CN109503553B - Light affinity probe molecule based on VEGFR-2 inhibitor B14 and preparation method thereof - Google Patents
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Abstract
A light affinity probe molecule based on VEGFR-2 inhibitor B14 and a preparation method thereof are disclosed, 4-bromopyridine-2-carboxylic acid and 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea are subjected to Suzuki coupling reaction to obtain an intermediate product with monocarboxylic acid; the linker containing the photoaffinity group bis-aziridine and alkynyl and the intermediate product with monocarboxylic acid are condensed under EDC & HCl to obtain the photoaffinity probe molecule based on VEGFR-2 inhibitor B14. The preparation method of the B14 photoaffinity probe molecule is simple, easy to realize and high in yield. The B14 small-molecule photoaffinity probe can be used for confirming the action target of B14 and verifying the feasibility of photoaffinity labeling technology in confirming the small-molecule target.
Description
Technical Field
The invention relates to a light affinity probe molecule based on VEGFR-2 inhibitor B14 and a preparation method thereof.
Background
The light affinity labeling technology (PAL) combines the advantages of modern molecular biology, cell biology, medicinal chemistry, analytical chemistry and other subjects, and applies the synthesized light affinity probe molecules to generate high-activity intermediates under the irradiation of light with specific wavelength, which can be directly irreversibly covalently crosslinked with the protein specifically combined with the medicinal molecules to realize the capture of the medicinal target protein molecules. It is one of the core tools for researching the interaction between ligand and receptor on molecular level, and has great promoting effect on the elucidation of the interaction mechanism between ligand and receptor and the discovery of new target of medicine.
In recent years, the technology is mainly applied to the confirmation of drug molecular target protein, and the capture and confirmation of the drug molecular target are mainly realized based on a photo-crosslinking technology, a bio-orthogonal technology, a related biological analysis technology and the like. The light affinity probe molecule is designed and synthesized by directly carrying out structural modification on the basis of not influencing the medicinal activity of a target, respectively introducing a light reaction active group and designing and synthesizing the light affinity probe molecule by a bioorthogonal handle, and the light affinity probe molecule is subjected to specific irreversible covalent capture of a target protein of a target compound under the irradiation of light with specific wavelength. And then the identification confirmation of the captured target protein is realized through a bio-orthogonal reaction.
Vascular endothelial growth factor receptor (vascular endothelial growth factor receptor) is a membrane protein expressed by VEGF gene, belongs to tyrosine family protein, and is a macromolecular protein closely related to malignant tumor. Vascular endothelial growth factor and its receptor are overexpressed in a range of tumor cells, and the receptor family includes three subtypes: VEGFR-1, VEGFR-2, VEGFR-3. Among them, VEGFR-2 is mainly involved in the proliferation of vascular endothelial cells and is most widely distributed among cancer cells. A series of studies have demonstrated that it can be an effective drug target.
Photoactive groups reported in the literature have been four main classes of aryl azides, benzophenones, substituted bis-aziridines, since last five years. Among them, the preferable photocrosslinking activity is bisaziridine. It is therefore necessary to design a photoaffinity probe molecule by selecting bisaziridine as the photoactive group.
Disclosure of Invention
The invention aims to provide a light affinity probe molecule based on a VEGFR-2 inhibitor B14 and a preparation method thereof, wherein the B14 light affinity probe molecule is used for confirming a target protein of B14 and an action mode of the target protein and can be used for verifying feasibility of a light affinity labeling technology in target protein confirmation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a VEGFR-2 inhibitor B14-based photoaffinity probe molecule has a structural formula as follows:
wherein X is O or NH.
A preparation method of a VEGFR-2 inhibitor B14-based photoaffinity probe molecule is characterized by comprising the following steps:
1) carrying out Suzuki coupling reaction on 4-bromopyridine-2-carboxylic acid and 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea to obtain an intermediate product with monocarboxylic acid;
2) the linker containing the photoaffinity group bis-aziridine and alkynyl and the intermediate product with monocarboxylic acid are condensed under EDC & HCl to obtain the photoaffinity probe molecule based on VEGFR-2 inhibitor B14, and the structural formula is as follows:
wherein X ═ O or NH.
The further improvement of the invention is that the specific process of the step 1) is as follows: dissolving 4-bromopyridine-2-carboxylic acid, 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, cesium carbonate and tetrakis (triphenylphosphine) palladium in a mixed solution of acetonitrile and water, and dissolving N in the mixed solution2Protecting, reacting at 100 ℃ for 16h, and then treating to obtain an intermediate product with monocarboxylic acid.
A further improvement of the present invention resides in that 4.95mmol of 4-bromopyridine-2-carboxylic acid, 5.94mmol of 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, 9.90mmol of cesium carbonate and 0.25mmol of tetrakis (triphenylphosphine) palladium are dissolved in 50mL of a mixed solution of acetonitrile and water, N2Protecting, reacting at 100 ℃ for 16h, and then treating to obtain an intermediate product with monocarboxylic acid.
The invention further improves that the volume ratio of the acetonitrile to the water in the mixed solution of the acetonitrile and the water is 4: 1.
The further improvement of the invention is that the specific process of the step 2) is as follows: dissolving the intermediate product with the monocarboxylic acid obtained in the step 1) in an anhydrous tetrahydrofuran solution, adding EDC & HCl and HOBt, then dropwise adding DIPEA, stirring at 0 ℃ for 1h, then adding a linker containing a photoaffinity group bisaziridine and alkynyl, stirring at room temperature for 24h, and then processing to obtain the photoaffinity probe molecule based on the VEGFR-2 inhibitor B14.
The further improvement of the invention is that 0.145mmol of intermediate product with monocarboxylic acid is dissolved in 3mL of anhydrous tetrahydrofuran solution, 0.218mmol of EDC. HCl and 0.174mmol of HOBt are added and stirred evenly at 0 ℃, then 0.725mmol of DIPEA is added drop by drop and stirred for 1h, 0.145mmol of linker containing the photoaffinity group biaziridine and alkynyl is added and stirred for 24h at room temperature, and the photoaffinity probe molecule based on VEGFR-2 inhibitor B14 is obtained.
An application of a VEGFR-2 inhibitor B14-based photoaffinity probe molecule in the aspect of photoaffinity labeling technology.
A further improvement of the present invention resides in the use of a VEGFR-2 inhibitor B14-based photoaffinity probe molecule for the confirmation of protein targets.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains the B14 photoaffinity probe molecule by connecting a linker which simultaneously contains a photoaffinity group diazirine and an alkynyl with a VEGFR-2 protein inhibitor B14. The photoaffinity probe molecule can be specifically and irreversibly combined with a target protein molecule of B14, and then a bioorthogonal handle in the probe molecule is subjected to click reaction with another bioorthogonal handle containing fluorescein or biotin so as to confirm the target protein. The preparation method of the B14 photoaffinity probe molecule is simple, easy to realize and high in yield.
The B14 photoaffinity probe molecule can carry out specific covalent binding on VEGFR-2 protein, and further realize the subsequent confirmation analysis of the captured target protein through click reaction. The use of photoaffinity labeling technology to achieve the validation of drug action target proteins and may improve some of the deficiencies of the original validation target technology. In the original target confirmation technology, probe molecules cannot be stably combined with drug target molecules, and false positive results are easily caused. Furthermore, the prior art usually requires a large volume of fluorophore attached to the drug molecule for analysis, which results in the disadvantages of reduced activity, poor solubility, poor cell permeability, etc. of the probe molecule. The photoaffinity labeling technology is used for target confirmation by combining a synthesized small-molecule photoaffinity probe with a bioorthogonal technology, and makes up for the defects of the prior art. The B14 small-molecule photoaffinity probe can be used for confirming the action target of B14 and verifying the feasibility of photoaffinity labeling technology in confirming the small-molecule target.
Drawings
FIG. 1 is a synthetic scheme of a VEGFR-2 inhibitor B14-based photoaffinity probe molecule according to the present invention;
wherein, compound 1 is 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, compound 2 is 4-bromopyridine-2-carboxylic acid, compound 3 is 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenyl) picolinic acid (B14), compound 4 is a linker containing a photoaffinity group diazirine and an alkynyl group, and compound (X) is B14 photoaffinity probe molecule.
The specific notations in the figures are:
a.CsCO3,Pd[P(C6H5)3]4,Acetonitrile,N2,80℃；b.EDC·HCl,HOBt,DIPEA,THF,rt。
Detailed Description
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
The present invention obtains photoaffinity probe molecules by linking VEGFR-2 protein inhibitors using linkers containing the photoaffinity group diazirine and alkynyl. The photoaffinity probe molecule can be used for confirming the target protein of B14.
In the invention, the double aziridine and alkyne containing photoaffinity groupsThe structural formula of the linker of the group is:
wherein X is O or NH.
The chemical structural formula of the photoaffinity probe molecule with the confirmed drug molecule target provided by the invention is as follows:
wherein X is O or NH.
The photoaffinity probe molecule with the target confirmation function comprises:
2- (3- (but-3-yn-1-yl) -3H-bisaziridin-3-yl) ethyl 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenyl) picolinic acid.
N- (2- (3- (but-3-yn-1-yl) -3H-bisaziridin-3-yl) ethyl) -4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenyl) picolinamide.
The following will describe in detail the preparation and activity screening method of the photoaffinity probe molecule with target confirmation provided by the present invention with reference to the synthetic route and specific synthetic example shown in fig. 1.
Referring to fig. 1, a method for preparing a VEGFR-2 inhibitor B14-based photoaffinity probe molecule comprises the following steps:
1) carrying out Suzuki coupling reaction on 4-bromopyridine-2-carboxylic acid and 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea to obtain an intermediate product with monocarboxylic acid;
the specific operation of the step 1) is as follows: dissolving 4-bromopyridine-2-carboxylic acid, 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, cesium carbonate and tetrakis (triphenylphosphine) palladium in a mixed solution of acetonitrile and water (the volume ratio of the acetonitrile to the water is 4:1), and dissolving N in the mixed solution2Protecting, and reacting for 16h at 100 ℃. After the reaction is finished, filtering, removing filtrate, adding a proper amount of ethyl acetate and water for extraction, combining organic phases, washing with saturated saline solution, and using anhydrous sodium sulfateDrying and separating by column chromatography to obtain intermediate product with monocarboxylic acid.
2) Condensing the intermediate product with monocarboxylic acid and a linker containing a photoaffinity group bisaziridine and an alkynyl under EDC & HCl as a condensing agent to obtain a compound represented by a general formula (X);
the specific operation of the step 2) is as follows: dissolving the intermediate product with the monocarboxylic acid obtained in the step 1), EDC & HCl and HOBT in anhydrous THF, dropwise adding DIPEA under ice bath, stirring for 1h, adding a linker containing a photoaffinity group bisaziridine and alkynyl, stirring for 24h at room temperature, after the reaction is finished, decompressing and rotatably removing the solvent, adding a proper amount of ethyl acetate, washing with water, washing with saturated sodium bicarbonate, washing with saturated saline, combining organic phases, drying with anhydrous sodium sulfate, and separating by column chromatography to obtain the compound represented by the general formula (X) based on the photoaffinity probe of VEGFR-2 inhibitor B14.
The VEGFR-2 inhibitor B14-based photoaffinity probe molecule is applied to preparation of an anti-tumor drug taking VEGFR-2 kinase as a target spot.
Example 1
In the structural formula of the B14 photoaffinity probe molecule with the target confirmation effect, X is O or NH and is prepared by the following steps (see figure 1):
carrying out Suzuki coupling reaction on 4-bromopyridine-2-carboxylic acid and 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea to obtain an intermediate product with monocarboxylic acid; the specific process is as follows:
dissolving 4-bromopyridine-2-carboxylic acid, 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, cesium carbonate and tetrakis (triphenylphosphine) palladium in a mixed solution of acetonitrile and water (the volume ratio of the acetonitrile to the water is 4:1), and dissolving N in the mixed solution2Protecting, and reacting for 16h at 100 ℃. After the reaction, the reaction mixture was filtered, the filtrate was removed by suction, and the mixture was extracted with ethyl acetate and water, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography, and the target compound was eluted with dichloromethane/methanol (V/V. 10/1) to obtain an intermediate product with monocarboxylic acid in a yield of 17% and a weight of 0.36 g.
LC-MS(ESI,m/z):436.79[M+H]+，434.79[M-H]-
(probe molecule I, X ═ O); the specific process is as follows:
dissolving intermediate 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenyl) picolinic acid with monocarboxylic acid (0.063g,0.145mmol) in 3mL of anhydrous tetrahydrofuran solution, adding EDC-HCl (0.04g,0.218mmol), HOBT (0.024g,0.174mmol) and stirring at 0 deg.C, then adding DIPEA (0.094g, 725mmol) dropwise, stirring for 1h, adding a linker (0.02g,0.145mmol) containing the photoaffinity group bisaziridine and alkynyl, stirring at room temperature for 24h, after the reaction is finished, removing the solvent under reduced pressure, adding a proper amount of ethyl acetate, washing with water, saturated sodium bicarbonate, washing with saturated brine, combining the organic phases, drying over anhydrous sodium sulfate, separating by column chromatography, eluting with petroleum ether/ethyl acetate (V/V: 1/1) to obtain the target compound, weighing 0.03g, and obtaining 41.66% yield. The compound represented by the general formula (X) of the light affinity probe based on the VEGFR-2 inhibitor B14 was obtained.
The structure of the obtained target compound is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows:1H NMR(400MHz,DMSO-D6)9.28(s,1H),9.17(s,1H),8.75-8.76(D,1H),8.34-8.35(d,1H),8.14(d,1H),7.97-7,99(d,1H),7.85-7.87(d,2H),7.65-7.68(m,4H),4.23-4.27(t,2H),2.83-2.84(t,1H),2.05-2.09(m,2H),1.91-1.94(t,2H),1.69-1.73(t,2H).
LC-MS(ESI,m/z):556.94[M+H]+。
example 2
In the structural formula of the photoaffinity probe molecule II, X is NH.
The synthesis procedure is as in example 1
The structure of the obtained target probe molecule II is as follows:
the hydrogen spectrum nuclear magnetic resonance data is as follows:1H NMR(400MHz,DMSO-D6)9.28(s,1H),9.17(s,1H),8.88-8.91(t,1H),8.66-8.68(d,1H),8.27-8.28(d,1H),8.14(d,1H),7.91-7,92(q,1H),7.86-7.84(d,2H),7.65-7.67(m,4H),3.22-3.27(q,2H),2.85-2.86(t,1H),2.01-2.06(m,2H),1.68-1.71(t,2H),1.62-1.66(t,2H).
LC-MS(ESI,m/z):555.96[M+H]+。
example 3
And B14 screening the inhibitory activity of the photoaffinity probe molecule on VEGFR-2 kinase.
The ADP-Glo luminescence method is adopted to measure the inhibitory activity of the B14 photoaffinity probe molecule on VEGFR-2 kinase.
With Buffer (Tris 80mM, MgCl)220mM, BSA 0.2mg/mL, DTT 2mM) diluted ATP (10mM) to 250. mu.M; preparing ATP (125 mu M) -Poly (4:1Glu, Tyr) Peptide (0.5 mu g/mu L) mixed solution by ATP and substrate Poly (4:1Glu, Tyr) Peptide according to the volume of 1: 1; the kinase was diluted with Buffer to 1.5 ng/. mu.L. Preparing a compound to be detected into 6 concentration gradient solutions, and sequentially adding 2 mu L of ATP-Poly (4:1Glu, Tyr) Peptide solution, 1 mu L of sample solution and 2 mu L of enzyme solution on a 384-well plate to start reaction. After incubation at 30 ℃ for 60min, 5. mu.L of ADP-Glo reagent was added to stop the reaction. Then 10 mu L of KinaseDedetection reagent is added to convert ADP into ATP, the ATP is incubated for 30min at 25 ℃, the chemiluminescence module of a PerkinElmer multifunctional microplate reader is used for measuring the luminescence value, and the inhibition rate is calculated.
Numerical value processing: inhibition rate ═ 100% (positive value-dosing group value)/(positive value-negative value);
the results of the compound experiments are shown in table 1:
TABLE 1B14 results of inhibitory Activity of photoaffinity probe molecules on VEGFR-2 kinase
As can be seen from Table 1, the B14 photoaffinity probe molecule prepared by the invention has better inhibitory activity on VEGFR-2 kinase.
Claims (1)
1. A preparation method of a VEGFR-2 inhibitor B14-based photoaffinity probe molecule is characterized by comprising the following steps:
1) carrying out Suzuki coupling reaction on 4-bromopyridine-2-carboxylic acid and 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea to obtain an intermediate product with monocarboxylic acid;
2) the linker containing the photoaffinity group bis-aziridine and alkynyl and the intermediate product with monocarboxylic acid are condensed under EDC & HCl to obtain the photoaffinity probe molecule based on VEGFR-2 inhibitor B14, and the structural formula is as follows:
wherein X ═ O or NH;
the specific process of the step 1) is as follows: dissolving 4-bromopyridine-2-carboxylic acid, 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, cesium carbonate and tetrakis (triphenylphosphine) palladium in a mixed solution of acetonitrile and water, and dissolving N in the mixed solution2Protecting, reacting at 100 ℃ for 16h, and then treating to obtain an intermediate product with monocarboxylic acid;
4.95mmol of 4-bromopyridine-2-carboxylic acid, 5.94mmol of 1- (4-chloro-3- (trifluoromethyl) phenyl) -3- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea, 9.90mmol of cesium carbonate and 0.25mmol of tetrakis (triphenylphosphine) palladium were dissolved in 50mL of a mixed solution of acetonitrile and water, and N was added2Protecting, reacting at 100 ℃ for 16h, and then treating to obtain an intermediate product with monocarboxylic acid; the volume ratio of acetonitrile to water in the mixed solution of acetonitrile and water is 4: 1;
the specific process of the step 2) is as follows: dissolving the intermediate product with the monocarboxylic acid obtained in the step 1) in an anhydrous tetrahydrofuran solution, adding EDC & HCl and HOBt, then dropwise adding DIPEA, stirring at 0 ℃ for 1h, then adding a linker containing a photoaffinity group bisaziridine and alkynyl, stirring at room temperature for 24h, and then processing to obtain a photoaffinity probe molecule based on a VEGFR-2 inhibitor B14;
dissolving 0.145mmol of intermediate product with monocarboxylic acid in 3mL of anhydrous tetrahydrofuran solution, adding 0.218mmol of EDC HCI and 0.174mmol of HOBt, stirring at 0 ℃, then dropwise adding 0.725mmol of DIPEA, stirring for 1h, adding 0.145mmol of linker containing photoaffinity group biaziridine and alkynyl, and stirring for 24h at room temperature to obtain the photoaffinity probe molecule based on VEGFR-2 inhibitor B14.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532197A (en) * | 2010-12-24 | 2012-07-04 | 中国科学院上海药物研究所 | First glass photoaffinity labeling difunctional probe molecule and preparation method and application thereof |
| CN104817493A (en) * | 2015-03-11 | 2015-08-05 | 西安交通大学 | Aromatic heterocyclic amide substituted diarylurea compound, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and Tissue-Based Proteome Profiling;Zhengqiu Li等;《Angew. Chem. Int. Ed.》;20130610;第52卷;第8551-8556页 * |
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