CN107501240A

CN107501240A - EGFR tyrosine kinase inhibitors BF with antitumor activity3AZD9291 and its preparation method and application

Info

Publication number: CN107501240A
Application number: CN201710729974.7A
Authority: CN
Inventors: 申宝忠; 孙夕林; 杨丽丽; 刘杨; 张重庆; 韩兆国
Original assignee: Harbin Medical University
Current assignee: Harbin Engineering University; Harbin Medical University
Priority date: 2017-08-23
Filing date: 2017-08-23
Publication date: 2017-12-22
Also published as: CN108033950B; CN108033950A

Abstract

The invention discloses the EGFR tyrosine kinase inhibitors BF with antitumor activity₃AZD9291 and its preparation method and application, belong to biomedicine field.The present invention has obtained brand-new EGFR TKI, has been named as BF by the way that AZD9291 chemical constitution is optimized and improved₃AZD9291 (Formulas I), research show EGFR TKI-BF that the present invention synthesizes₃AZD9291 can effectively suppress the resistance of tumour cell, compared with AZD9291, have stronger suppression drug-resistant effect to mutation EGFR lung cancer cell lines H1975T790M, therefore can clinically be used as antitumor agent application.In addition, the invention also discloses the epidermal growth factor recipient tyrosine kinase inhibitor BF for preparing the tool antitumor activity₃AZD9291 method.The it is proposed of the present invention is that the treatment of oncotherapy, particularly non-small cell lung cancer provides a kind of effective technological means.

Description

EGFR tyrosine kinase inhibitors BF with antitumor activity3- AZD9291 and its system Preparation Method and application

Technical field

The present invention relates to pyrimidine acrylamides, more particularly to EGFRTKI-BF with antitumor activity₃- AZD9291 and preparation method thereof, the invention further relates to application of the medicine as antitumor agent, belong to biomedicine field.

Background technology

The energy-rich phosphate bond that the EGFR EGFR-TKs (EGFR TK) having now been found that can be catalyzed ATP is transferred to EGFR's Several tyrosine sites, make its phosphorylation, so as to activate the signal paths such as PI3K/Akt, the Ras/MAPK in downstream, promote cell Enter growing multiplication, metabolism, suppress Apoptosis.When EGFR is over-expressed and is mutated in cell, meeting block cell programmed cell death, make The growth regulating of cell is out of control, and obtains infinite multiplication and invasive ability, that is, develops into malignant cell.EGFR TKI lead to The binding site with ATP competition intracellular EGFR-TKs is crossed, the autophosphorylation by internal tyrosine is prevented, suppresses EGFR junket ammonia Acid kinase activates, so as to suppress growth of tumour cell, accelerate apoptosis of tumor cells, suppress angiogenesis and tumor cell invasion, Transfer.Small molecule EGFR TKI basic structure is quinazoline amine.EGFR TKI can be divided into：1) invertibity TKI mainly has：Ji Fei For Buddhist nun (gefitinib), Tarceva (erlotinib), Lapatinib (Lapatinib), 2) irreversibility TKI mainly has： Afatinib (Afatinib), up to Buddhist nun (Dacomitinib) can be replaced.At present, invertibity and irreversibility EGFR TKI are to mutation There is the effect of notable in the EGFR non-small cell lung cancer short time, but finally cause treatment to be lost because producing acquired resistance Lose, be not obviously prolonged the life cycle of patient.Therefore, develop and develop and be with stronger, more longlasting antitumor activity new EGFR TKI will likely be that mutation EGFR Patients with Non-small-cell Lung brings life.

The content of the invention

An object of the present invention is to provide brand-new epidermal growth factor recipient tyrosine kinase inhibitor (EGFRTKI)—BF₃- AZD9291 and preparation method thereof.

The second object of the present invention is to provide BF₃Purposes of-the AZD9291 in apoptosis of tumor cells is promoted, its mechanism are Given birth to by suppressing epidermal growth factor recipient tyrosine kinase phosphorylation so as to promote apoptosis of tumor cells, suppress tumor vessel Into, invasion and attack and transfer.

The above-mentioned purpose of the present invention is achieved through the following technical solutions：

The present invention to irreversibility molecular targeted anti-tumor agentses-osimertinib (AZD9291) chemical constitution by entering Row optimization and improvement, have obtained brand-new EGFR TKI-BF₃- AZD9291, it is due to introducing the organic trifluoroborate of amphion Group, to improve inhibitor hydrophily, the uptake ratio of liver is reduced, improves tumor uptake rate and smaller toxic side effect, simultaneously Have mutation EGFR non-small cell lung cancers compared with strong inhibitory activity, and it is by suppressing tumour cell epidermal growth to illustrate its mechanism Factor receptor tyrosine tyrosine phosphorylation, downstream signaling pathway activation is hindered, so as to suppress growth of tumour cell, propagation, promoted Apoptosis of tumor cells.

A kind of epidermal growth factor recipient tyrosine kinase inhibitor provided by the invention or its pharmaceutically useful salt, are named as BF₃- AZD9291, described epidermal growth factor recipient tyrosine kinase inhibitor have the structure shown in Formulas I:

Of course, it is possible to prepare the salt of the compounds of this invention acid addition, these salt are included in the present invention.

The acid-addition salts of the compounds of this invention are preferably pharmaceutically acceptable, with appropriate acid (such as hydrochloric acid, acetic acid, Sulfuric acid) nontoxic salt is formed, in addition to pharmaceutically acceptable salt, other salt are also included in the present invention.

Further, the invention also provides described epidermal growth factor recipient tyrosine kinase inhibitor or its can medicine Purposes of the salt in the medicine for promoting apoptosis of tumor cells is prepared.

Wherein, described promotion apoptosis of tumor cells is by suppressing epidermal growth factor recipient tyrosine kinase phosphorylation And realize.

Wherein, it is preferred that described tumour cell is the non-small cell lung cancer cell of EGFR mutation.

Further, the invention also provides one kind prepares above-described epidermal growth factor recipient tyrosine kinase The method of inhibitor or its pharmaceutically useful salt, comprises the following steps：

(1) nitrogen protection is lower by (2- chlorine pyrimidine-4-yl) -1- methyl -3a, 7a- dihydro -1H- indoles, the fluoro- 2- methoxies of 4- Base -5- nitroanilines and p-methyl benzenesulfonic acid are mixed in 2- amylalcohols, and mechanic whirl-nett reaction 2-3h, is cooled to room at 100-110 DEG C Wen Hou, is filtered, and filter cake is washed with 2- amylalcohols, drains to obtain light tan solid, purified by column chromatography for separation, using methanol and two Chloromethanes is according to volume ratio 1:100 eluents being mixed to get, finally give pale yellow needles solid, i.e. compound one；

(2) by compound one, N, N, N'- trimethyls ethylenediamine and DIPEA is miscible is stirred in DMF solvent Mix, 115-125 DEG C of reaction 3-5h, solution is poured into frozen water after cooling room temperature and diluted, with dichloromethane extraction three times, is associated with Machine phase is simultaneously washed three times with saturated common salt, and organic phase is used into anhydrous sodium sulfate drying, filters, passes through column chromatography for separation after being spin-dried for Purification, using methanol and dichloromethane according to volume ratio 3:100 eluents being mixed to get, finally give rufous needle-like Solid, i.e. compound two；

(3) compound two, iron powder and ammonium chloride are added in round-bottomed flask, it is molten with the mixing of the second alcohol and water in flask Liquid is miscible, heating reflux reaction 2-4h, and cooling filters, and filtrate concentration is spin-dried for, purified by SCX post separations, eluent is first Alcohol ammonia solution, obtain brown solid, i.e. compound three；

(4) compound three and diisopropylethylamine are dissolved in dichloromethane, the agitation and dropping acryloyl chloride under ice salt bath Dichloromethane solution, react 0.5-1.5h, then at room temperature hydrogenation aqueous solution of sodium oxide continue react 3-5h, filter, collect Solid is dried to obtain product, i.e. compound four；

(5) compound four is placed in round-bottomed flask under room temperature under nitrogen protection, adds anhydrous tetrahydro furan dissolving；Separately take Anhydrous tetrahydro furan and iodomethyl pinacol borate are added in round-bottomed flask, and rinses pipette tips repeatedly, by iodomethyl boric acid frequency Which alcohol ester tetrahydrofuran solution is slowly added in the solution of compound four, and suction filtration obtains white solid after stirring 10-14h, that is, changes Compound five；

(6) compound five is put into round-bottomed flask, sequentially adds KHF₂The aqueous solution, HCl/water solution, deionized water and DMF Solution, 2h is reacted at 40-50 DEG C, NH is added after cooling₄The OH aqueous solution is quenched, and is added drop-wise on small silicagel column with dropper and used respectively Water and ethyl acetate washing, collect cleaning solution and are extracted with absolute ether, merge organic phase, with anhydrous sodium sulfate drying, filtering rotation It is dry, obtain solid product, the epidermal growth factor recipient tyrosine kinase inhibitor as shown in Formulas I.

The present invention has obtained brand-new EGFR by the way that osimertinib (AZD9291) chemical constitution is optimized and improved TKI—BF₃- AZD9291, through biological evaluation, EGFR TKI-BF of the present invention₃It is thin that-AZD9291 can effectively suppress tumour The propagation of born of the same parents, there is outstanding antitumor activity, can clinically be used as antitumor agent application.

Present invention also offers a kind of antitumor medicine composition, the pharmaceutical composition by effective dose chemical combination of the present invention Thing or its pharmaceutically useful salt coordinate with pharmaceutically acceptable carrier to be formed, in other words, by the sheet of pharmaceutically acceptable dosage After invention compound coordinates with pharmaceutically acceptable carrier or diluent, it is prepared into by the conventional formulation method in this area Any one suitable pharmaceutical composition.The compounds of this invention of effective dose is matched somebody with somebody with pharmaceutically acceptable carrier or diluent After conjunction, any one suitable pharmaceutical composition is prepared into by the conventional formulation method in this area.Usual said composition is fitted Together in oral administration and drug administration by injection, other medications also are adapted for.Said composition can be tablet, capsule, pulvis, Granula, lozenge, suppository, or the liquid forms such as oral liquid.According to different medications, pharmaceutical composition of the present invention can be with The compounds of this invention containing 0.1%-99% weight, preferably 10-60% weight.

Brief description of the drawings

Fig. 1 is EGFR TKI-BF of the present invention₃The disjunctive path figure of-AZD9291 compounds；

Fig. 2 is BF₃- AZD9291 high performance liquid chromatography (HPLC) result；

Fig. 3 is BF₃- AZD9291 nuclear magnetic spectrum result；

(1)1H-NMR；(2)19F-NMR

Fig. 4 is BF₃- AZD9291 mass spectral results.

Embodiment

The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention Row modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.

EGFR TKI-the BF of embodiment 1₃- AZD9291 preparation

Described BF₃- AZD9291 molecular structural formula is shown in formula I:

Preparation flow figure is as shown in figure 1, specific preparation method is as follows：

(1) by 50g (2- chlorine pyrimidine-4-yl) -1- methyl -3a, 7a- dihydro -1H- indoles, 38.03g 4- under nitrogen protection Fluoro- 2- methoxyl groups -5- nitroanilines and 37.09g p-methyl benzenesulfonic acid are mixed in 580ml 2- amylalcohols, and mechanical agitation is anti-at 105 DEG C 2.5h is answered, after being cooled to room temperature, is filtered, filter cake is washed with 2- amylalcohols, drains to obtain light tan solid, carried by column chromatography for separation It is pure, using methanol and dichloromethane according to volume ratio 1:100 eluents being mixed to get, finally give pale yellow needles and consolidate Body is compound one, yield 85%；

(2) it is 65g compounds one, 19.65gN, N, N'- trimethyls ethylenediamine and 26.93N, N- diisopropylethylamine is miscible Stirred in 600mlDMF solvents, 120 DEG C of reaction 4h, solution is poured into frozen water after cooling room temperature and diluted, with 50ml dichloromethanes Alkane extracts three times, merges organic phase and is washed three times with saturated common salt, by organic addition anhydrous sodium sulfate drying, filters, be spin-dried for Purified afterwards by column chromatography for separation, using methanol and dichloromethane according to volume ratio 3:100 eluents being mixed to get, most Rufous needle-like solid, i.e. compound two, yield 62% are obtained eventually；

(3) 3.22g compounds two, 9.62g iron powders and ammonium chloride are added in round-bottomed flask, with the 120mL in flask The mixed solution of ethanol and 30ml water is miscible, heating reflux reaction 3h, and cooling filters, and filtrate concentration is spin-dried for, and passes through SCX posts point From purification, eluent is 0.35M methanolic ammonia solution, obtains brown solid, i.e. compound three, yield 85%；

(4) 1.7g compounds three and 0.73ml diisopropylethylamine are dissolved in 50ml dichloromethane, stirred under ice salt bath The 1ml dichloromethane solutions that 0.34ml acryloyl chlorides are added dropwise are mixed, react 1h, then add 30ml 0.5g sodium hydroxide water at room temperature Solution continues to react 4h.Filter, collect solid and be dried to obtain product, i.e. compound four, yield 39%；

(5) 9.2mg compounds four are placed in 25ml round-bottomed flasks by nitrogen protection at room temperature, add the anhydrous tetrahydrochysene furans of 2ml Mutter dissolving；Another take adds 2ml anhydrous tetrahydro furans and 3 μ l iodomethyl pinacol borate in 25ml round-bottomed flasks, and repeatedly Rinse pipette tips.Iodomethyl pinacol borate tetrahydrofuran solution is slowly added in the solution of compound four in 5min or so, stirred Suction filtration obtains white solid, i.e. compound five, yield 49% after mixing 12h；

(6) compound five is put into round-bottomed flask, sequentially adds 3M, 300 μ l KHF₂The aqueous solution, 4M, 300 μ l HCl The aqueous solution, 200 μ l deionized waters and 600 μ l DMF solutions, react 2h at 45 DEG C, and 10 μ l NH are added after cooling₄The OH aqueous solution Quenching, it is added drop-wise to dropper on small silicagel column with being washed respectively with water and ethyl acetate, collects cleaning solution and extracted with absolute ether, Merge organic phase, with anhydrous sodium sulfate drying, filtering is spin-dried for, and obtains solid product, i.e. compound six, i.e. described epidermal growth Factor receptor tyrosine kinase inhibitor BF₃- AZD9291, yield 85%.

BF₃- AZD9291 high performance liquid chromatography (HPLC) result from HPLC results as shown in Fig. 2 can be seen that this hair The bright BF being prepared₃- AZD9291 pharmaceutical purities are single, free from admixture.BF₃- AZD9291 nuclear magnetic spectrum and mass spectral results point Not as shown in Figure 3 and 4.

The BF3-AZD9291 of embodiment 2 is tested the inhibitory action for being mutated EGFR Lines H1975

1st, test compound：BF3-AZD9291, prepared by the method for embodiment 1

2nd, test method：

Take the logarithm growth period EGFR21 exons point mutation (L858R) merge 20 exon point mutation (T790M) Lines H1975 cells, be inoculated in 10000/hole in 96 orifice plates, in 37 DEG C, 5%CO₂Under the conditions of After culture 24 hours, administration group adds test compound, make its final concentration be respectively 0.01,0.1,1.25,2.5,5,10,20, 40 μM, blank control group adds 0.08% dimethyl sulfoxide (DMSO), after being cultivated 24,48 hours under the conditions of 37 DEG C of 5%CO2, using MTT Colorimetric method, absorbance A value is determined at wavelength 490nm.Half inhibiting rate of the medicine to growth of tumour cell is calculated according to A values (IC50)。

3rd, result of the test

Result of the test shows that the compounds of this invention BF3-AZD9291 has to lung cancer cell line H1975 suppresses its propagation Effect.

Claims

1. epidermal growth factor recipient tyrosine kinase inhibitor or its pharmaceutically useful salt, are named as BF₃- AZD9291, its feature It is that described epidermal growth factor recipient tyrosine kinase inhibitor has the structure shown in Formulas I:

2. the epidermal growth factor recipient tyrosine kinase inhibitor or its pharmaceutically useful salt described in claim 1 are preparing promotion Purposes in the medicine of apoptosis of tumor cells.

3. purposes as claimed in claim 2.It is characterized in that described promotion apoptosis of tumor cells is given birth to by suppressing epidermis Growth factor receptor body EGFR-TK phosphorylation and realize.

4. purposes as claimed in claim 2 or claim 3, it is characterised in that described tumour cell is the non-small cell lung of EGFR mutation Cancer cell.

5. a kind of epidermal growth factor recipient tyrosine kinase inhibitor prepared described in claim 1 or its pharmaceutically useful salt Method, it is characterised in that comprise the following steps：

(1) nitrogen protection is lower by (2- chlorine pyrimidine-4-yl) -1- methyl -3a, 7a- dihydro -1H- indoles, the fluoro- 2- methoxyl groups -5- of 4- Nitroaniline and p-methyl benzenesulfonic acid are mixed in 2- amylalcohols, mechanic whirl-nett reaction 2-3h at 100-110 DEG C, after being cooled to room temperature, Filter, filter cake is washed with 2- amylalcohols, drains to obtain light tan solid, purified by column chromatography for separation, using methanol and dichloromethane Alkane is according to volume ratio 1:100 eluents being mixed to get, finally give pale yellow needles solid, i.e. compound one；

(2) by compound one, N, N, N'- trimethyls ethylenediamine and DIPEA is miscible is stirred in DMF solvent, 115-125 DEG C is reacted 3-5h, is poured into frozen water solution after cooling room temperature and is diluted, and with dichloromethane extraction three times, is merged organic Mutually and with saturated common salt washing three times, organic phase is used into anhydrous sodium sulfate drying, filters, is carried after being spin-dried for by column chromatography for separation It is pure, using methanol and dichloromethane according to volume ratio 3:100 eluents being mixed to get, finally give rufous needle-like and consolidate Body, i.e. compound two；

(3) compound two, iron powder and ammonium chloride are added in round-bottomed flask, mixed with the mixed solution of the second alcohol and water in flask Molten, heating reflux reaction 2-4h, cooling is filtered, and filtrate concentration is spin-dried for, purified by SCX post separations, eluent is methanol ammonia Solution, obtain brown solid, i.e. compound three；

(4) compound three and diisopropylethylamine are dissolved in dichloromethane, the two of agitation and dropping acryloyl chloride under ice salt bath Chloromethanes solution, 0.5-1.5h is reacted, then hydrogenation aqueous solution of sodium oxide continues to react 3-5h at room temperature, filters, and collects solid It is dried to obtain product, i.e. compound four；

(5) compound four is placed in round-bottomed flask under room temperature under nitrogen protection, adds anhydrous tetrahydro furan dissolving；Separately take round bottom Anhydrous tetrahydro furan and iodomethyl pinacol borate are added in flask, and rinses pipette tips repeatedly, by iodomethyl boric acid pinacol Ester tetrahydrofuran solution is slowly added in the solution of compound four, and suction filtration obtains white solid, i.e. compound after stirring 10-14h Five；

(6) compound five is put into round-bottomed flask, sequentially adds KHF₂The aqueous solution, HCl/water solution, deionized water and DMF solution, 2h is reacted at 40-50 DEG C, NH is added after cooling₄The OH aqueous solution is quenched, with dropper be added drop-wise on small silicagel column respectively with water and Ethyl acetate washs, and collects cleaning solution and is extracted with absolute ether, merges organic phase, with anhydrous sodium sulfate drying, filtering is spin-dried for, and is obtained To solid product, the epidermal growth factor recipient tyrosine kinase inhibitor as shown in Formulas I.

6. a kind of antineoplastic pharmaceutical compositions, it is characterised in that as the epidermal growth factor receptor described in the claim 1 of effective dose Body tyrosine kinase inhibitor or its pharmaceutically useful salt and pharmaceutically acceptable carrier or auxiliary material composition.

7. antineoplastic pharmaceutical compositions as claimed in claim 6, it is characterised in that described pharmaceutical composition is tablet, capsule Agent, pulvis, granule, lozenge, suppository or oral liquid.

8. antineoplastic pharmaceutical compositions as claimed in claim 6, it is characterised in that the right containing 0.1%-99% weight will Seek the epidermal growth factor recipient tyrosine kinase inhibitor or its pharmaceutically useful salt described in 1.

9. antineoplastic pharmaceutical compositions as claimed in claim 6, it is characterised in that the claim 1 containing 10-60% weight Described epidermal growth factor recipient tyrosine kinase inhibitor or its pharmaceutically useful salt.

10. purposes of the pharmaceutical composition described in claim any one of 6-9 in antineoplastic is prepared.