CN109503553B - Light affinity probe molecule based on VEGFR-2 inhibitor B14 and preparation method thereof - Google Patents

Abstract

A kind of photoaffinity probe molecule based on VEGFR-2 inhibitor B14 and preparation method thereof, 4-bromopyridine-2-carboxylic acid and 1-(4-chloro-3-(trifluoromethyl)phenyl)-3 ‑(4‑(4,4,5,5‑tetramethyl‑1,3,2‑dioxaborolane‑2‑yl)phenyl)urea was subjected to Suzuki coupling reaction to obtain a Intermediate product of acid; linker containing photoaffinity group bis-aziridine and alkynyl group and intermediate product with monocarboxylic acid under the condensation of EDC·HCl to obtain photoaffinity based on VEGFR-2 inhibitor B14 and probe molecules. The preparation method of the B14 photoaffinity probe molecule of the present invention is simple, easy to realize, and has a high yield. The B14 small molecule photoaffinity probe of the present invention can be used to confirm the action target of B14 and to verify the feasibility of the photoaffinity labeling technology in confirming the small molecule target.

Description

A kind of photoaffinity probe molecule based on VEGFR-2 inhibitor B14 and preparation method thereof

technical field

The present invention relates to a photoaffinity probe molecule based on VEGFR-2 inhibitor B14 and a preparation method thereof.

Background technique

Photoaffinity labeling technology (PAL) combines the advantages of modern molecular biology, cell biology, medicinal chemistry, analytical chemistry and other disciplines, and uses synthetic photoaffinity probe molecules to produce high activity under the irradiation of specific wavelengths of light. It can be directly irreversibly covalently cross-linked with the protein specifically bound to the drug molecule to realize the capture of the drug target protein molecule. It is one of the core tools for studying the interaction between ligands and receptors at the molecular level.

In recent years, this technology has been mainly used in the confirmation of drug molecular target proteins. It is mainly based on photocrosslinking technology, bioorthogonal technology and related bioanalytical technologies, etc., to achieve the capture and confirmation of drug molecular targets. Among them, the design and synthesis of photoaffinity probe molecules is to directly modify the structure without affecting the activity of the target drug, and introduce photoreactive groups and bioorthogonal handles to design and synthesize photoaffinity probe molecules. Under the irradiation of wavelength light, the target protein of the target compound is specifically and irreversibly captured covalently. Next, the identification of the captured target protein is confirmed by bioorthogonal reaction.

Vascular endothelial growth factor receptor (vascular endothelial growth factor receptor) is a membrane protein expressed by the VEGF gene, belonging to the tyrosine family of proteins, and is a macromolecular protein closely related to malignant tumors. Vascular endothelial growth factor and its receptors are overexpressed in a series of tumor cells, and the receptor family includes three subtypes: VEGFR-1, VEGFR-2, and VEGFR-3. Among them, VEGFR-2 is mainly involved in the proliferation of vascular endothelial cells and is most widely distributed in cancer cells. A series of studies have confirmed that it can be an effective drug target.

In the past five years, the photoactive groups reported in the literature include aryl azides, benzophenones, substituted diaziridines, and diaziridines. Among them, the photocrosslinking activity is preferably diaziridine. Therefore, it is necessary to choose diaziridine as the photoactive group to design and synthesize photoaffinity probe molecules.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a photo-affinity probe molecule based on VEGFR-2 inhibitor B14 and a preparation method thereof. The B14 photo-affinity probe molecule is used to confirm the target protein of B14 and its mode of action with the target protein, And it can be used to verify the feasibility of photoaffinity labeling technology in the confirmation of target protein.

To achieve the above object, the present invention adopts the following technical solutions:

A photoaffinity probe molecule based on VEGFR-2 inhibitor B14, its structural formula is as follows:

where X is O or NH.

A preparation method based on a VEGFR-2 inhibitor B14 photoaffinity probe molecule, characterized in that it comprises the following steps:

1) 4-Bromopyridine-2-carboxylic acid and 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1) , 3,2-dioxaborolane-2-yl) phenyl) urea carries out Suzuki coupling reaction, obtains the intermediate product with monocarboxylic acid;

2) Under the condensation of EDC·HCl between the linker containing photoaffinity group bisaziridine and alkynyl group and the intermediate product with monocarboxylic acid, a photoaffinity probe based on VEGFR-2 inhibitor B14 is obtained Molecules, the structural formula is as follows:

where X=O or NH.

A further improvement of the present invention is that the specific process of the step 1) is: 4-bromopyridine-2-carboxylic acid, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-( 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)urea, cesium carbonate and tetrakis(triphenylphosphine)palladium In a mixed solution of acetonitrile and water, protected by N ₂ , reacted at 100 °C for 16 h, and then treated to obtain an intermediate product with a monocarboxylic acid.

The further improvement of the present invention is that 4.95 mmol of 4-bromopyridine-2-carboxylic acid, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5 ,5-tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)urea 5.94mmol, cesium carbonate 9.90mmol and tetrakis(triphenylphosphine)palladium 0.25mmol dissolved in acetonitrile In 50 mL of a mixed solution with water, protected by _N2 , reacted at 100 °C for 16 h, and then treated to obtain an intermediate product with a monocarboxylic acid.

A further improvement of the present invention is that the volume ratio of acetonitrile to water in the mixed solution of acetonitrile and water is 4:1.

A further improvement of the present invention is that the specific process of the step 2) is as follows: the intermediate product with the monocarboxylic acid obtained in the step 1) is dissolved in anhydrous tetrahydrofuran solution, EDC·HCl, HOBt are added, and then dropwise added DIPEA was stirred at 0 °C for 1 h and then added with a linker containing a photoaffinity group of bis-aziridine and an alkynyl group. After stirring at room temperature for 24 h, it was treated to obtain a photoaffinity probe molecule based on VEGFR-2 inhibitor B14. .

A further improvement of the present invention is that 0.145 mmol of the intermediate product with monocarboxylic acid is dissolved in 3 mL of anhydrous tetrahydrofuran solution, and 0.218 mmol of EDC·HCl and 0.174 mmol of HOBt are added to stir evenly at 0°C, and then 0.725 mmol of DIPEA is added dropwise. , stirred for 1 h, added 0.145 mmol of a linker containing a photoaffinity group bisaziridine and an alkynyl group, and stirred at room temperature for 24 h to obtain a photoaffinity probe molecule based on VEGFR-2 inhibitor B14.

Application of a photoaffinity probe molecule based on VEGFR-2 inhibitor B14 in photoaffinity labeling technology.

A further improvement of the present invention is based on the application of the VEGFR-2 inhibitor B14 photoaffinity probe molecule in confirming the protein target.

Compared with the prior art, the present invention has the following beneficial effects:

In the present invention, a B14 photoaffinity probe molecule is obtained by using a linker containing both a photoaffinity group, diaziridine and an alkynyl group, and connecting it with the VEGFR-2 protein inhibitor B14. The photoaffinity probe molecule can specifically and irreversibly bind to the target protein molecule of B14, and then the bioorthogonal handle in the probe molecule performs a click reaction with another bioorthogonal handle containing fluorescein or biotin to Confirm the target protein. The preparation method of the B14 photoaffinity probe molecule of the present invention is simple, easy to realize, and has a high yield.

The B14 photoaffinity probe molecule in the present invention can specifically covalently bind to the VEGFR-2 protein, and then the subsequent confirmatory analysis of the captured target protein can be achieved through a click reaction. The use of photoaffinity labeling technology to confirm the target protein of drug action can improve some deficiencies of the original target confirmation technology. In the original target confirmation technology, the probe molecule cannot stably bind to the drug target molecule, which may easily lead to false positive results. Moreover, the original technology usually needs to attach a large volume of fluorescent groups to the drug molecules for analysis, which will lead to the disadvantages of reduced probe molecule activity, poor solubility, and poor cell permeability. The photoaffinity labeling technology uses the synthesis of small molecule photoaffinity probes combined with bioorthogonal technology to confirm the target, which makes up for the shortcomings of the original technology. The B14 small molecule photoaffinity probe of the present invention can be used to confirm the action target of B14 and to verify the feasibility of the photoaffinity labeling technology in confirming the small molecule target.

Description of drawings

Fig. 1 is a synthetic roadmap based on the VEGFR-2 inhibitor B14 photoaffinity probe molecule provided by the present invention;

Wherein, compound 1 is 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxo) Borolin-2-yl)phenyl)urea, compound 2 is 4-bromopyridine-2-carboxylic acid, compound 3 is 4-(4-(3-(4-chloro-3-(trifluoromethane) group) phenyl) ureido) phenyl) picolinic acid (B14), compound 4 is a linker containing a photoaffinity group bis-aziridine and an alkynyl group, and compound (X) is a B14 photoaffinity probe molecule.

Specifically marked in the figure are:

a. CsCO ₃ , Pd[P(C ₆ H ₅ ) ₃ ] ₄ , Acetonitrile, N ₂ , 80° C.; b. EDC·HCl, HOBt, DIPEA, THF, rt.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, which are to explain rather than limit the present invention.

In the present invention, the photoaffinity probe molecule is obtained by linking the VEGFR-2 protein inhibitor by using the linker containing the photoaffinity group diaziridine and the alkynyl group. The photoaffinity probe molecule involved in the present invention can be used to confirm the target protein of B14.

其中，X为O或NH。In the present invention, the structural formula of the linker containing the photoaffinity group diaziridine and the alkynyl 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 present invention is as follows:

Wherein, X is O or NH.

The photoaffinity probe molecule with target confirmation function of the present invention includes:

2-(3-(But-3-yn-1-yl)-3H-bisaziridine-3-yl)ethyl 4-(4-(3-(4-chloro-3-(trifluoromethyl) )phenyl)ureido)phenyl)picolinic acid.

N-(2-(3-(But-3-yn-1-yl)-3H-bisaziridine-3-yl)ethyl)-4-(4-(3-(4-chloro-3- (Trifluoromethyl)phenyl)ureido)phenyl)picolinamide.

The preparation and activity screening method of the photoaffinity probe molecule with target confirmation provided by the present invention will be described in detail below in conjunction with the synthetic route shown in FIG. 1 and specific synthetic examples.

Referring to Figure 1, a preparation method based on VEGFR-2 inhibitor B14 photoaffinity probe molecule, comprising the following steps:

1) 4-Bromopyridine-2-carboxylic acid and 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1) , 3,2-dioxaborolane-2-yl) phenyl) urea carries out Suzuki coupling reaction, obtains the intermediate product with monocarboxylic acid;

The specific operation of the step 1) is: 4-bromopyridine-2-carboxylic acid, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4, 5,5-Tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)urea, cesium carbonate and tetrakis(triphenylphosphine)palladium dissolved in a mixed solution of acetonitrile and water (The volume ratio of acetonitrile to water is 4:1), protected by N ₂ , and reacted at 100 °C for 16 h. After the reaction, suction filtration, spin off the filtrate, add an appropriate amount of ethyl acetate and water for extraction, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, and separate by column chromatography to obtain an intermediate product with monocarboxylic acid.

2) with the intermediate product with monocarboxylic acid and the linker containing photoaffinity group bis-aziridine and alkynyl under EDC·HCl as the condensation action of condensing agent, obtain the compound represented by general formula (X);

The specific operation of the step 2) is as follows: dissolving the intermediate product with monocarboxylic acid obtained in step 1), EDC·HCl, and HOBT in anhydrous THF, adding DIPEA dropwise under an ice bath, stirring for 1 h, adding a mixture containing The linker of the photoaffinity group bis-aziridine and the alkynyl group was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. After adding an appropriate amount of ethyl acetate, washed with water, saturated sodium bicarbonate, and saturated common salt. Wash with water, combine the organic phases, dry with anhydrous sodium sulfate, and separate by column chromatography to obtain a compound represented by the general formula (X) based on the VEGFR-2 inhibitor B14 photoaffinity probe.

The application of the above-mentioned VEGFR-2 inhibitor B14 photoaffinity probe molecule in the preparation of an antitumor drug targeting VEGFR-2 kinase.

Example 1

In the structural formula of the B14 photoaffinity probe molecule with target confirmation function, X is O or NH, and is prepared by the following steps (see Figure 1):

4-Bromopyridine-2-carboxylic acid and 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1,3) , 2-dioxaborolane-2-yl) phenyl) urea carries out Suzuki coupling reaction, obtains the intermediate product with monocarboxylic acid; Concrete process is as follows:

4-Bromopyridine-2-carboxylic acid, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1, A mixed solution of 3,2-dioxaborol-2-yl)phenyl)urea, cesium carbonate and tetrakis(triphenylphosphine)palladium dissolved in acetonitrile and water (the volume ratio of acetonitrile to water is 4: 1), protected by N ₂ , and reacted at 100 °C for 16 h. After the reaction, suction filtration, spin off the filtrate, add appropriate amount of ethyl acetate and water for extraction, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, and separate by column chromatography using dichloromethane/methanol (V/V= 10/1) Elution to obtain the target compound, weighing 0.36 g, and the yield is 17% to obtain an intermediate product with a monocarboxylic acid.

LC-MS (ESI, m/z): 436.79 [M+H] ⁺ , 434.79 [MH] ^-

(probe molecule I, X=O); the specific process is as follows:

The intermediate with monocarboxylic acid, 4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenyl)picolinic acid (0.063 g, 0.145 mmol) was dissolved in To 3mL of anhydrous tetrahydrofuran solution, add EDC·HCl (0.04g, 0.218mmol), HOBT (0.024g, 0.174mmol) and stir evenly at 0°C, then add DIPEA (0.094g, .725mmol) dropwise, stir for 1h, Add the linker (0.02g, 0.145mmol) containing the photoaffinity group bis-aziridine and alkynyl group, stir at room temperature for 24h, after the reaction is over, remove the solvent under reduced pressure, add an appropriate amount of ethyl acetate, wash with water, Washed with saturated sodium bicarbonate and saturated brine, combined the organic phases, dried over anhydrous sodium sulfate, separated by column chromatography, and eluted with petroleum ether/ethyl acetate (V/V=1/1) to obtain the target compound, weighing 0.03 g , the yield is 41.66%. The compound represented by the general formula (X) based on the VEGFR-2 inhibitor B14 photoaffinity probe is obtained.

The structure of the obtained target compound is as follows:

The hydrogen spectrum nuclear magnetic resonance data are: ¹ H NMR (400MHz, DMSO-D6)δ9.28(s,1H),δ9.17(s,1H),δ8.75-8.76(D,1H),δ8.34- 8.35(d,1H),δ8.14(d,1H),δ7.97-7,99(d,1H),δ7.85-7.87(d,2H),δ7.65-7.68(m,4H) ,δ4.23-4.27(t,2H),δ2.83-2.84(t,1H),δ2.05-2.09(m,2H),δ1.91-1.94(t,2H),δ1.69-1.73 (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.

Synthesis steps are the same as in Example 1

The structure of the obtained target probe molecule II is as follows:

The hydrogen spectrum nuclear magnetic resonance data are: ¹ H NMR (400MHz, DMSO-D6)δ9.28(s,1H),δ9.17(s,1H),δ8.88-8.91(t,1H),δ8.66- 8.68(d,1H),δ8.27-8.28(d,1H),δ8.14(d,1H),δ7.91-7,92(q,1H),δ7.86-7.84(d,2H) ,δ7.65-7.67(m,4H),δ3.22-3.27(q,2H),δ2.85-2.86(t,1H),δ2.01-2.06(m,2H),δ1.68-1.71 (t,2H),δ1.62-1.66(t,2H).

LC-MS (ESI, m/z): 555.96 [M+H] ⁺ .

Example 3

Screening of the inhibitory activity of B14 photoaffinity probe molecules on VEGFR-2 kinase.

The ADP-Glo luminescence method was used to determine the inhibitory activity of B14 photoaffinity probe molecules on VEGFR-2 kinase.

Dilute ATP (10 mM) to 250 μM with Buffer (Tris 80 mM, MgCl ₂ 20 mM, BSA 0.2 mg/mL, DTT 2 mM); ATP and substrate Poly (4:1 Glu, Tyr) Peptide are prepared by volume 1:1 to prepare ATP ( 125μM)-Poly(4:1Glu,Tyr)Peptide(0.5μg/μL) mixed solution; Dilute the kinase with Buffer to 1.5ng/μL. The compounds to be tested were prepared into 6 concentration gradient solutions, and 2 μL of ATP-Poly (4:1Glu,Tyr) Peptide solution, 1 μL of sample solution, and 2 μL of enzyme solution were sequentially added to the 384-well plate to initiate the reaction. After incubation at 30°C for 60 min, 5 μL of ADP-Glo reagent was added to stop the reaction. Then add 10 μL of KinaseDetection reagent to convert ADP into ATP, incubate at 25°C for 30min, use the chemiluminescence module of PerkinElmer multi-function microplate reader to measure the luminescence value and calculate the inhibition rate.

Numerical processing: inhibition rate=(positive value-administration group value)/(positive value-negative value)×100%;

The experimental results of the compounds are shown in Table 1:

Table 1B14 Results of inhibitory activity of photoaffinity probe molecules on VEGFR-2 kinase

It can be seen from Table 1 that the B14 photoaffinity probe molecule prepared by the present invention has a good inhibitory activity on VEGFR-2 kinase.

Claims (1)

1. a preparation method based on VEGFR-2 inhibitor B14 photoaffinity probe molecule, is characterized in that, comprises the following steps: 1) 4-Bromopyridine-2-carboxylic acid and 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl-1) , 3,2-dioxaborolane-2-yl) phenyl) urea carries out Suzuki coupling reaction, obtains the intermediate product with monocarboxylic acid; 2) Under the condensation of EDC·HCl between the linker containing photoaffinity group bisaziridine and alkynyl group and the intermediate product with monocarboxylic acid, a photoaffinity probe based on VEGFR-2 inhibitor B14 is obtained Molecules, the structural formula is as follows:

wherein X=O or NH; The specific process of the step 1) is: 4-bromopyridine-2-carboxylic acid, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4, 5,5-Tetramethyl-1,3,2-dioxaborol-2-yl)phenyl)urea, cesium carbonate and tetrakis(triphenylphosphine)palladium dissolved in a mixed solution of acetonitrile and water , _N2 protected, reacted at 100 °C for 16 h and treated to obtain an intermediate product with a monocarboxylic acid; 4-Bromopyridine-2-carboxylic acid 4.95mmol, 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(4-(4,4,5,5-tetramethyl- 5.94 mmol of 1,3,2-dioxaborol-2-yl)phenyl)urea, 9.90 mmol of cesium carbonate and 0.25 mmol of tetrakis(triphenylphosphine)palladium were dissolved in 50 mL of a mixed solution of acetonitrile and water , protected by N ₂ , reacted at 100 °C for 16 h and treated to obtain an intermediate product with monocarboxylic acid; the volume ratio of acetonitrile to water in the mixed solution of acetonitrile and water was 4:1; The specific process of the step 2) is: dissolving the intermediate product with monocarboxylic acid obtained in step 1) in anhydrous tetrahydrofuran solution, adding EDC·HCl, HOBt, then adding DIPEA dropwise, stirring at 0 ° C for 1 h Then, a linker containing a photo-affinity group bis-aziridine and an alkynyl group was added, and after stirring at room temperature for 24 hours, the treatment was performed to obtain a photo-affinity probe molecule based on VEGFR-2 inhibitor B14; Dissolve 0.145 mmol of the intermediate product with monocarboxylic acid in 3 mL of anhydrous tetrahydrofuran solution, add 0.218 mmol of EDC·HCl, 0.174 mmol of HOBt, stir evenly at 0 °C, then add 0.725 mmol of DIPEA dropwise, stir for 1 h, add containing light The linker of the affinity group bis-aziridine and the alkynyl group was 0.145 mmol, and stirred at room temperature for 24 h to obtain a photoaffinity probe molecule based on the VEGFR-2 inhibitor B14.

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