, and is preferably tetrahydrofuran;

the reaction temperature is 0-50 ℃;

the method 2 comprises the following steps:

wherein R, R' is as defined in formula (I);

the compound of formula II is reacted with a dicarbonate under alkaline conditions

Reacting to obtain a compound shown as a general formula (I);

the alkali is selected from one or more of sodium hydride, potassium tert-butoxide, potassium carbonate, triethylamine and 4-dimethylaminopyridine, preferably triethylamine and dimethylaminopyridine,

the solvent used in the reaction is one or more selected from tetrahydrofuran, dichloromethane, N-dimethylformamide, acetonitrile, dioxane and toluene, preferably dichloromethane;

the reaction temperature is 0-40 ℃;

the method 3 comprises the following steps:

wherein R, R' is as defined in formula (I);

the compound of formula II and a carbonate

Under the condition that ionic liquid is used as a catalyst, reacting to obtain a compound shown in a general formula (I);

the ionic liquid is 1-butyl-3-methylimidazol [ Bmim ] OH hydroxide or tetrabutylammonium hydroxide;

the temperature of the reaction is 0-90 ℃.

In a preferred embodiment, the method for preparing the indole-1-carbonate compound shown in formula (i) comprises the following steps:

preparation of intermediate 3: reacting the intermediate 1 with deuterated iodomethane or iodomethane under an alkaline condition to obtain an intermediate 2, and removing a Boc protecting group through acid catalysis to obtain an intermediate 3;

the alkali is selected from one or more of sodium hydride, potassium carbonate and triethylamine, and potassium carbonate or triethylamine is preferred;

the acid is selected from one or more of hydrochloric acid, hydrobromic acid or trifluoroacetic acid, and is preferably hydrochloric acid;

in a preferred embodiment, the method for preparing the indole-1-carbonate compound shown in formula (i) comprises the following steps 1 to 5:

wherein R, R' is as defined in formula (I);

step 1: carrying out nucleophilic substitution on the intermediate 4 and the intermediate 5 under the acid catalysis condition to obtain an intermediate 6;

the acid is selected from one or more of p-toluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid or hydrochloric acid, and is preferably p-toluenesulfonic acid;

the reaction solvent is selected from one or more of 2-pentanol, tert-butanol, 1, 4-dioxane or tetrahydrofuran, and is preferably 2-pentanol;

the reaction temperature is selected from 80-150 ℃;

step 2: nucleophilic substitution is carried out on the intermediate 6 and the intermediate 3 under the alkaline condition to obtain an intermediate 7;

the alkali is selected from one or more of triethylamine, diisopropylethylamine, potassium carbonate or sodium carbonate, and is preferably diisopropylethylamine;

the reaction solvent is one or more selected from N, N-dimethylformamide, N-dimethylacetamide and N-methylpyrrolidone, and is preferably N, N-dimethylacetamide;

the reaction temperature is 80-180 ℃;

and step 3: the nitro group of the intermediate 7 is reduced by iron powder or metal catalytic hydrogen to obtain an intermediate 8;

the iron powder reduction is selected from the reaction in the presence of iron powder and acetic acid or in the presence of iron powder, ammonium chloride and ethanol;

the reaction temperature of the iron powder reduction is 60-100 ℃;

the metal catalyzing the reduction of hydrogen is selected from Pd/C,Pd(OH)₂raney nickel (Raney Ni) or PtO₂Preferably Pd/C;

the solvent for metal catalytic hydrogen reduction is selected from one or more of ethanol, ethyl acetate, tetrahydrofuran or dioxane, preferably ethanol;

the reaction temperature of the metal catalytic hydrogen reduction is 20-60 ℃;

and 4, step 4: 8 and acryloyl chloride react in a solvent to obtain an intermediate 9;

the solvent for the reaction is selected from one or more of dichloromethane, tetrahydrofuran, ethyl acetate or N, N-dimethylformamide, and is preferably dichloromethane;

the reaction temperature is-10-50 ℃;

and 5: the intermediate 9 reacts with an acylation reagent to obtain the indole-1-carbonate compound shown in the formula (I),

the solvent for the reaction is selected from one or more of dichloromethane, tetrahydrofuran, ethyl acetate or N, N-dimethylformamide, and is preferably dichloromethane;

the reaction temperature is-10-50 ℃.

In a third aspect, the present invention also provides a pharmaceutical composition comprising a safe and effective amount of one or more selected from the group consisting of the indole-1-carbonate compounds, deuterated compounds, pharmaceutically acceptable salts and solvates thereof as an active ingredient, and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical composition further comprises other pharmaceutically acceptable therapeutic agents, in particular other anti-tumor drugs. Such therapeutic agents include, but are not limited to: an antitumor drug acting on a DNA chemical structure, such as cisplatin, an antitumor drug affecting nucleic acid synthesis, such as Methotrexate (MTX), 5-fluorouracil (5FU) and the like, an antitumor drug affecting nucleic acid transcription, such as doxorubicin, epirubicin, aclarubicin, mithramycin and the like, an antitumor drug affecting tubulin synthesis, such as paclitaxel, vinorelbine and the like, an aromatase inhibitor, such as aminoglutethimide, landetron, letrozole, ryanodine and the like, a cell signaling pathway inhibitor, such as epidermal growth factor receptor inhibitor Imatinib (Imatinib), Gefitinib (Gefitinib), Erlotinib (Erlotinib), Lapatinib (Lapatinib) and the like.

"pharmaceutically acceptable salt" refers to salts of inorganic or organic acids that are relatively non-toxic. Suitable inorganic acids include hydrochloric, hydrobromic, sulphuric or phosphoric acid and suitable organic acids include trifluoromethanesulphonic, methanesulphonic, trifluoroacetic, citric, maleic, fumaric, succinic or tartaric acid.

In a fourth aspect, the invention also provides an application of the indole-1-carbonate compound represented by the general formula (I), a deuterated compound, a pharmaceutically acceptable salt or a solvate thereof in preparing a medicament for selectively inhibiting the active EGFR resistance mutation T790M and the activating mutation.

The selective inhibitory activity EGFR resistance mutation T790M and the activating mutation include: lung cancer, breast cancer, ovarian cancer, liver cancer, melanoma, prostate cancer, colon cancer, rectal cancer, glial cell carcinoma, head and neck cancer, gastric cancer, etc.; the compound of the general formula (I) and the pharmaceutically acceptable salt thereof can be independently administered or combined with other pharmaceutically acceptable compounds, and the administration route can be selected from oral administration, rectal administration and parenteral administration (intravenous, intramuscular or subcutaneous).

Advantageous effects

The general formula (I) compound provided by the invention introduces a carbonate structure on indole nitrogen innovatively in design, the obtained compound shows good activity and selectivity at a cell level, the exposure amount in an animal body is obviously superior to that of Oxecitinib, and the compound has the characteristic of excellent drug forming property. In conclusion, the patent innovatively obtains the selective inhibitory activity EGFR resistance mutation T790M and the activating mutated small molecule entity with more medicinal and novel structures.

The preparation method of the compound of the general formula (I) provided by the invention has the obvious advantages of one-step reaction of commercially available raw materials, simple operation and high reaction efficiency (only 10min is needed for part of compound reaction). Meanwhile, the compounds can be synthesized by a de novo method, and the synthetic route has better flexibility and wider applicability.

Drawings

FIG. 1 shows the effect of compound I-1 on the phosphorylation levels of EGFR and its downstream signals AKT and ERK mutated at L858R/T790M.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention only and are not intended to limit the invention. The specific experimental procedures are not specified in the specific examples, and are generally carried out under conventional conditions.

N- (5- ((4- (1H-indol-3-yl) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide and the like were prepared from Tokyo Longitude technology Co., Ltd. (J.Med.chem.2014,57, 8249-8267);

¹h NMR is recorded by a nuclear magnetic resonance apparatus of the Varian Mercury-500 or Varian Mercury-400 type, chemical shifts are expressed in (ppm); mass spectra were recorded on a Finnigan/MAT-95(EI) and Finnigan LCQ/DECAandMicromass Ultra Q-TOF (ESI) type mass spectrometer; the solvents and reagents used were purchased from national pharmaceutical group chemical reagents, Inc. and Bailingwei technologies, Beijing.

Described below "¹H-NMR(CDCl₃400MHz) "refers to the nuclear magnetic resonance of hydrogen nuclei measured under 400MHz conditions using deuterated chloroform as a solvent, as chemical shift, in ppm.

Examples of preparation of Compound (I)

Example 1

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid methyl ester (I-1)

The synthesis method 1:

dissolving N- (5- ((4- (1H-indol-3-yl) pyrimidine-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide (namely AZ5104, 3.0g and 6.18mmol) in 200mL of anhydrous tetrahydrofuran, adding slow sodium hydride (wrapped by mineral oil with the content of 60 percent, 740mg and 18.53mmol) under ice bath conditions, stirring for 2min, slowly dissolving methyl chloroformate (1.75g and 18.53mmol) in 50mL of tetrahydrofuran, removing the ice bath, slowly pouring the reaction liquid into 200mL of saturated ammonium chloride solution, adding 200mL of ethyl acetate, dropwise adding an organic phase for extraction, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, drying with a dry solvent, and purifying by column chromatography to obtain 2- (2- ((5-acrylamide-4- ((2- (dimethylamino) ethyl) ((10 mL of ethyl acetate) is obtained Methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid methyl ester, 2.61g, 77.0% yield.

¹H NMR(400MHz,CDCl₃)10.11(s,1H),9.65(s,1H),8.52(d,J＝5.2Hz,1H),8.46(s,1H),8.32(d,J＝7.8Hz,1H),8.26(d,J＝8.1Hz,1H),7.62(s,1H),7.36(dt,J＝20.0,7.2Hz,2H),7.12(d,J＝5.2Hz,1H),6.80(s,1H),6.41(d,J＝16.5Hz,1H),6.31(dd,J＝16.9,10.0Hz,1H),5.67(d,J＝10.6Hz,1H),4.06(s,3H),3.89(s,3H),2.88(t,J＝5.4Hz,2H),2.71(s,3H),2.31–2.28(m,2H),2.26(s,6H)。

LR-Mass(ESI)：544.3(M+1，C₂₉H₃₄N₇O₄)。

The synthesis method 2 comprises the following steps:

dissolving N- (5- ((4- (1H-indol-3-yl) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide (namely AZ5104, 3.0g and 6.18mmol) in 100mL of anhydrous dichloromethane, sequentially adding triethylamine (1.88g and 18.53mmol), 4-dimethylaminopyridine (0.15g and 1.24mmol) and dimethyl dicarbonate (1.08g and 8.03mmol), reacting at room temperature for 6H, adding 100mL of dichloromethane and 100mL of water to the reaction solution for dilution, separating, drying an organic phase by using anhydrous sodium sulfate, drying a solvent by spinning, and purifying by column chromatography to obtain the 2- (2- ((5-acrylamide-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidine-4- Methyl) -1H-indole-1-carbonate, 2.40g, yield 71.6%. The nuclear magnetic and mass spectra were the same as before.

The synthesis method 3:

dissolving N- (5- ((4- (1H-indol-3-yl) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide (namely AZ5104, 3.0g and 6.18mmol) in 30mL dimethyl carbonate, adding [ Bmim ] OH (88mg and 0.618mmol), heating to 90 ℃ for reaction for 1H, slowly pouring the reaction solution into 200mL saturated ammonium chloride solution, adding 200mL ethyl acetate to extract an organic phase, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, spinning to dry a solvent, and purifying by column chromatography to obtain 2- (2- ((5-acrylamide-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indol-1 Methyl carbonate, 2.12g, yield 63.2%. The nuclear magnetic and mass spectra were the same as before.

Example 2

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid ethyl ester (I-2)

The synthesis was performed in the same manner as in synthesis 1 of example 1, except that ethyl chloroformate was used instead of methyl chloroformate.

¹H NMR(400MHz,CDCl₃)10.08(s,1H),9.64(s,1H),8.53(d,J＝5.2Hz,1H),8.42(s,1H),8.38(d,J＝7.5Hz,1H),8.26(d,J＝8.1Hz,1H),7.64(s,1H),7.37(ddd,J＝15.1,14.0,6.7Hz,2H),7.14(d,J＝5.2Hz,1H),6.81(s,1H),6.41(d,J＝2.8Hz,2H),5.72–5.64(m,1H),4.53(q,J＝7.1Hz,2H),3.90(s,3H),2.94–2.86(m,2H),2.72(s,4H),2.36(s,2H),2.31(s,8H),1.49(d,J＝7.1Hz,3H)。

LR-Mass(ESI)：558.8(M+1，C₃₀H₃₆N₇O₄)。

Example 3

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carbonic acid propyl ester (I-3)

The synthesis method was the same as synthesis method 1 in example 1, except that methyl chloroformate was replaced with propyl chloroformate.

¹H NMR(500MHz,CDCl₃)9.91(s,1H),9.50(s,1H),8.73(d,J＝14.8Hz,1H),8.55(dd,J＝14.8,3.1Hz,1H),8.11(dd,J＝14.9,3.1Hz,1H),7.43–7.06(m,4H),6.43(s,1H),6.17(ddd,J＝26.6,24.4,12.2Hz,2H),5.69(dd,J＝33.3,4.6Hz,1H),4.69(s,1H),4.21(t,J＝14.7Hz,2H),3.86(s,3H),3.53(dt,J＝21.9,14.6Hz,2H),2.75(s,3H),2.50(t,J＝14.6Hz,2H),2.21(s,6H),1.91–1.49(m,2H),1.01(t,J＝13.4Hz,3H)。

LR-Mass(ESI)：572.8(M+1，C₃₁H₃₈N₇O₄)。

Example 4

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid isopropyl ester (I-4)

The synthesis was the same as in example 1, except that methyl chloroformate was replaced with isopropyl chloroformate.

¹H NMR(400MHz,DMSO-d₆)10.08(s,1H),8.63(s,1H),8.59(s,1H),8.46(d,J＝8.0Hz,1H),8.43–8.35(m,2H),8.15(d,J＝8.3Hz,1H),7.43(d,J＝5.3Hz,1H),7.38(t,J＝7.3Hz,1H),7.23(t,J＝7.4Hz,1H),7.04(s,1H),6.45(s,1H),6.19(dd,J＝16.9,1.9Hz,1H),5.77–5.69(m,1H),5.29–5.15(m,1H),3.81(s,3H),2.95(s,2H),2.74(s,3H),2.40(s,2H),2.28(s,6H),1.45(d,J＝6.2Hz,6H)。

LR-Mass(ESI)：572.8(M+1，C₃₁H₃₈N₇O₄)。

Example 5

2- (2- ((5-acrylamide-4- ((2- (deuterated dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid methyl ester (I-5)

Step 1:

dissolving N-methyl-N-tert-butoxycarbonylethylenediamine (1.74g, 10mmol) in 50mL of anhydrous dichloromethane, and sequentially adding potassium carbonate (4.15g, 30mmol) and deuterated iodomethane (CD) under ice-bath condition₃I, 3.19g and 22mmol) and reacting at room temperature for 12h, drying the solvent by spinning and purifying by column chromatography to obtain the N-methyl-N-tert-butyloxycarbonyl-N ', N' -di (deuterated methyl) -ethylenediamine, 1.5g and the yield of 72 percent.

¹H NMR(500MHz,CDCl₃)3.38(t,J＝14.6Hz,1H),3.23(s,3H),3.20–3.10(m,1H),2.53(t,J＝14.6Hz,2H),1.42(s,9H)。

LR-Mass(ESI)：209.3(M+1，C₁₀H₁₇D₆N₂O₂)。

Dissolving N-methyl-N-tert-butoxycarbonyl-N ', N ' -di (deuterated methyl) -ethylenediamine (1.5g, 7.2mmol) in 30mL of dioxane solution of 1M hydrogen chloride, reacting at room temperature for 1h, and spin-drying the solvent to obtain 1.3g of dihydrochloride of N, N-di (deuterated methyl) -N ' -methyl-ethylenediamine with the yield of 100%.

¹H NMR(500MHz,CDCl₃)3.25(s,3H),2.54–2.45(m,2H),2.45–2.33(m,2H)。

LR-Mass(ESI)：109.2(M+1，C₅H₉D₆N₂)。

Step 2:

dissolving the intermediate 4(23g, 100mmol) in 500mL of 2-pentanol, adding p-toluenesulfonic acid (1.72g, 10mmol), heating to 105 ℃ for reaction for 4h, cooling the reaction solution to precipitate yellow solid, filtering, washing a filter cake with 50mL of 2-pentanol, and drying to obtain 635g of the intermediate with the yield of 92%.

¹H NMR(500MHz,CDCl₃)8.65–8.50(m,2H),8.23–8.06(m,3H),7.59(dd,J＝11.1,7.0Hz,1H),7.33(ddd,J＝18.1,13.0,5.7Hz,4H),5.02(s,1H),3.86(s,3H)。

LR-Mass(ESI)：380.4(M+1，C₁₉H₁₅N₅O₃)。

Dissolving the intermediate 6(19g, 50mmol) in 200mL of trifluoroethanol, adding diisopropylethylamine (25.9g, 200mmol) and the intermediate 3(9g, 50mmol) in sequence, refluxing for 12h, drying the solvent by spinning, and purifying by column chromatography to obtain an intermediate 7(15g, light yellow solid, 64%).

¹H NMR(500MHz,CDCl₃)8.65–8.44(m,2H),8.27–8.06(m,2H),7.94(s,1H),7.59(dd,J＝11.1,7.0Hz,1H),7.43–7.20(m,3H),6.59(s,1H),5.04(s,1H),3.86(s,3H),3.63(t,J＝14.6Hz,1H),3.49(t,J＝14.6Hz,1H),2.75(s,3H),2.50(t,J＝14.5Hz,2H)。

LR-Mass(ESI)：468.4(M+1，C₂₄H₂₂D₆N₇O₃)。

Dissolving intermediate 7(10g, 21.4mmol), iron powder (11.9g, 214mmol) and ammonium chloride (11.44g, 214mmol) in 200mL of ethanol/water mixed solution (ethanol/water ═ 3:1, v/v), heating and refluxing for 4h, adding 200mL of ethyl acetate into the reaction solution for dilution, filtering with diatomite, spin-drying the filtrate, and purifying by column chromatography to obtain intermediate 8(8.3g, pale yellow viscous liquid, yield 88%).

¹H NMR(500MHz,CDCl₃)8.64–8.46(m,2H),8.14(q,J＝6.5Hz,2H),7.66–7.19(m,4H),6.15(d,J＝20.1Hz,2H),5.01(s,1H),3.84(d,J＝22.3Hz,5H),3.50(dt,J＝20.7,14.6Hz,2H),2.75(s,3H),2.50(t,J＝14.6Hz,2H)。

LR-Mass(ESI)：438.7(M+1,C₂₄H₂₄D₆N₇O)。

Dissolving the intermediate 8(4.4g and 10mmol) in 100mL of anhydrous dichloromethane, slowly and dropwise adding acryloyl chloride (0.9g and 10mmol) dissolved in 50mL of anhydrous dichloromethane under an ice bath condition, reacting for 2h in an ice bath, adding 100mL of 1M sodium carbonate solution to quench the reaction, separating an organic phase, drying the organic phase with anhydrous sodium sulfate, spin-drying the solvent, and purifying by column chromatography to obtain an intermediate 9(3.5g, pale yellow solid, 71%).

¹H NMR(500MHz,CDCl₃)9.89(s,1H),8.73–8.51(m,2H),8.19–8.03(m,2H),7.59(dd,J＝11.1,7.0Hz,1H),7.41–7.24(m,3H),7.12(s,1H),6.49–6.18(m,2H),6.05(dd,J＝20.0,4.6Hz,1H),5.69(dd,J＝33.3,4.6Hz,1H),5.15(s,1H),3.86(s,3H),3.56(dt,J＝28.9,14.7Hz,2H),2.75(s,3H),2.50(t,J＝14.7Hz,2H).

LR-Mass(ESI)：492.8(M+1,C₂₇H₂₆D₆N₇O₂)。

Example 5 was obtained by reacting intermediate 9 with methyl chloroformate using synthesis method 1 in example 1.

¹H NMR(500MHz,CDCl₃)9.90(s,1H),9.46(s,1H),8.68–8.38(m,2H),8.10(dd,J＝15.0,3.1Hz,1H),7.41–7.01(m,4H),6.42(s,1H),6.11(ddd,J＝26.4,24.9,12.5Hz,2H),5.68(dd,J＝32.8,4.9Hz,1H),4.98(s,1H),3.86(s,3H),3.74–3.48(m,5H),2.75(s,3H),2.50(t,J＝14.6Hz,2H)。

LR-Mass(ESI)：550.8(M+1,C₂₉H₂₈D₆N₇O₄)。

Example 6

2- (2- ((5-acrylamide-4- ((2- (deuterated dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid ethyl ester (I-6)

The procedure was as in example 5 except that methyl chloroformate was replaced with ethyl chloroformate.

¹H NMR(500MHz,CDCl₃)9.83(s,1H),8.80–8.59(m,2H),8.51(dd,J＝14.8,3.1Hz,1H),8.07(dd,J＝14.9,3.1Hz,1H),7.42–7.01(m,4H),6.40(s,1H),6.10(ddd,J＝26.5,24.9,12.5Hz,2H),5.66(dd,J＝32.9,4.9Hz,1H),4.98(s,1H),4.19(q,J＝11.8Hz,2H),3.84(s,3H),3.55(dt,J＝38.8,14.3Hz,2H),2.74(s,3H),2.49(t,J＝14.1Hz,2H),1.26(t,J＝11.8Hz,3H)。

LR-Mass(ESI)：564.8(M+1,C₃₀H₃₀D₆N₇O₄)。

Example 7

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carbonic acid cyclopropyl ester (I-7)

The synthesis was the same as in example 1, except that methyl chloroformate was replaced with cyclopropyl chloroformate.

¹H NMR(500MHz,CDCl₃)8.80–8.42(m,3H),8.11(dd,J＝14.9,3.0Hz,1H),7.48–7.06(m,4H),6.49–6.24(m,2H),6.05(dd,J＝20.0,4.4Hz,1H),5.69(dd,J＝33.3,4.4Hz,1H),4.92(s,1H),4.20(p,J＝16.5Hz,1H),3.86(s,3H),3.58(dd,J＝45.2,35.1Hz,2H),2.75(s,3H),2.50(t,J＝10.1Hz,2H),2.21(s,6H),0.93–0.54(m,2H),0.39–0.20(m,2H)。

LR-Mass(ESI)：570.7(M+1,C₃₁H₃₆N₇O₄)。

Example 8

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carbonic acid phenyl ester (I-8)

The synthesis was performed in the same manner as in synthesis 1 of example 1, except that phenyl chloroformate was used instead of methyl chloroformate.

¹H NMR(400MHz,CDCl₃)9.97(s,1H),9.63(s,1H),8.60(s,1H),8.53(d,J＝5.2Hz,1H),8.40–8.34(m,1H),8.34–8.29(m,1H),7.67(s,1H),7.47(ddd,J＝5.5,2.9,1.3Hz,2H),7.40(td,J＝7.4,1.5Hz,2H),7.37–7.30(m,4H),7.17(d,J＝5.2Hz,1H),6.79(s,1H),6.42(dd,J＝16.5,9.3Hz,1H),6.32(dd,J＝16.9,1.9Hz,1H),5.58(dd,J＝9.8,2.0Hz,1H),3.89(s,3H),3.00–2.89(m,2H),2.70(s,3H),2.45(s,2H),2.33(s,7H)。

LR-Mass(ESI)：606.7(M+1,C₃₄H₃₆N₇O₄)。

Example 9

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carbonic acid benzyl ester (I-9)

The synthesis was performed in the same manner as in synthesis 1 of example 1, except that methyl chloroformate was replaced with benzyl chloroformate.

¹H NMR(400MHz,CDCl₃)10.08(s,1H),9.63(s,1H),8.54(d,J＝5.2Hz,1H),8.39(dd,J＝9.7,2.0Hz,2H),8.26(d,J＝8.0Hz,1H),7.64(s,1H),7.57–7.50(m,2H),7.48–7.33(m,5H),7.12(d,J＝5.2Hz,1H),6.82(s,1H),6.48–6.29(m,2H),5.68(dd,J＝9.3,2.6Hz,1H),5.51(s,2H),3.91(s,3H),2.95–2.89(m,2H),2.73(s,3H),2.36(s,2H),2.31(s,6H)。

LR-Mass(ESI)：620.7(M+1,C₃₅H₃₈N₇O₄)。

Example 10

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid 2' -methylbenzyl ester (I-10)

The synthesis was performed in the same manner as in synthesis 1 of example 1, except that methyl chloroformate was replaced with o-methylbenzyl chloroformate.

¹H NMR(500MHz,CDCl₃)9.88(s,1H),8.78–8.69(m,2H),8.55(dd,J＝14.8,3.1Hz,1H),8.11(dd,J＝14.9,3.0Hz,1H),7.47–6.99(m,8H),6.43(s,1H),6.12(dd,J＝53.0,19.0Hz,2H),5.69(dd,J＝33.0,4.9Hz,1H),5.13(s,2H),4.92(s,1H),3.86(s,3H),3.55(dt,J＝25.1,14.6Hz,2H),2.75(s,3H),2.50(t,J＝14.6Hz,2H),2.29(s,3H),2.21(s,6H)。

LR-Mass(ESI)：634.8(M+1,C₃₆H₄₀N₇O₄)。

Example 11

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-carboxylic acid 2' -acetoxybenzyl ester (I-11)

The synthesis was performed in the same manner as in 1, except that methyl chloroformate was replaced with o-acetoxybenzyl chloroformate.

¹H NMR(500MHz,CDCl₃)9.81(s,1H),9.10(s,1H),8.80–8.37(m,2H),8.11(dd,J＝15.0,3.1Hz,1H),7.42–7.01(m,8H),6.43(s,1H),6.30–5.95(m,2H),5.69(dd,J＝33.2,4.8Hz,1H),5.13(s,2H),4.94(s,1H),3.86(s,3H),3.53(dt,J＝16.1,14.5Hz,2H),2.75(s,3H),2.55–2.26(m,5H),2.21(s,6H)。

LR-Mass(ESI)：678.8(M+1,C₃₇H₄₀N₇O₆)。

Example 12

2- (2- ((5-acrylamido-4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indole-1-allyl carbonate (I-12)

The synthesis method was the same as synthesis method 1 in example 1, except that methyl chloroformate was replaced with allyl chloroformate.

¹H NMR(400MHz,CDCl₃)9.56(s,1H),9.54–9.40(m,1H),8.52(d,J＝5.2Hz,1H),8.42(s,1H),8.38(d,J＝6.8Hz,1H),8.27(d,J＝8.3Hz,1H),7.63(s,1H),7.46–7.35(m,2H),7.15(d,J＝5.2Hz,1H),6.74(s,1H),6.42(dd,J＝16.9,1.9Hz,1H),6.18–6.05(m,1H),6.20–6.05(m,1H),5.72(d,J＝12.0Hz,1H),5.51(dd,J＝17.1,1.3Hz,1H),5.40(d,J＝10.3Hz,1H),4.98(d,J＝5.9Hz,2H),3.92(s,3H),3.25(s,2H),2.75(s,4H),2.72(s,4H)。

LR-Mass(ESI)：570.8(M+1,C₃₁H₃₆N₇O₄)。

(II) examples of measurement of biological Activity

The first test example: cell level inhibition assay

The experimental method comprises the following steps:

the in vitro proliferation inhibition effect of the compound on a human epidermal carcinoma A431 cell line with high expression of EGFR WT and a human non-small cell lung cancer NCI-H1975 cell line with high expression of EGFRT790M/L858R mutation is detected. A431 cells and NCI-H1975 cell line were purchased from the American Collection of Standard biologies (ATCC). The detection is performed by using a sulforhodamine B (SRB) method, which specifically comprises the following steps: inoculating a certain number of different tumor cells in a logarithmic growth phase to a 96-well culture plate, culturing for 24h, adding the tested compound of the invention with different concentrations, setting three or more wells for each concentration, and setting DMSO solution control and cell-free zeroing wells with corresponding concentrations. After treating the cells with the drug for 72h, the culture medium was decanted, 100. mu.L of ice-pre-cooled 10% trichloroacetic acid solution was added to fix the cells, the cells were left at 4 ℃ for 1h, washed 5 times with distilled water, and air-dried. Then 100. mu.L of SRB (4mg/mL) (Sigma, St Louis, MO, USA) solution was added, stained at room temperature for 15min, destained, washed 5 times with 1% glacial acetic acid, and air dried. Finally, 150. mu.L of 10mM Tris solution (pH 10.5) and an adjustable wavelength microplate reader (VERSAmax) are added^TMMolecular Device Corporation, Sunnyvale, Calif., USA) at a wavelength of 515 nm. The inhibition rate of the drug on cell growth was calculated by the following formula: inhibition (%) ═ OD control-OD dosing)/OD control × 100%.

Calculating half Inhibitory Concentration (IC) of the compound based on its cell growth inhibitory effect₅₀) Values, see table 1.

TABLE 1 analysis of cell growth inhibition by Compounds

And (4) experimental conclusion: in parallel, some indole-1-carbonates had IC's comparable to AZD9291 (available from Bingchun information technology (Beijing) Ltd.) for EGFR mutant cell line NCI-H1975₅₀And simultaneously, the method shows good selectivity and may have better safety in animal bodies.

Test example two: effect of I-1 and I-12 on phosphorylation of EGFR and its downstream signals

The experimental method comprises the following steps:

detection was performed using conventional immunoblotting (Western Blot). Respectively planting A431 cells and NCI-H1975 cells in logarithmic growth phase on a 6-well plate according to a certain quantity, after culturing overnight in an incubator in a wall-mounted manner, starving for 24H by replacing serum-free culture solution, adding a compound with a certain concentration for acting for 2H, adding 50ng/mL EGF stimulation factor for acting for 10min, and cracking the cells by using a lysate for sampling. Then, an appropriate amount of sample is subjected to SDS-PAGE electrophoresis, after the electrophoresis is finished, the protein is transferred to a nitrocellulose membrane by a semi-dry electrotransfer system, the nitrocellulose membrane is placed in a blocking solution (5% skimmed milk powder is diluted in TBS containing 0.1 wt.% of Tween 20) and is blocked for 2 hours at room temperature, and then the membranes are respectively placed in a primary antibody solution (1: 500 is diluted in TBS containing 0.1 wt.% of Tween 20) and are incubated overnight at 4 ℃. Washed three times with Triethanolamine Buffered Saline (TBS) containing 0.1 wt.% tween 20 for 15min each time. The membrane was placed in a secondary antibody solution (horseradish peroxidase-labeled goat anti-rabbit IgG, diluted 1: 2000 in TBS with 0.1 wt.% Tween 20) and reacted for 1h at room temperature. After washing three times with TBS containing 0.1 wt.% Tween 20, 15min each time, color was developed with ECLplus reagent and photographed with Image Quant LAS 4000, see FIG. 1.

And (4) experimental conclusion: in a parallel experiment, I-1 can obviously inhibit the phosphorylation of double mutations of EGFR L858R/T790M under the conditions of 10nM and 100nM concentration, and has obvious inhibition effect on the phosphorylation of AKT and ERK of a downstream signal channel, and the overall effect is similar to that of AZD 9291.

Test example three: i-1 pharmacokinetic testing in beagle dogs

The administration mode comprises the following steps: single administration by intragastric administration.

Administration dose: 2 mg/5 ml/kg.

The preparation prescription is as follows: suspensions were made with 0.5% CMC-Na.

Experimental animals: strain: beagle dogs; sex: male; weight: 8-11 Kg; fasted for 12h before the test, water was freely available. Food was consumed uniformly 4h after dosing.

Collecting samples: before and after administration, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 and 24h blood is taken from the veins of the limbs by 0.6 mL.

Before blood collection, 20. mu.L of 1M esterase inhibitor (BNPP) was added to the labeled EDTA anticoagulation tube and stored in a refrigerator at 4 ℃. In the sample collection, 600 μ L of whole blood was collected into an anticoagulation tube to which 20 μ L of 1M esterase inhibitor (BNPP) had been added, and after gently mixing the tube upside down 5 to 6 times (without vigorous shaking to avoid hemolysis), the plasma was obtained by centrifugation at 4 ℃ for 10min (3500rpm) within 1h and stored in a refrigerator at-70 ℃. (Note: preparation method of 1M dinitrophenol phosphodiester (BNPP) solution: 1.36g of BNPP was weighed and then sufficiently dissolved in 4mL of acetonitrile/water (1: 1, v/v).)

Pretreatment of a plasma sample: to 25.0. mu.L of the plasma sample were added 25.0. mu.L of the internal standard solution (50.0ng/mL of the verapamil internal standard solution) and 150. mu.L of acetonitrile, vortexed for 1min, centrifuged for 5min (14000rpm, 4 ℃), and the supernatant was subjected to LC-MS/MS analysis.

Liquid phase-mass spectrometry analysis:

1. instrument for measuring the position of a moving object

Liquid chromatography system: WatersACQUITYI Class system, Waters corporation

Autosampler watersacuityftn autosampler, Waters corporation

MS/MS system: triple Quad 5500 Triple quadrupole tandem mass spectrometer equipped with electrospray ionization source (ESI source), applied biosystems, Inc., USA

Data processing: analyst 1.6.2 quantitative processing software (applied biosystems, USA)

2. Chromatographic and mass spectral conditions

Chromatographic conditions

And (3) analyzing the column: YMC-Triat C₁₈Column, 2.0X 50mm, 5.0. mu.M, Japan YMC

Pre-column: c₁₈Guard post, 4.0X 3.0mm I.D., Philomen, USA

Column temperature: 40 deg.C

Mobile phase: phase A: 5mM ammonium acetate in water (containing 0.1% formic acid); phase B: acetonitrile

Flow rate: 0.600mL/min

Sample introduction amount: 2 μ L

Gradient elution:

conditions of Mass Spectrometry

The ion source is an electrospray ionization source (ESI source), and positive ion detection is carried out; ion spray voltage 5500V; the ion source temperature is 500 ℃; ion source gas 1 (N)₂)50 psi; ion source gas 2 (N)₂)50 psi; gas curtain gas (N)₂)30psi。

TABLE 2 analysis of the results of the Metabolic kinetics of the Compounds in beagle dogs

And (4) experimental conclusion: the results of the pharmacokinetic test of beagle dogs show that the plasma exposure Cmax of I-1 in beagle dogs is 1.7 times that of AZD9291, the AUC is 1.6 times that of AZD9291, the half-life period is 7h and the pharmaceutical parameters are reasonable when 20mg is administered, and the results are shown in Table 2. Compared with AZD9291, the compound preparation has better drug property.

From the above description, numerous modifications and alterations of the above-described embodiments of the invention are possible in light of the common general knowledge in the art or in common usage without departing from the basic technical idea of the invention.

Claims (6)

1. Indole-1-carbonate compounds shown in a general formula (I) or pharmaceutically acceptable salts thereof,

wherein the content of the first and second substances,

r is selected from methyl, ethyl, propyl and isopropyl;

r' is methyl.

2. The method for preparing the indole-1-carbonate compound shown in the general formula (I) or the pharmaceutically acceptable salt thereof comprises the step of subjecting the compound shown in the formula II and an acylation reagent to acylation reaction to obtain the compound shown in the general formula (I), which is shown in the following formula:

wherein R, R' is as defined in claim 1;

the acylating agent is selected from haloformate XCOOR and dicarbonate

Or carbonates

Wherein X is halogen.

3. A pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the indole-1-carbonate compounds or pharmaceutically acceptable salts according to claim 1 as an active ingredient, and optionally a pharmaceutically acceptable carrier.

4. A pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the indole-1-carbonate compounds or pharmaceutically acceptable salts according to claim 1 as an active ingredient together with other pharmaceutically acceptable therapeutic agents, and optionally a pharmaceutically acceptable carrier.

5. Use of the indole-1-carbonate compound or pharmaceutically acceptable salt according to claim 1 or the pharmaceutical composition according to claim 3 or 4 for the preparation of a medicament for selectively inhibiting the active EGFR resistance mutation T790M and the activating mutation.

6. The use of claim 5, wherein said selective inhibitory activity of EGFR resistant mutation T790M and activating mutation is selected from lung cancer, breast cancer, ovarian cancer, liver cancer, melanoma, prostate cancer, colon cancer, rectal cancer, glial cell carcinoma, head and neck cancer and gastric cancer.

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