Near-infrared light activated tandem PROTAC prodrug responsive to tumor marker as well as preparation method and application thereof

Abstract

The invention discloses a tumor marker responsive near-infrared light activated tandem PROTAC prodrug as well as a preparation method and application thereof, and belongs to the technical field of protein targeted degradation, the structural formula is as follows: # imgabs0 #, the invention provides a new way, the in-vivo extratumoral risk of PROTAC can be reduced to the greatest extent, and an important step is made towards accurate PROTAC treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein targeted degradation, and specifically relates to a tumor biomarker-driven near-infrared photoprotein hydrolysis tandemly activated PROTAC prodrug, and a preparation method and application thereof. Background Art

[0002] Proteolysis-targeting chimeras (PROTACs) are an emerging anti-cancer modality that has entered late-stage clinical trials. Their working mechanism differs from traditional drugs, which induce degradation of protein targets rather than inhibition. This mode of action of PROTACs stems from their unique bispecific structure, which contains a target protein ligand of interest and an E3 ubiquitin ligase ligand. Therefore, PROTACs can degrade oncoproteins through the ubiquitin-proteasome system, thereby providing event-driven pharmacology. Although many difficult-to-degrade tumor-associated proteins, such as bromodomain and extra-terminal (BET) family proteins, Bruton's tyrosine kinase (BTK), and Kirsten rat sarcoma viral oncogene homolog (KRAS) proteins, have been shown to be accessible via the PROTAC approach, their therapeutic application is limited by potential systemic toxicity issues caused by extra-tumor proteolysis. Therefore, minimizing PROTAC activity in non-cancerous tissues is crucial for achieving safe and effective cancer therapy.

[0003] Prodrugs use chemical derivatization to inactivate the parent drug until stimulus-mediated restoration of its function, which is an effective approach to reduce side effects outside the tissue. Prodrug-type PROTACs, which become active from inactive upon reaching the tumor, have been proposed to overcome the problem of toxicity outside the tumor. Activation of PROTAC prodrugs can be achieved by endogenous or exogenous stimuli. For example, prodrugs that respond to internal tumor biomarker stimulation (such as enzymes and redox substances) or external stimuli (including light, X-rays, ultrasound, and bioorthogonal chemicals) have been developed. These PROTAC prodrugs have shown great promise in localizing proteolytic activity within the tumor area with the help of stimuli. However, this single activatable PROTAC prodrug design may still cause tumor problems because non-cancerous tissues also moderately express the corresponding biomarkers, and bond cleavage reactions triggered by physicochemical stimuli may occur nonspecifically in complex biological environments.

[0004] In view of the above limitations, here we report a tandemly activatable PROTAC prodrug (TAP) that is activated by integrating endogenous and exogenous stimuli. TAP consists of five consecutive units: PROTAC, singlet oxygen ( 1O2) cleavable linker, near-infrared (NIR) photosensitizer (PS), cathepsin B (CatB) cleavable moiety and tumor homing ligand. CatB is a biomarker enzyme that is often overexpressed in cancer cells. Initially, PROTAC is inactivated by chemical blocking, and the photosensitivity of PS is also quenched by CatB substrate modification. After systemic administration, TAP can actively target and enter cancer cells with the action of tumor-targeting ligand. The internalized TAP then encounters CatB, which cleaves its substrate moiety to release the PS photosensitivity. Thereafter, the tumor tissue is exposed to deep near-infrared light that penetrates the tissue, resulting in the production of PS. 1 O2, by cutting off 1 O2-sensitive ligation activates PROTAC. The restored PROTAC further recruits E3 ligase to degrade the BET family member BRD4, which synergizes with PS photodynamic therapy (PDT) to promote cancer cell apoptosis. Therefore, this biomarker enzyme and NIR light tandem controllable method allows PROTAC to accurately hydrolyze tumor proteins. Summary of the Invention

[0005] Technical problem to be solved: The present invention provides a tumor biomarker-driven near-infrared photoprotein hydrolysis tandem activation PROTAC prodrug and its preparation method and application, which is suitable for the research of activating target proteins and synergistic tumor treatment.

[0006] Technical solution: Tumor marker-responsive near-infrared light-activated tandem PROTAC prodrug, with the following structural formula:

[0007]

[0008] The preparation method of the tumor marker-responsive near-infrared light-activated tandem PROTAC prodrug comprises the following steps: (1) dissolving 2,2′-(propane-2,2-diylbis(sulfonamide))diethanol, 4-nitrophenyl chloroformate and triethylamine (TEA) in anhydrous tetrahydrofuran (THF) at a molar ratio of 1:(3-5):(4-7); stirring the mixture at room temperature for 8-12 hours, and then concentrating under reduced pressure; the resulting residue is purified by short silica gel column chromatography to obtain an esterified intermediate; and under nitrogen protection, ARV-771 is added to the mixture. , esterification intermediate, 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) in a molar ratio of 1: (2-4): (6-8) in anhydrous dichloromethane (DCM) are stirred at room temperature for 12-24 hours, and then the solvent is removed under reduced pressure, and the esterification intermediate compound 1 is obtained by column chromatography separation; (2) the compound 1,2-(2-azidoethoxy)ethylamine acetate prepared in step (1) and TEA in a molar ratio of 1: (0.5-2): (3-6) are dissolved in anhydrous N, N-dimethylformamide (DMF), stirred at room temperature 8 to 16 hours, then the solvent was removed under vacuum, and the resulting residue was purified by HPLC to obtain compound 3; (3) 2,3,3-trimethylindoline and 5-iodo-1-yne were dissolved in acetonitrile at a molar ratio of 1:(0.5 to 3), stirred at 75-85°C, and the resulting residue was diluted with DCM, washed with petroleum ether (PE), dried, filtered and concentrated by evaporation under reduced pressure to obtain compound 5; compound 5, (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carboxaldehyde and sodium acetate were dissolved in a molar ratio of 1:(1 to 2 .5):(0.5~2) in acetic anhydride solution at 70-80℃, stir evenly, remove the solvent under vacuum, dilute the residue with DCM, precipitate into excess ether, and filter to obtain compound 6; dissolve compound 6, resorcinol and K2CO3 in CH3CN at a molar ratio of 1:(1~3):(1.5~4.5), stir evenly at 45-55℃, and separate by column chromatography to obtain compound 7; (4) Val-Cit-PAB-OH, DBCO-NHS and DIPEA at a molar ratio of 1:(0.5-2): (3-6) are dissolved in anhydrous DMF, stirred evenly at room temperature, and quickly purified with a short silica gel column to obtain a crude product compound 9, compound 9, bis (4-nitrophenyl) carbonate and N, N-diisopropylethylamine (DIPEA) are dissolved in anhydrous DMF at a molar ratio of 1: (2-5): (2-4) at room temperature, and the residue is purified by HPLC to obtain an esterification intermediate, followed by adding the compound 7 prepared in step (3), the esterification intermediate, DMAP and DIPEA at a molar ratio of 1: (4-7): (1-3): (4-7) and dissolving them in anhydrous DMF at room temperature for 18-24h, and the obtained residue is purified by HPLC to obtain compound 11; (5) step (4) The prepared compound 11 and the small molecule c(RGDfk)-N3 were dissolved in anhydrous dimethyl sulfoxide (DMSO) at a molar ratio of 1:(0.5-2), and stirred at room temperature. The obtained residue was purified by HPLC to obtain compound 13; (6) CuSO4·5H2O, trihydroxypropyltriazolemethylamine (THPTA) and sodium ascorbate (Vc) were dissolved in water at a molar ratio of 1:(0.5-1):(1-3), the compound 3 prepared in step (2) and the compound 13 prepared in step (5) were dissolved in DMF at a molar ratio of 1:(1-3), added to the above aqueous solution, stirred at room temperature for 12-18 hours, and the obtained mixture was immediately purified by HPLC to obtain TAP.

[0009] In the above step (1), 2,2'-(propane-2,2-diylbis(sulfonamide))diethanol, 4-nitrophenyl chloroformate and TEA are added in a molar ratio of 1:4:6 and stirred at room temperature for 8 to 12 hours; then concentrated under reduced pressure to obtain a residue, which is purified by short silica gel column chromatography to obtain a crude product esterification intermediate; ARV-771, the esterification intermediate, DMAP and TEA are added in a molar ratio of 1:3:6 and stirred at room temperature for 18 hours; the compound 1,2,2-(2-azidoethoxy)ethylamine acetate and TEA in step (1) are added in a molar ratio of 1:1.5:4, dissolved in anhydrous DMF and stirred for 12 hours, and then the solvent is removed under vacuum. The resulting residue is purified by HPLC to obtain compound 3.

[0010] In the above step (3), 2,3,3-trimethylindoline and 5-iodine-1-yne are added in a molar ratio of 1:1.5 and stirred evenly in acetonitrile at 80°C; compound 5, (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carboxaldehyde and sodium acetate are added in a molar ratio of 1:1.5:1 to acetic anhydride solution and stirred evenly at 70-80°C; the solvent is removed under vacuum to obtain compound 6; compound 6, resorcinol and K2CO3 are added in a molar ratio of 1:2:2 to CH3CN, stirred evenly at 45-55°C, and separated by column chromatography to obtain compound 7.

[0011] Val-Cit-PAB-OH, DBCO-NHS and DIPEA were added to anhydrous DMF in a molar ratio of 1:1.2:4, stirred at room temperature, and then concentrated under reduced pressure. The mixture was purified by a short silica gel column to obtain a crude product, compound 9, which was directly used in the next step; compound 9 in step (4), bis(4-nitrophenyl) carbonate and DIPEA were added to anhydrous DMF in a molar ratio of 1:3:3 and dissolved at room temperature for 12 hours; the residue was purified by HPLC to obtain Esterification intermediate; then, compound 7 prepared in step (3), esterification intermediate, DMAP and DIPEA were added in a molar ratio of 1:6:2:6 and dissolved in anhydrous DMF at room temperature for 24 hours, and the obtained residue was purified by HPLC to obtain compound 11; compound 11 in step (4) and small molecule c(RGDfk)-N3 were added and dissolved in anhydrous DMSO in a molar ratio of 1:1.2, and stirred evenly at room temperature; the obtained residue was purified by HPLC to obtain compound 13.

[0012] CuSO4·5H2O, THPTA and Vc were added to water in a molar ratio of 1:1:2; compound 3 in step (2) and compound 13 in step (5) were added to DMF in a molar ratio of 1:2, and stirred at room temperature overnight; the resulting mixture was immediately purified by HPLC to obtain the product TAP.

[0013] The structural formula of compound 1 in step (1) is as follows:

[0014]

[0015] The structural formula of compound 3 is as follows:

[0016]

[0017] The structural formula of compound 5 is as follows:

[0018]

[0019] The structural formula of compound 6 is as follows:

[0020]

[0021] The structural formula of compound 7 is as follows:

[0022]

[0023] The structural formula of compound 9 is as follows:

[0024]

[0025] The structural formula of compound 11 is as follows:

[0026]

[0027] The structural formula of compound 13 is as follows:

[0028]

[0029] The use of the above-mentioned near-infrared light-activated tandem PROTAC prodrug in the preparation of BRD4 selective degradation drugs in U87MG cells.

[0030] Application of the above-mentioned near-infrared light-activated tandem PROTAC prodrug in the preparation of anti-tumor drugs.

[0031] An anti-tumor drug containing the above-mentioned near-infrared light-activated tandem PROTAC prodrug.

[0032] The preparation method of the above tumor biomarker-driven near-infrared light protein hydrolysis tandem activation PROTAC prodrug, the standard chemical synthesis process synthesizes the protein hydrolysis targeting chimera ARV-771 targeting BRD4 protein, singlet oxygen ( 1 O2) cleavable linker, near-infrared (NIR) photosensitizer (PS), cathepsin B (CatB) cleavable moiety and tumor homing ligand.

[0033]

[0034] Beneficial Effects: This study developed TAP as a PROTAC prodrug that can be sequentially unlocked by tumor biomarker enzymes and near-infrared light. Previous studies have focused on single-stimulus activation, either by internal cancer-associated markers or external physicochemical triggers. Recently, dual-activation PROTAC prodrugs targeting two endogenous enzymes have been reported to further improve the specificity of protein degradation in vitro. However, to our knowledge, this study represents the first example of a tandemly activated PROTAC prodrug that integrates activation by both endogenous and exogenous stimuli for precise spatiotemporal proteolysis in vivo. It was demonstrated that TAP can only respond to singlet oxygen generated by near-infrared light in the presence of CatB to release the active PROTAC. This binding to a tumor-targeting ligand limits protein degradation outside the tumor and further synergizes with PDT for safe and effective tumor inhibition. Since tandem activation has been incorporated into VHLE3 ligase ligands for PROTAC development, TAP design is also a general strategy. Collectively, this invention provides a novel approach to minimize the extratumoral risk of PROTACs in vivo and represents an important step toward precision PROTAC therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Synthetic approach for tandemly activated PROTAC prodrugs (TAPs) for tumor biomarker-driven near-infrared photoproteolysis.

[0036] Figure 2 Mass spectrometric characterization of compound 1 in the tandemly activated PROTAC prodrug.

[0037] Figure 3 Mass spectrometric characterization of compound 3 in the tandemly activated PROTAC prodrug.

[0038] Figure 4 This is the hydrogen spectrum characterization of compound 5 in the tandem activated PROTAC prodrug.

[0039] Figure 5 This is the hydrogen spectrum characterization of compound 7 in the tandem activated PROTAC prodrug.

[0040] Figure 6 Hydrogen spectrum and mass spectrometry characterization of compound 11 in the tandemly activated PROTAC prodrug.

[0041] Figure 7 Mass spectrometric characterization of compound 13 in the tandemly activated PROTAC prodrug.

[0042] Figure 8 Mass spectrometric characterization of the tandemly activated PROTAC prodrug TAP.

[0043] Figure 9Mass spectrometric characterization of NCP in PROTAC prodrug.

[0044] Figure 10 Mass spectrometric characterization of NUP in PROTAC prodrug.

[0045] Figure 11 HPLC chart of PROTAC release in solution after the above drug treatment.

[0046] Figure 12 This is the detection of singlet oxygen release in the solution after treatment with the above drugs.

[0047] Figure 13 BRD4 expression in U87MG cells before and after treatment with the above drugs.

[0048] Figure 14 This is the inhibition of U87MG cell proliferation after treatment with the above drugs.

[0049] Figure 15 The figure shows the growth inhibition of U87MG ectopic tumor after treatment with the above drugs.

[0050] Figure 16 Figure 2 shows the expression of BRD4 in U87MG ectopic tumor cells after treatment with the above drugs.

[0051] Figure 17 Schematic diagram of the method for preparing the protein targeted degradation compound of the present invention. DETAILED DESCRIPTION

[0052] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0053] Example 1. Synthesis of a tandemly activated PROTAC prodrug (TAP) driven by near-infrared photoproteolysis by tumor biomarkers

[0054] like Figure 1 As shown, the standard chemical synthesis process synthesizes the tandemly activated PROTAC prodrug TAP based on tumor biomarker-driven near-infrared photoprotein hydrolysis, the tandemly activated PROTAC prodrug compound NCP targeting BRD4 protein without singlet oxygen response, and the tandemly activated PROTAC prodrug compound NUP targeting BRD4 protein without CatB response. The specific method is: (1) Synthesis of compound 1:

[0055]

[0056] 2,2′-(Propane-2,2-diylbis(sulfonamide))diethanol, 4-nitrophenyl chloroformate and TEA were added in a molar ratio of 1:4:6 and stirred at room temperature for 10 hours. The residue was then concentrated under reduced pressure and purified by short silica gel column chromatography to obtain the esterification intermediate. ARV-771, the esterification intermediate, DMAP and TEA were added in a molar ratio of 1:3:6, stirred at room temperature for 18 hours, and concentrated under reduced pressure to obtain compound 1. The mass spectrometry results are shown in FIG. Figure 2 , calculated results [M+H] + 1373.4265, actual measurement 1373.4248, [M+Na] + 1395.4085, actual measurement 1395.4045.

[0057] (2) Synthesis of compound 3:

[0058]

[0059] Compound 1,2-(2-azidoethoxy)ethylamine acetate and TEA were added in a molar ratio of 1:1.5:4, dissolved in anhydrous DMF and stirred for 12 hours, then the solvent was removed under vacuum, and the resulting residue was purified by HPLC to obtain compound 3. The mass spectrometry results are shown in Figure 3 , calculated results [M+H] + 1364.4850, actual measurement 1364.4830, [M+Na] + 1386.4670, actual measurement 1386.4606.

[0060] (3) Synthesis of compound 7:

[0061]

[0062] 2,3,3-Trimethylindoline and 5-iodo-1-yne were added in a molar ratio of 1:1.5 and stirred in acetonitrile at 80°C for 12 hours. The product was used directly in the next step without further purification. Figure 4 , 1H NMR (400 MHz, DMSO-d6) δ 7.98-7.94 (m, 1H), 7.87-7.83 (m, 1H), 7.66-7.62 (m, 2H), 4.53-4.48 (m, 2H), 2.96 (t, J = 2.6 Hz, 1H), 2.85 (s, 3H), 2.42 (td, J = 7.2, 2.7 Hz, 2H), 2.08 (p, J = 7.3 Hz, 2H), 1.55 (s, 6H). Compound 5, (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carbaldehyde and sodium acetate were added to the acetic anhydride solution at a molar ratio of 1:1.5:1 and stirred at 75°C for 12 h. The solvent was removed under vacuum to obtain compound 6, which was used directly in the next reaction; compound 6, resorcinol and K2CO3 were added to CH3CN in a molar ratio of 1:2:2, and stirred at 50°C for 10 hours, followed by column chromatography to obtain compound 7. The hydrogen spectrum results are shown in Figure 5 , 1 H NMR (400MHz, DMSO-d6) δ7.90 (d, J=13.2Hz, 1H), 7.63 (d, J=1.0Hz, 1H), 7.41 (d, J=6.3Hz, 1H), 7. 34 (d, J=9.2Hz, 1H), 7.26 (t, J=7.7Hz, 1H), 7.08-6.98 (m, 2H), 6.42 (dd, J=9.2, 2.0Hz, 1H), 6.13 (dd, J=1.9, 1.0Hz, 1H), 5.81 (d, J=13.3Hz, 1H), 3.95 (t, J=7.3Hz, 2H), 2.95 (t, J=2.6Hz, 1H), 2. 62 (q, J=7.1, 6.7Hz, 3H), 2.30 (dt, J=7.2, 3.5Hz, 2H), 1.82 (dp, J=12.1, 6.6Hz, 4H), 1.63 (s, 5H).

[0063] (4) Synthesis of compound 13:

[0064]

[0065] Compound Val-Cit-PAB-OH, DBCO-NHS and DIPEA were added to anhydrous DMF in a molar ratio of 1:1.2:4, stirred at room temperature for 12 hours, and then the mixture was concentrated under reduced pressure and purified with a short silica gel column to obtain a crude product, compound 9, which was directly used in the next step; compound 9, bis(4-nitrophenyl) carbonate and DIPEA were added to anhydrous DMF in a molar ratio of 1:3:3 and dissolved at room temperature for 12 hours. The residue was purified by HPLC to obtain an esterification intermediate; then compound 7, esterification intermediate, DMAP and DIPEA were added to anhydrous DMF in a molar ratio of 1:6:2:6 and dissolved at room temperature for 24 hours. The resulting residue was purified by HPLC to obtain compound 11; the hydrogen spectrum results are shown in FIG. Figure 6 , 1 H NMR (400MHz, DMSO-d6) δ9.55 (s, 1H), 8.58 (s, 1H), 8.35-8.24 (m, 1H), 8.05 (s, 1H),7.84-7.71(m,6H)v7.71-7.45(m,11H),7.45-7.34(m,6H),7.34-7.29(m,2 H), 7.29-7.19 (m, 3H), 7.11-6.99 (m, 2H), 6.96 (s, 1H), 6.66 (dd, J=15.1, 4.7Hz , 2H), 6.02 (s, 1H), 5.29 (s, 3H), 4.87 (d, J = 14.2Hz, 1H), 4.48 (t, J = 7.4Hz, 2H), 4.39 (td, J=9.3, 4.4Hz, 2H), 4.03 (dd, J=7.5, 5.8Hz, 2H), 3.03-2.98 (m, 3H), 2 .77-2.60 (m, 7H), 2.38 (tq, J=5.5, 3.0Hz, 5H), 2.28 (t, J=7.4Hz, 2H), 2.15-2.0 1 (m, 5H), 1.99 (d, J = 7.0 Hz, 4H), 1.78 (s, 2H), 1.76 (s, 6H), 1.74 (d, J = 6.1 Hz, 1H), 1.59-1.36 (m, 6H), 1.24 (s, 7H), 0.81 (dd, J = 11.5, 6.9 Hz, 8H). Mass spectrometry results, calculated results [M] + 1128.52, actual measurement 1128.80. Compound 11 and compound 8 were dissolved in anhydrous DMSO at a molar ratio of 1:1.2 and stirred at room temperature for 12 hours. The resulting residue was purified by HPLC to obtain compound 13. The mass spectrometry results are shown in Figure 7 , calculation results [M] + 1961.9455, actual measurement 1961.9424.

[0066] (5) Synthesis of compound TAP:

[0067] CuSO4·5H2O, THPTA and Vc were added to water in a molar ratio of 1:1:2. Compound 3 in step (2) and compound 13 in step (5) were added to DMF in a molar ratio of 1:2 and stirred at room temperature overnight. The resulting mixture was immediately purified by HPLC to obtain the product TAP. The mass spectrometry results are shown in Figure 8 Calculated results [M+3H] 3+ 1109.4816, measured 1109.4822, calculated [M+2H] 2+ 1663.7189, actual measurement 1663.7150; NCP and NUP were obtained under the same conditions. NCP mass spectrum results are shown in Figure 9 Calculated results [M+3H] 3+ 1088.1670, measured 1088.1679; calculated [M+2H] 2+ 1631.7468, actual measurement 1631.7416; NUP mass spectrum results see Figure 10 Calculated results [M+3H] 3+ 1062.1115, measured 1062.1117; calculated [M+2H] 2+ 1592.6636, actual measurement 1592.6544.

[0068] Example 2. Release of tandemly activated protein-targeted degradation drugs by PROTAC in solution

[0069] TAP (5 μM) in PBS was irradiated with 670 nm (200 mW / cm 2 ) irradiated with light for 5 min, placed at room temperature for 24 h, and then analyzed by high performance liquid chromatography (mobile phase: A: CH3CN, B: H2O, 0.1% TFA; flow rate: 1 mL / min; elution gradient: 0-40 min, from 15% A to 85% A). The results are as follows Figure 11 As shown, ARV-771 was released at 22.3 min in the TAP plus CatB and light group, while no ARV-771 release was observed in the other control groups.

[0070] Example 3. Detection of Singlet Oxygen Release from Tandemly Activated PROTAC Prodrugs in Solution

[0071] To determine the singlet oxygen production capacity of TAP, SOSG was used as a singlet oxygen fluorescence indicator. The cells were pretreated with CatB (2 μg / mL) for 3 h, and then SOSG (2.5 μM) was added. The solution was illuminated with 670 nm light (200 mW / cm2 ) irradiate for 5 min, measure the fluorescence at 520 nm, and the results are as follows Figure 12 The fluorescence of TAP shown here is significantly enhanced after light treatment, indicating the generation of singlet oxygen.

[0072] Example 4. Application of tandem activated PROTAC prodrugs in targeting BRD4 degradation in cells

[0073] After adding different concentrations of compounds TAP, NCP and NUP to U87MG cells and incubating for 3 h, the 670 nm (200 mW / cm 2 ) irradiate with light for 5 min and then incubate for 24 h. Figure 13 As shown, Western blotting detected that BRD4 in the cells was significantly degraded only in the TAP illumination group, and the degradation effect increased with the increase of its concentration. The results showed that the compound TAP degraded the BRD4 protein in U87MG cells under light conditions.

[0074] Example 5. Inhibition of U87MG Cell Proliferation by Tandemly Activated PROTAC Prodrugs

[0075] After adding different ARV-771, TAP, NCP and NUP to U87MG cells and incubating for 3 h, the irradiation was performed at 670 nm (200 mW / cm 2 ) After 5 min of light irradiation and 45 h of incubation, Figure 14 As shown, the proliferation rate of cells detected by CCK-8 was significantly downregulated with the increase of incubation concentration, and the effect of TAP illumination group was the most obvious, verifying that the protein degradation of targeted BRD4 after TAP illumination can simultaneously cooperate with photodynamic therapy to inhibit the proliferation of cancer cells.

[0076] Example 6. Application of tandem activated PROTAC prodrugs in tumor treatment

[0077] 1×10 7 MCF-7 cells. When the tumor size reaches about 100 mm 3 The same dose of PROTAC (5 mg / kg ARV-771) was injected intravenously every 2 days. 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. Mice were sacrificed on day 14, and tumor tissues and major normal tissues were collected for analysis. The tumor inhibition rate was as follows: Figure 15 As shown in Figure 2, the tumor growth in the TAP plus light group was the slowest, so the near-infrared light-activated protein targeted degradation chimera can inhibit tumor growth. Figure 16As shown, histone degradation occurred after treatment with ARV-771 alone, but the histone degradation effect of TAP plus light was the most obvious, verifying that the chimera can achieve targeted BRD4 protein degradation in vivo.

[0078] The above specific implementation manner does not limit the technical solution of the present invention in any form. Any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A tumor marker-responsive near-infrared light-activated tandem PROTAC prodrug, characterized in that: The structural formula is as follows:

2. The method for preparing the tumor marker-responsive near-infrared light-activated tandem PROTAC prodrug according to claim 1, characterized in that: Here are the steps: (1) 2,2′-(propane-2,2-diylbis(sulfonamide))diethanol, 4-nitrophenyl chloroformate and triethylamine (TEA) were dissolved in anhydrous tetrahydrofuran (THF) at a molar ratio of 1:(3-5):(4-7); the mixture was stirred at room temperature for 8-12 hours and then concentrated under reduced pressure. The resulting residue was purified by short silica gel column chromatography to obtain an esterification intermediate; under nitrogen protection, ARV-771, the esterification intermediate, 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) were mixed at a molar ratio of 1:(2-4): (6-8) The reaction mixture was stirred in anhydrous dichloromethane (DCM) at room temperature for 12-24 h, and then the solvent was removed under reduced pressure. The esterified intermediate compound 1 was separated by column chromatography; (2) The compound 1,2-(2-azidoethoxy)ethylamine acetate prepared in step (1) and TEA were dissolved in anhydrous N,N-dimethylformamide (DMF) at a molar ratio of 1:(0.5-2):(3-6), stirred at room temperature for 8-16 hours, and then the solvent was removed under vacuum. The resulting residue was purified by HPLC to obtain compound 3; (3) 2,3,3-Trimethylindoline and 5-iodo-1-yne were dissolved in acetonitrile at a molar ratio of 1: (0.5-3), stirred at 75-85°C, and the resulting residue was diluted with DCM, washed with petroleum ether (PE), dried, filtered, and concentrated by evaporation under reduced pressure to obtain compound 5; compound 5, (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carboxaldehyde, and sodium acetate were dissolved in a molar ratio of 1: (1-2.5): (0.5-2) in acetic anhydride solution at 70-80°C, stirred evenly, the solvent was removed under vacuum, the residue was diluted with DCM, precipitated into excess ether, and filtered to obtain compound 6; compound 6, resorcinol and K2CO3 were dissolved in CH3CN at a molar ratio of 1: (1-3): (1.5-4.5), and stirred evenly at 45-55°C, and separated by column chromatography to obtain compound 7; (4) Val-Cit-PAB-OH, DBCO-NHS and DIPEA were dissolved in anhydrous DMF at a molar ratio of 1: (0.5-2): (3-6), stirred at room temperature, and quickly purified by a short silica gel column to obtain a crude product compound 9. Compound 9, bis(4-nitrophenyl) carbonate and N,N-diisopropylethylamine (DIPEA) were dissolved in anhydrous DMF at a molar ratio of 1: (2-5): (2-4) at room temperature, and the residue was purified by HPLC to obtain an esterification intermediate. Compound 7 prepared in step (3), the esterification intermediate, DMAP and DIPEA were then added at a molar ratio of 1: (4-7): (1-3): (4-7) was dissolved in anhydrous DMF at room temperature for 18-24 h, and the resulting residue was purified by HPLC to give compound 11; (5) Compound 11 and the small molecule c(RGDfk)-N3 prepared in step (4) were dissolved in anhydrous dimethyl sulfoxide (DMSO) at a molar ratio of 1:(0.5-2), and stirred at room temperature. The resulting residue was purified by HPLC to obtain compound 13; (6) CuSO4·5H2O, trihydroxypropyltriazolemethylamine (THPTA) and sodium ascorbate (Vc) were mixed in a molar ratio of 1: (0.5-1): (1-3) was dissolved in water, and the compound 3 prepared in step (2) and the compound 13 prepared in step (5) were dissolved in DMF at a molar ratio of 1: (1-3), added to the above aqueous solution, and stirred at room temperature for 12-18 hours. The resulting mixture was immediately purified by HPLC to obtain TAP.

3. The method for preparing a near-infrared light-activated tandem PROTAC prodrug according to claim 2, characterized in that: In the step (1), 2,2′-(propane-2,-diylbis(sulfonamide))diethanol, 4-nitrophenyl chloroformate and TEA are added in a molar ratio of 1:4:6 and stirred at room temperature for 8 to 12 hours; then the residue is concentrated under reduced pressure and purified by short silica gel column chromatography to obtain a crude product esterification intermediate; ARV-771, the esterification intermediate, DMAP and TEA are added in a molar ratio of 1:3:6 and stirred at room temperature for 18 hours; the compound 1,2,2-(2-azidoethoxy)ethylamine acetate and TEA in step (1) are added in a molar ratio of 1:1.5:4, dissolved in anhydrous DMF and stirred for 12 hours, then the solvent is removed under vacuum, and the resulting residue is purified by HPLC to obtain compound 3.

4. The method for preparing a near-infrared light-activated tandem PROTAC prodrug according to claim 2, wherein: In the step (3), 2,3,3-trimethylindoline and 5-iodine-1-yne are added in a molar ratio of 1:1.5 and stirred evenly in acetonitrile at 80°C; compound 5, (E)-2-chloro-3-(hydroxymethyl)cyclohex-1-ene-1-carboxaldehyde and sodium acetate are added in a molar ratio of 1:1.5:1 to acetic anhydride solution and stirred evenly at 70-80°C; the solvent is removed under vacuum to obtain compound 6; compound 6, resorcinol and K2CO3 are added in a molar ratio of 1:2:2 to CH3CN, stirred evenly at 45-55°C, and separated by column chromatography to obtain compound 7.

5. The method for preparing a near-infrared light-activated tandem PROTAC prodrug according to claim 2, wherein: Val-Cit-PAB-OH, DBCO-NHS and DIPEA were added to anhydrous DMF in a molar ratio of 1:1.2:4, stirred at room temperature, and then concentrated under reduced pressure. The mixture was purified by a short silica gel column to obtain a crude product, compound 9, which was directly used in the next step; compound 9 in step (4), bis(4-nitrophenyl) carbonate and DIPEA were added to anhydrous DMF in a molar ratio of 1:3:3 and dissolved at room temperature for 12 hours; the residue was purified by HPLC to obtain Esterification intermediate; then, compound 7 prepared in step (3), esterification intermediate, DMAP and DIPEA were added in a molar ratio of 1:6:2:6 and dissolved in anhydrous DMF at room temperature for 24 hours, and the obtained residue was purified by HPLC to obtain compound 11; compound 11 and small molecule c(RGDfk)-N3 in step (4) were added and dissolved in anhydrous DMSO in a molar ratio of 1:1.2, and stirred evenly at room temperature; the obtained residue was purified by HPLC to obtain compound 13.

6. The method for preparing a near-infrared light-activated tandem PROTAC prodrug according to claim 2, wherein: CuSO4·5H2O, THPTA and Vc were added to water in a molar ratio of 1:1:2; compound 3 in step (2) and compound 13 in step (5) were added to DMF in a molar ratio of 1:2, and stirred at room temperature overnight; the resulting mixture was immediately purified by HPLC to obtain the product TAP.

7. The method for preparing a near-infrared light-activated tandem PROTAC prodrug according to claim 2, wherein: The structural formula of compound 1 in step (1) is as follows: The structural formula of compound 3 is as follows: The structural formula of compound 5 is as follows: The structural formula of compound 6 is as follows: The structural formula of compound 7 is as follows: The structural formula of compound 9 is as follows: The structural formula of compound 11 is as follows: The structural formula of compound 13 is as follows:

8. Use of the near-infrared light-activated tandem PROTAC prodrug according to claim 1 in the preparation of a drug for selective degradation of BRD4 in U87MG cells.

9. Use of the near-infrared light-activated tandem PROTAC prodrug according to claim 1 in the preparation of anti-tumor drugs.

10. An anti-tumor drug, characterized in that: Contains the near-infrared light-activated tandem PROTAC prodrug according to claim 1.

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