Near-infrared light-activated protein-targeting degradation chimera, and preparation method and application thereof

Abstract

Near-infrared light activated protein targeted degradation chimera, preparation method and application thereof, and structure is as follows: the present application first describes a near-infrared (NIR) light activated nano protein targeted degradation chimera (NAP) for remote controllable protein hydrolysis in vivo.NAP is self-assembled by connecting protein targeted degradation chimera ARV771 capable of specifically degrading BRD4 protein with near-infrared photosensitizer through singlet oxygen ( 1 O2) cleavable linker.The effect of disease research, cancer cell growth inhibition and tumor treatment is achieved by regulating the level of pathogenic protein.The present application uses chemical method to prepare singlet oxygen activated protein targeted degradation drug synthesis and apply it to the combination of targeted degradation of protein in cells and photodynamic therapy to effectively inhibit tumor growth.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of protein targeted degradation, and particularly relates to a near-infrared light activated protein targeted degradation chimera and a preparation method and application thereof. BACKGROUND

[0002] The protein hydrolysis-targeted chimera (PROTAC) technology has become a promising anticancer approach. PROTAC contains a ligand of a target protein and an E3 ubiquitin ligase, and induces targeted protein degradation through the ubiquitin-proteasome pathway. So far, PROTAC has been applied to degrade various oncogenic proteins, such as androgen receptor (AR), estrogen receptor (ER), and bromodomain-containing protein 4 (BRD4). However, the off-target / toxicity caused by PROTAC activity in normal tissues after systemic administration is still a key safety problem. Therefore, tumor-specific treatment strategies should be considered during treatment. The protein targeted degradation chimera is not active before being exposed to the tumor site, and its protein hydrolysis activity is restored by triggering a response group, providing a practical method for manipulating the degradation process. Among them, light has safety and high spatiotemporal resolution. At present, light cage-type and light switch-type PROTACs have been used to temporarily mask the protein hydrolysis activity by using photolysis and photoisomerization groups, respectively. These light-activated PROTACs show great prospects in the optical control of targeted protein degradation in living cells. However, their activation depends on ultraviolet or visible light irradiation, which greatly hinders their application in vivo. Here, we report a near-infrared (NIR) light-activated nano-formulated PROTAC (NAP) for remotely controllable protein hydrolysis in tumor cells in vivo (Scheme 1). Due to covalent cross-linking, PROTAC is initially inactive in protein hydrolysis. After systemic administration, NAP accumulates in tumor tissues and enters tumor cells through endocytosis. Then, the released PROTAC restores its activity for protein degradation. At the same time, the photosensitizer can be used for photodynamic therapy to achieve synergistic cancer inhibition. SUMMARY

[0003] The application provides a near-infrared light activated protein targeted degradation chimera and a preparation method and application thereof, which is suitable for the research on activation of target proteins and synergistic tumor treatment.

[0004] Technical scheme: The near-infrared light activated protein targeted degradation chimera has the following structure:

[0005]

[0006] The preparation method of the near-infrared light activated protein targeted degradation chimera comprises the following steps: 1 eq of mercaptoethanol, 1.2 eq of potassium fluoride are mixed in acetic acid, and the mixture is reacted at 80 DEG C for 18 hours; the reaction product is extracted with ethyl acetate, and then column chromatography is performed to obtain purified product 1; 1 eq of the purified product 1 is reacted with 2.2 eq of acetone and 1 eq of trifluoroacetic acid at room temperature for 24 hours; column chromatography is performed to obtain purified product 2; 1 eq of the purified product 2 is dissolved in methanol with 4 eq of potassium hydroxide at room temperature for 12 hours; the reaction product is extracted with ethyl acetate, and then column chromatography is performed to obtain purified product 3; 1 eq of the purified product 3 is dissolved in dichloromethane with 4 eq of p-nitrochloroformic acid phenyl ester and 8 eq of TEA at room temperature for 12 hours; column chromatography is performed to obtain intermediate product molecule 4; 1 eq of PA and 1.2 eq of single BOC-ethylenediamine are stirred in 8 eq of TEA at room temperature for 12 hours; column chromatography is performed to obtain PA intermediate 7; 1 eq of ARV-771 and 2 eq of the intermediate product molecule 4 are stirred in 2 eq of TEA at room temperature for 24 hours under nitrogen protection; column chromatography is performed to purify the product; 1 eq of the purified product, 1 eq of the PA intermediate 7 and 8 eq of TEA are dissolved in dichloromethane (DCM) at room temperature for 12 hours; high performance liquid chromatography is performed to obtain product molecule NAP.

[0007] The chimera is used for preparing a drug for selectively degrading BRD4 in MCF-7 cells.

[0008] The chimera is used for preparing an antitumor drug.

[0009] An antitumor drug comprises the near-infrared light activated protein targeted degradation chimera.

[0010] The preparation method of the near-infrared light activated protein targeted degradation chimera comprises the following steps: 1 eq of mercaptoethanol, 1.2 eq of potassium fluoride are mixed in acetic acid, and the mixture is reacted at 80 DEG C for 18 hours; the reaction product is extracted with ethyl acetate, and then column chromatography is performed to obtain purified product 1; 1 eq of the purified product 1 is reacted with 2.2 eq of acetone and 1 eq of trifluoroacetic acid at room temperature for 24 hours; column chromatography is performed to obtain purified product 2; 1 eq of the purified product 2 is dissolved in methanol with 4 eq of potassium hydroxide at room temperature for 12 hours; the reaction product is extracted with ethyl acetate, and then column chromatography is performed to obtain purified product 3; 1 eq of the purified product 3 is dissolved in dichloromethane with 4 eq of p-nitrochloroformic acid phenyl ester and 8 eq of TEA at room temperature for 12 hours; column chromatography is performed to obtain intermediate product molecule 4; 1 eq of PA and 1.2 eq of single BOC-ethylenediamine are stirred in 8 eq of TEA at room temperature for 12 hours; column chromatography is performed to obtain PA intermediate 7; 1 eq of ARV-771 and 2 eq of the intermediate product molecule 4 are stirred in 2 eq of TEA at room temperature for 24 hours under nitrogen protection; column chromatography is performed to purify the product; 1 eq of the purified product, 1 eq of the PA intermediate 7 and 8 eq of TEA are dissolved in dichloromethane (DCM) at room temperature for 12 hours; high performance liquid chromatography is performed to obtain product molecule NAP.

[0011]

[0012] Beneficial effects: The present application first uses near-infrared light to activate and induce targeted protein degradation and photodynamic therapy, and achieves the effects of disease research, cancer cell growth inhibition and tumor treatment through the level regulation of pathogenic proteins. The present application uses chemical methods to prepare a near-infrared light activated protein targeted degradation drug and apply it to the research of targeted degradation of proteins in cells and photodynamic therapy. (1) Synthesis of a near-infrared light activated protein targeted degradation drug: a protein proteolysis-targeting chimera ARV771 capable of degrading BRD4 protein is selected and synthesized with a photosensitizer PA, and the near-infrared light activated protein targeted degradation drug is prepared mainly through amide condensation reaction. (2) Release of the near-infrared light activated protein targeted degradation drug: the synthesized near-infrared light activated protein targeted degradation drug is irradiated in a solution, and the release of the PROTAC molecule ARV771 is detected by high performance liquid chromatography, and the generation of singlet oxygen is verified in a spectrum experiment. (3) Application of the release of the near-infrared light activated protein targeted degradation drug in targeted protein degradation in cells: the near-infrared light activated protein targeted degradation drug is co-incubated with tumor cells and irradiated, and then the expression of BRD4 in the cells is detected. The protein degradation and the killing effect of the PDT effect on tumor tissues are verified by in vivo experiments. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a synthesis method of a near-infrared light activated protein targeted degradation drug;

[0014] Figure 2 It is mass spectrometry characterization of the near-infrared light activated protein targeted degradation drug;

[0015] Figure 3 It is an HPLC chart of the release of the PROTAC in a solution after the above drug treatment;

[0016] Figure 4 It is a spectrum chart of the release of singlet oxygen in a solution after the above drug treatment;

[0017] Figure 5 It is the expression of BRD4 in MCF-7 cells before and after the above drug treatment;

[0018] Figure 6 It is the proliferation inhibition of MCF-7 cells after the above drug treatment;

[0019] Figure 7 It is the growth inhibition of MCF-7 ectopic tumors after the above drug treatment;

[0020] Figure 8 It is the expression of BRD4 in MCF-7 ectopic tumor cells after the above drug treatment;

[0021] Figure 9This is a schematic diagram of the method for preparing the protein-targeted degradation compound of the present invention. Detailed Implementation

[0022] Example 1: Structure and Synthesis Method of Near-Infrared Photoactivated Protein-Targeted Degradation Drug

[0023] like Figure 1 As shown, the standard chemical synthesis procedure synthesizes napp, a near-infrared photoactivated protein-targeting degradation molecule that targets BRD4 protein based on singlet oxygen response, which can self-assemble into NAP; and ncpp, a near-infrared photoactivated protein-targeting degradation molecule that does not contain singlet oxygen response and targets BRD4 protein, which can self-assemble into NCP.

[0024] The specific method is as follows: First, mercaptoethanol (5g) and potassium fluoride (4.6g) are mixed with acetic acid and reacted at 80℃ for 18 hours. After extraction with ethyl acetate three times and purification by column chromatography with petroleum ether:ethyl acetate (1:1), the product is reacted with acetone (4.0g) and trifluoroacetic acid (140μL) at room temperature for 24 hours. After purification by column chromatography with petroleum ether:ethyl acetate (3:1), 2g of the product is added to potassium hydroxide (1.8g), dissolved in methanol, and left at room temperature for 12 hours. After extraction with ethyl acetate and purification by column chromatography with dichloromethane:methanol (30:1), 1.0g of the product is added to phenyl p-nitrochloroformate (6.17g), and TEA (200μL) is dissolved in dichloromethane (10mL) and reacted at room temperature. After 12 hours, intermediate molecule 4 was purified by column chromatography using dichloromethane:methanol (50:1). PA (100 mg) and mono-BOC-ethylenediamine (32.43 mg) were stirred in TEA at room temperature for 12 hours, and column chromatography was used to obtain PA intermediate 7. ARV-771 (100 mg) and intermediate molecule 4 (213.3 mg) were stirred in TEA at room temperature for 24 hours under nitrogen protection. After purification by dichloromethane:methanol (20:1) column chromatography, the product (20 mg), PA intermediate 7 (11.1 mg), and TEA (10 μL) were dissolved in dichloromethane (DCM), reacted at room temperature for 12 hours, and purified by high-performance liquid chromatography to obtain the product molecule NAP. Mass spectrometry results are shown below. Figure 2 The calculation result is [M+Na]. + 1891.7172, actual measurement 1891.8150; calculated result [M+K]+1907.6861, actual measurement 1907.7853. NCP is obtained under the same conditions.

[0025] Example 2: Release of PROTAC, a near-infrared light-activated protein-targeting degradation drug, in solution.

[0026] NAP (5 μM) or NCP (5 μM) in PBS was reacted at 670 nm (300 mW / cm²). 2) light irradiation for 5 min, and incubation at room temperature for 12 h, followed by high performance liquid chromatography (mobile phase: A: CH3CN, B: H2O, 0.1% TFA; flow rate: 1 mL / min; elution gradient: 0-30 min, from 40% A to 95% A), the results are shown in Figure 3 Figure 6, where the NAP plus light irradiation group showed ARV-771 release at 30 min, while no ARV-771 release was observed in the other control groups.

[0027] To determine the singlet oxygen production ability of NAP and NCP, SOSG was used as a singlet oxygen fluorescent indicator. SOSG (10 mM) was added to 5 mM NAP or NCP solution, and the solution was irradiated with 670 nm (300 mW / cm 2 ) light for 5 min, and the fluorescence at 520 nm was measured. The results are shown in Figure 4 Figure 7, where the fluorescence of NAP and NCP was significantly enhanced after light irradiation, indicating the production of singlet oxygen.

[0028] Example 3, Application of Protein Targeted Degradation Chimeras in Targeting BRD4 Degradation in Cells

[0029] Different concentrations of compound NAP, NCP were added to MCF-7 cells, and after incubation for 12 h, the cells were irradiated with 670 nm (300 mW / cm 2 ) light for 5 min and then incubated for another 12 h. Western blotting was used to detect BRD4 in the cells, and the results are shown in Figure 5 Figure 8, where BRD4 degradation was only obvious in the NAP light irradiation group, and the degradation effect increased with increasing concentration of NAP. This result indicates that compound NAP can degrade BRD4 protein in MCF-7 cells under light irradiation.

[0030] Different ARV-771, NAP, NCP were added to A549 cells, and after incubation for 12 h, the cells were irradiated with 670 nm (300 mW / cm 2 ) light for 5 min and then incubated for another 36 h. CCK-8 was used to detect the proliferation rate of the cells, and the results are shown in Figure 6 Figure 9, where the proliferation rate of the cells was significantly down-regulated with increasing incubation concentration, and the effect was most obvious in the NAP light irradiation group. This verifies that targeted BRD4 protein degradation after NAP light irradiation can inhibit cancer cell proliferation in combination with photodynamic therapy.

[0031] Example 4, Application of Protein Targeted Degradation Chimeras in Tumor Therapy

[0032] 4-6 week old female BALB / c (BALB / c-nude) mice were subcutaneously transplanted with 1 x 10 7 MCF-7 cells. When the tumor size reached about 120 mm 3Every 2 days, the same dose (10 mg / kg ARV-771) of the PROTAC was injected intravenously. The same dose of control agent (normal saline) was also injected as a negative control. Tumor size and body weight were measured every 2 days. The mice were sacrificed on day 16, and tumor tissues and major normal tissues were taken for analysis. The tumor inhibition rate was calculated as Figure 7 As shown in FIG. 6, the tumor growth of the NAP plus light group was the slowest, so the near-infrared light-activated protein targeted degradation chimera can achieve inhibition of tumor growth. The level of BRD4 in tumor tissues was as shown in FIG. 7, the histone was degraded after ARV-771 treatment alone, but the protein degradation effect of the NAP plus light group was the most obvious, verifying that the chimera can achieve targeted BRD4 protein degradation in vivo. Figure 8

[0033] The above specific embodiments do not limit the technical solutions of the present application in any form, and any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present application.​

Claims

1. A near-infrared light-activated protein targeted degradation chimera, characterized in that, The structure is as shown below:

2. The method for preparing the near-infrared light-activated protein-targeted degradation chimera according to claim 1, characterized in that, The steps are as follows: first, 1 eq of mercaptoethanol, 1.2 eq of potassium fluoride are mixed in acetic acid, and the reaction is carried out at 80°C for 18 hours; the reaction product is extracted with ethyl acetate, and then column chromatography is carried out to obtain the purified product 1; 1 eq of the purified product 1 is reacted with 2.2 eq of acetone and 1 eq of trifluoroacetic acid at room temperature for 24 hours, and then column chromatography is carried out to obtain the purified product 2; 1 eq of the purified product 2 is dissolved in methanol with 4 eq of potassium hydroxide at room temperature for 12 hours, the reaction product is extracted with ethyl acetate, and then column chromatography is carried out to obtain the purified product 3; 1 eq of the purified product 3 is dissolved in dichloromethane with 4 eq of p-nitrochloroformic acid phenyl ester and 8 eq of TEA at room temperature for 12 hours, and then column chromatography is carried out to obtain the intermediate product molecule 4; 1 eq of PA and 1.2 eq of single BOC-ethylenediamine are stirred in 8 eq of TEA at room temperature for 12 hours, and then column chromatography is carried out to obtain the PA intermediate 7; 1 eq of ARV-771 is stirred in 2 eq of TEA with 2 eq of the intermediate product molecule 4 at room temperature for 24 hours, and then column chromatography is carried out to purify the purified product; 1 eq of the purified product, 1 eq of the PA intermediate 7 and 8 eq of TEA are dissolved in dichloromethane (DCM) and reacted at room temperature for 12 hours, and then high-performance liquid chromatography is carried out to obtain the product molecule.

3. The use of the chimera of claim 1 in the preparation of a drug for selective degradation of BRD4 in MCF-7 cells.

4. The use of the chimera of claim 1 in the preparation of an anti-tumor drug.

5. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The near-infrared light-activated protein-targeted degradation chimera of claim 1.

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