CN104109163B

CN104109163B - With IKK-β be target spot 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, application and preparation method thereof

Info

Publication number: CN104109163B
Application number: CN201410033587.6A
Authority: CN
Inventors: 叶发青; 郭平; 谢自新; 刘剑敏; 王跃武; 宋晓琴; 陈梁芳
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2014-01-24
Filing date: 2014-01-24
Publication date: 2016-06-15
Anticipated expiration: 2034-01-24
Also published as: CN104109163A

Abstract

The invention discloses with IKK-β be target spot 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, application and preparation method thereof, the compound of the present invention to the inhibitory activity of A549 cell along with the increase of administration concentration, its suppression ratio also can increase, but this compounds is to b16-f10, the basic unrestraint effect of H460 and U251 cell strain, this selective inhibitory action has special meaning for it as specific antitumor drug.

Description

With IKK-β be target spot 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, application and preparation method thereof

Technical field

The invention belongs to antitumor drug and organic synthesis field, in particular to a kind of with IKK-β be target spot 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, application and preparation method thereof.

Background technology

Protein tyrosine kinase (proteintyrosinekinases, PTKs) it is that on a class catalysis ATP, γ-phosphoric acid transfers to the kinases on protein-tyrosine residue, energy catalysis multiple substrate protein white matter tyrosine residue phosphorylation, has important function in Growth of Cells, propagation, differentiation. Therefore, in including the Therapy study of cancer and other numerous diseases, tyrosine kinase is all target and the object of research. Block by developing selective tyrosine kinase inhibitor (tyrosinekinaseinhibitor, TKI) or regulate and control the promising strategy having been become antitumor research by the abnormal disease produced of these signal paths.

Fibroblast growth factor acceptor (FibroblastGrowthFactorReceptor, FGFRs) is the tyrosine kinase receptor that a class wears film, and research shows, the gene variation of FGFRs and the change of expression thereof are relevant with the generation of multiple disease. Such as: normal mammary epithelial and breast cancer cell are expressed FGFR-1 and has detected, it has been found that it is high more than normal breast cell that breast cancer cell expresses FGFR-1;RitaS etc. find that the cause of disease of achondroplastic dwarf disease (A-ehondroplasia, ACH) is relevant with the point mutation of FGFR-3 gene; Have been reported that, forming the expression all having FGFR on the fibroblast of Proliferative vetreoretinopathy, neurogliocyte and retinal pigment epithelium; It addition, in the kinds of tumor cells such as bladder cancer, gastric cancer, colon cancer, carcinoma of endometrium, carcinoma of prostate, fibroma, rhabdomyosarcoma, glioma and melanoma, all find sudden change or the high level expression of FGF/FGFR. Therefore, FGFR is considered one of therapeutic targets of disease such as tumor and receives much concern.

And FGFR and fibroblast growth factor (FibroblastGrowthFactor, FGFs) combine the FGF/FGFR signal transmission constituted and can mediate a plurality of signal transduction pathway, to normal cell and the propagation of tumor cell, differentiation, and the aspect such as the growth of tumor and transfer, angiogenesis, neural formation all plays important adjustment effect. In FGF/FGFR signal transduction pathway, main signal pathway is ras → MAPK and PI3K/AKT approach. After part FGF is combined with receptor, trigger receptor is Dimerized, make receptor generation autophosphorylation, thus being combined with the SH2 domain of the adaptor protein Grb2 in downstream, then be combined with SOS, ras successively, so that ras activates, then activating Raf, MEK, MAPK etc. successively, the activation of this approach is main relevant with cell proliferation and differentiation. After receptor phosphorylation, moreover it is possible to be combined with PLC γ, activating the signal pathways such as PI3K and AKT, or activate PI3K/AKT approach by ras coupling, the activation of this approach is chiefly to facilitate cells survival, opposing apoptosis.

If high selective fibroblast growth factor tyrosine kinase micromolecular inhibitor (FGFRTKI) therefore can be researched and developed, FGFR signal then can be suppressed to transmit, suppressing the injury repairing of tumor cell, making cell division be arrested in G1 phase, induction and maintenance apoptosis, anti-angiogenesis etc., thus reaching antineoplastic effect. Therefore, research and develop high selectivity, high efficiency FGFR inhibitor is pursuing a goal and urgent task of current medical personal.

Summary of the invention

It is an object of the invention to provide a kind of 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, application and preparation method thereof, in order to realize the purpose of the present invention, intend adopting the following technical scheme that.

One aspect of the present invention relates to a kind of with the IKK-β 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant being target spot, it is characterised in that described 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant has the structure being shown below:

In a preferred embodiment of the present invention, described R is selected from: phenyl, 2', 4'-3,5-dimethylphenyl, 2'-methoxyphenyl, indole-3'-base, 2', 3'-Dimethoxyphenyl, 4'-bromo indole-3'-base.

The preparation method that another aspect of the present invention further relates to above-mentioned 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, it is characterised in that undertaken by reaction as follows:

Another aspect of the present invention further relates to the application of above-mentioned 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, and described application is the application in Selective depression human lung adenocarcinoma cell growth in vitro.

In another aspect of this invention, further relating to the application of above-mentioned 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, described application is the application in the medicine preparing selective therapy adenocarcinoma of lung.

In another aspect of this invention, further relating to the application of above-mentioned 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, described application is the application in the medicine of preparation and FGFR-1 receptor associated diseases.

The compound of the present invention to the inhibitory activity of A549 cell along with the increase of administration concentration, its suppression ratio also can increase, but this compounds is to b16-f10, the basic unrestraint effect of H460 and U251 cell strain, this selective inhibitory action has special meaning for it as specific antitumor drug.

Detailed description of the invention

Embodiment 1:

Synthetic route

G1:R=phenyl G4:R=2', 4'-3,5-dimethylphenyl

G2:R=2'-methoxyphenyl G5:R=indole-3'-base

G3:R=2', 3'-Dimethoxyphenyl G6:R=4'-bromo indole-3'-base

Experimental implementation

(1) the sulfate concrete operations of 2,4,5-triamido-6-hydroxy pyrimidine referring to document (Shao Weiqing, the synthesis of hair magnificence .2,4,5-triamido-6-hydroxy pyrimidine sulfate. Chinese Journal of Pharmaceuticals 2002,33 (12): 579.).

(2) 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivative synthesizing process: to synthesize M2: weigh 2,4,5-triamido-6-hydroxy pyrimidine sulfate 0.239g (1.0mmol), Benzaldehyde,2-methoxy 0.136g (1.0mmol), in 100ml round-bottomed flask, adds 40ml dimethyl sulfoxide. Being placed in 80 DEG C of oil baths, add reflux, temperature constant magnetic stirring reacts. Follow the tracks of reaction with tlc silica gel plate, after question response terminates, reactant liquor is cooled to room temperature, add water to Precipitation, sucking filtration (solvent DMSO is collected in waste liquid cylinder), wash with hot ethanol, be placed in exsiccator and dry to obtain thick product M2. Dissolving gained desciccate by NaOH solution, then adjust PH to 7 with hydrochloric acid, can obtain the product after recrystallization, product colour is light yellow. Therefore this experiment selects 80 DEG C is reaction condition, and after 5 hours, reaction is basically completed. Reactant liquor cooling adds water, and product can be made to precipitate out with precipitation form. After product is dissolved with NaOH, hydrochloric acid adjusts PH to neutral, gets final product purified product so as to again precipitate out, all has higher yields.

(3) the 8-9 substituted guanine analog derivative M1-6 of 10.0mmol, uses 45ml acetic anhydride, and under 120 DEG C of reaction conditions, productivity is up to about 84%, and the response time is 2.5 hours.

The productivity of G1-G6 and relevant sign are referring to table 1 and table 2

Table 1

Table 2

Compound G (1-6) is faint yellow solid; Compound G (1-6) is dissolved in DMSO, the methanol of heat, ethanol, water insoluble, ether, acetone.

Pharmacodynamic activity experimental technique

The cultivation of 1A549 cell strain, frozen, recovery

A549 cell is human lung adenocarcinoma cell, belongs to continuous cell line, can stably Secondary Culture.

The cultivation of A549 cell strain: A549 cell takes out from liquid nitrogen container, rapidly as in 37 DEG C of thermostatted waters, in in 2-3min, inside culture medium is melted completely, 800r/min is centrifuged 3-5min, add containing 10%FBS, 1% dual anti-RPMIMEDIUM1640 and DMEM culture medium, put 37 DEG C, 5%CO2 incubator cultivates 24 in cultivating make to change after cell attachment fresh culture, goes down to posterity in 1:3 ratio after 0.5% trypsinization in 3 days.

A549 cell strain frozen: digestion method is in the cell culture of exponential phase routinely, make single cell suspension, calculate total cellular score, living cells quantity can not less than 90%, by centrifugal for cell suspension 800r/min 5min, abandoning supernatant, it is divided in frozen vials after adding freezing liquid, classification is freezing, finally puts into Liquid nitrogen storage.

The recovery of A549 cell strain: take out the cryopreservation tube having A549 cell from liquid nitrogen container, puts in water-bath and melts. By centrifugal for cell suspension 800r/min 5min.Being removed by supernatant with 1ml rifle head, cell is dispelled out by the cell culture fluid adding 1ml preheating, adds fresh medium Eddy diffusion cell and proceeds to cultivation box, observes under inverted microscope, 37 DEG C, 5%CO2 cultivation. After 24h, the culture fluid more renewed. Routine passage is cultivated.

Said method is equally applicable to b16-f10 (mouse skin melanoma cell), H460 (human lung carcinoma cell), the cultivation of U251 (glioma cell), frozen and recovery.

The cytotoxicity experiment (mtt assay) of 2 gained compounds

A549 cell covers with to 80%-90%, with preparing into single cell suspension after 0.5% trypsinization, collects logarithmic (log) phase cell, adjusts concentration of cell suspension after cell counting. Point in 96 orifice plates, 5 × 103/ holes, it is placed in after CO2 incubator (37 DEG C, 5%CO2) cultivates 24h, cell attachment and well-grown, absorb whole culture fluid. Add the medicine of variable concentrations, each concentration sets 6 multiple holes, every hole adds 20ulMTT solution (5mg/ml, i.e. 0.5%MTT), after continuing cultivation 4h in CO2 incubator, carefully sucking culture fluid in hole, every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, make crystal fully dissolve. Measure the light absorption value (OD) in each hole, record result (DMSO is matched group) at enzyme-linked immunosorbent assay instrument 490nm place, process data with GraphpadPrims and obtain IC50. Experimental data all represents with mean ± standard deviation (x ± s); Acquired results is in Table 3.

The suppression ratio of FGFR-1 is tested by 3 screening gained compounds

In this experiment, by CaliperMobilityShiftAssay method, in vitro 12 compounds of gained have been carried out the inhibitory activity to FGFR-1 and tested. Testing drug is divided into tri-concentration of 0.1uM, 1uM, 10uM, and Staurosporine is reference substance.

Experimental technique is as follows:

(1) compound concentration being finally diluted to 50uM, 5uM and 0.5uM, standby in 384 orifice plates, with 100%DMSO for matched group, 250mMEDTA is lowcontrol.

(2) FGFR1 enzyme is added preparation 2.5x enzymatic solution in the buffer of 1.25x.

(3) ATP and the polypeptide containing FAM labelling are added preparation 2.5x polypeptide solution in 1.25x buffer.

(4) each hole adds the enzymatic solution of the 10 above-mentioned preparations of μ l2.5x, incubated at room 10min.

(5) then each hole adds the 2.5x polypeptide solution of the 10 above-mentioned preparations of μ l2.5x, after hatching a period of time at 28 DEG C, adds 25 μ lstopbuffer stopped reaction.

(6) on Caliper, read data, and transfer data to suppression ratio. Percent inhibition=(max-reads data)/(max-min) * 100. Max represents DMSO matched group, and min represents lowcontrol. Final acquired results is in Table 4.

Table 3 compound suppression ratio to A549 Growth of Cells

Due to the b16-f10 (mouse skin melanoma cell) calculated, H460 (human lung carcinoma cell), U251 (glioma cell) MTT result be negative value, illustrate that these three Growth of Cells is not had inhibitory action by such medicine, and A549 cell can only be produced growth inhibited effect. From table 3 it can be seen that increase with drug level, the growth inhibitory effect of A549 (human lung adenocarcinoma cell) is also increased by medicine simultaneously. IC50 according to the control drug calculated is 12.82 μm of ol/l, the compound that contrast is synthesized, and its IC50 is all higher than comparison medicine, illustrates that the growth inhibited effect to A549 cell of this compounds is not as positive control drug 5-fluorouracil.

Table 4 compound inhibitory activity to FGFR-1

From table 4, it can be seen that G1-6 shows the relatively good activity suppressing FGFR-1; 8 G5 and G6 for indole replacement show the relatively good activity suppressing FGFR-1.

The above is the preferred embodiments of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from principle of the present invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims

1. a 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant, it is characterised in that described 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant has the structure being shown below:

Described R is selected from: phenyl, 2', 4'-3,5-dimethylphenyl, 2'-methoxyphenyl, indole-3'-base, 2', 3'-Dimethoxyphenyl, 4'-bromo indole-3'-base.

2. the preparation method of the 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant described in claim 1, it is characterised in that undertaken by reaction as follows:

3. the application of the 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant described in claim 1, described application is the application in Selective depression human lung adenocarcinoma cell growth in vitro.

4. the application of the 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant described in claim 1, described application is the application in the medicine preparing selective therapy adenocarcinoma of lung.

5. the application of the 2-acetylaminohydroxyphenylarsonic acid 8-9 substituted guanine derivant described in claim 1, described application is the application in the medicine of preparation and FGFR-1 receptor associated diseases.