A phthalocyanine-based photo-PROTAC drug, its preparation method and application

Abstract

The application discloses a phthalocyanine-based photo-PROTAC drug and a preparation method and application thereof, and mainly adopts a chemical synthesis method of amino and carboxyl coupling, takes photosensitizer ZnPc and BRD4 ligand JQ1 (a BRD4 inhibitor) as a structural main body, selects a polyethylene glycol (PEG) chain with different lengths as a linker of ZnPc and JQ1, and constructs a photo-PROTAC drug with BRD4 as a target point. The raw material of the application has a wide source, and the preparation method is simple. The drug utilizes the expression of BRD4 in bladder cancer tumor tissues, and realizes efficient degradation of PROTAC independent of E3 ubiquitinase by combining selective tumor site light. Meanwhile, BRD4 degradation destroys the antioxidant and hypoxic inhibition barrier of PDT, and realizes synergistic effect of PDT and PROTAC.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a phthalocyanine-based photo-PROTAC drug, its preparation method, and its application. Background Technology

[0002] It is well known that the occurrence and development of cancer depend on the overexpression of proteins in multiple signaling pathways. In the past few decades, targeted therapy has become one of the most successful methods for cancer treatment. Among them, protein hydrolysis-targeted chimeric (PROTAC) technology has become a promising and attractive approach in cancer targeted therapy due to its advantage of selectively degrading proteins rather than directly inhibiting protein activity. A PROTAC molecule is a heterobifunctional molecule consisting of a ligand that recruits the target protein and another that recruits the E3 ubiquitin ligase. The two ligands are interconnected via a linker. This chemically induced connection between the POI and the E3 ligase leads to ubiquitination of the ubiquitin-proteasome system (UPS) and degradation of the POI. Compared with traditional "occupation-driven" small molecule inhibitors, PROTAC degraders have the advantages of low toxicity, low drug resistance, and low dosage.

[0003] Bromodomain protein 4 (BRD4) is a member of the bromodomain and superterminal domain (BET) family. It recognizes acetylated histones and plays an important role in transcription, replication, and DNA repair. Overexpression or aberrant activation of BRD4 is closely related to the development and progression of many tumors. In normal tissues, tightly regulated expression of BRD4 is crucial for maintaining cell cycle progression. Currently, a commercially available PROTAC degrader for BRD4 is dBET1. This degrader uses a thalidomide derivative as an E3 ligase recruiting ligand to hijack CRBN to degrade BRD4 protein. This degradation mechanism targets all BRD4 proteins in vivo and lacks selectivity for tumors and adjacent normal tissues.

[0004] PROTAC technology offers many potential advantages, but it also faces significant challenges in cancer treatment. PROTAC activity depends on its associated E3, whose expression may vary across different cell types, tissues, or species, and off-target effects with its E3 ligand are possible. Furthermore, precisely controlling the function of PROTACs in tumor regions within the body remains a challenge. Summary of the Invention

[0005] Purpose of the invention: To address the potential off-target effects of PROTAC drugs in tumor treatment, this invention designs a phthalocyanine-based photo-PROTAC drug. This drug directly degrades target proteins by generating reactive oxygen species through photoactivation, independent of E3 ubiquitinase. Given that BRD4 levels in most tumor tissues are higher than in adjacent normal tissues, this invention further enhances the selective degradation of tumor target proteins by Photo-PROTAC drugs using a photo-assisted targeting activation strategy with selective illumination of tumor sites. This approach holds promise for achieving safe and efficient tumor treatment.

[0006] The present invention also provides a method for preparing and applying the phthalocyanine-based photo-PROTAC drug.

[0007] Technical solution: To achieve the above objectives, the present invention provides a phthalocyanine-based photo-PROTAC drug, the structural formula of which is shown in Formula I or Formula II:

[0008] ,

[0009] Where n is any integer from 1 to 10.

[0010] Preferably, n is any integer of 1, 2, 3, 4 or 6.

[0011] The method for preparing the phthalocyanine-based photo-PROTAC compound of the present invention includes the following steps:

[0012] (1) Synthesis of intermediate 1:

[0013] 4-Nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate were uniformly mixed and reacted in an organic solvent to obtain intermediate 1;

[0014] (2) Synthesis of compound 2 (ZnPc): intermediate 1, phthalonitrile and anhydrous zinc acetate were dissolved in an organic solvent, and DBU was added under the protection of an inert gas to react and obtain ZnPc.

[0015] (3) Synthesis of compound 4 (ZnPc-JQ1): Compound 2, (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (JQ1) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were dissolved in an organic solvent and stirred. Then N,N-diisopropylethylamine (DIPEA) was added and reacted at room temperature to obtain compound 4.

[0016] (4) Synthesis of compound 5a (ZnPc-O1-JQ1): Compound 2, 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid and HATU were dissolved in an organic solvent. After stirring, DIPEA was added and the reaction was carried out at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added and stirred to remove ammonia BOC to obtain compound 3a. Compound 3a was reacted with JQ1 at room temperature to obtain compound 5a.

[0017] (5) Synthesis of compound 5b (ZnPc-O2-JQ1): Compound 2, N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid and HATU were dissolved in an organic solvent. After stirring, DIPEA was added and the reaction was carried out at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added and stirred to remove the ammonia BOC to obtain compound 3b. Compound 3b reacted with JQ1 to obtain compound 5b.

[0018] (6) Synthesis of compound 5c (ZnPc-O3-JQ1): Compound 2, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecane-17-acid and HATU were dissolved in an organic solvent. After stirring, DIPEA was added and the reaction was carried out at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added and stirred to remove the ammonia BOC, yielding compound 3c. 3c reacted with JQ1 to obtain compound 5c.

[0019] (7) Synthesis of compound 5d (ZnPc-O4-JQ1): Compounds 2, 5,8,11,14-tetraoxa-2-azaheptadecanoic acid 1-tert-butyl ester and HATU were dissolved in an organic solvent. After stirring, DIPEA was added and the reaction was carried out at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added and stirred to remove ammonia BOC to obtain compound 3d. 3d reacted with JQ1 to obtain compound 5d.

[0020] (8) Synthesis of compound 5e (ZnPc-O6-JQ1): Compound 2, 21-(Boc-amino)-4,7,10,13,16,19-hexaoxadocosuccinic acid and HATU were dissolved in an organic solvent. After stirring and reacting, DIPEA was added and reacted at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added and stirred to remove the ammonia BOC, giving compound 3e. 3e reacted with JQ1 to give compound 5e.

[0021] The reaction formula is shown below:

[0022] .

[0023] In step (1), the molar ratio of 4-nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate is 1:1.2~1.5:1.55~2, the solvent volume is 25~30 mL, the reaction temperature is 50~60 ℃, and the stirring reaction time is 6~7 h.

[0024] Preferably, in step (1), the molar ratio of 4-nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate is 1:1.3:1.55, the solvent volume is 25 mL, the reaction temperature is 50 °C, and the stirring reaction time is 6 h.

[0025] In step (2), the molar ratio of intermediate 1, phthalonitrile, and anhydrous zinc acetate is 1:8.5~9:3~4, the volume of the solvent is 25~30 mL, the volume of the catalyst DBU is 400~50 μL, the reaction temperature is 130~140 ℃, and the stirring reaction time is 9~10 h.

[0026] Preferably, in step (2), the molar ratio of phthalonitrile to anhydrous zinc acetate is 1:8.5:3.2, the volume of the solvent is 25 mL, the volume of the catalyst DBU is 400 μL, the reaction temperature is 140 ℃, and the stirring reaction time is 9 h.

[0027] In step (3), the molar ratio of compound 2, JQ1 and HATU is 1:1.0~1.3:1.8~3, the volume of solvent is 15~20 mL, the volume of DIPEA is 80~100 μL, the reaction temperature is room temperature, and the stirring reaction time is 4~5 h.

[0028] Preferably, in step (3), the molar ratio of compound 2, JQ1 and HATU is 1:1.1:1.92~2, the volume of solvent is 15 mL, the volume of DIPEA is 80~100 μL, the reaction temperature is room temperature, and the stirring reaction time is 4 h.

[0029] In step (4), the molar ratio of compound 2,3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid and HATU is 1:1.0~1.3:2~3, the volume of solvent is 20~25 mL, the volume of DIPEA is 150~180 μL, the reaction temperature is room temperature, and the stirring reaction time is 6~7 h.

[0030] Preferably, in step (4), the molar ratio of compound 2, 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid and HATU is 1:1.2:2, the volume of the solvent is 20 mL, the volume of DIPEA is 150 μL, the reaction temperature is room temperature, and the stirring reaction time is 6 h.

[0031] The reaction conditions described in steps (5), (6), (7) and (8) are the same as those in step (4).

[0032] The application of the phthalocyanine-based photo-PROTAC drug described in this invention in the preparation of anticancer drugs.

[0033] The application of the phthalocyanine-based photo-PROTAC drug as a photosensitizer in the preparation of anticancer drugs.

[0034] Furthermore, the photo-PROTAC compound (ZnPc-O3-JQ1) described in this invention has excellent photosensitizing activity, good physiological compatibility and selectivity, and is suitable as a photosensitizer for photodynamic therapy of tumors.

[0035] This invention utilizes a phthalocyanine-based photo-PROTAC compound synthesized primarily through a chemical synthesis method involving the coupling of amino and carboxyl groups. The raw materials are widely available, and the preparation method is simple. This invention uses the photosensitizer ZnPc and BRD4 ligand JQ1 (a BRD4 inhibitor) as the main structural components, selecting polyethylene glycol (PEG) chains of varying lengths as linkers for ZnPc and JQ1 to construct photo-PROTAC compounds targeting BRD4. The optimal photosensitizer, ZnPc-O3-JQ1, was selected. ZnPc-O3-JQ1 exhibits excellent BRD4 binding and phototriggered degradation activity. BRD4 is a transcription factor that directly or indirectly regulates the expression and activity of more than a variety of proteins, including hypoxia-inducible factor-1α (HIF-1α). Its degradation can downregulate HIF-1α levels in cells, thereby reducing tumor resistance to treatment induced by PDT-induced hypoxia. To cope with the oxidative stress caused by reactive oxygen species generated during photodynamic therapy (PDT), cells synthesize more glutathione, enhancing their antioxidant capacity. HIF-1α can negatively regulate glutathione (GSH) synthesis by downregulating GCL (a key enzyme in the de novo synthesis of glutathione), thereby disrupting the antioxidant defense capabilities of tumor cells. Therefore, ZnPc-O3-JQ1, while achieving efficient photo-PROTAC degradation of BRD4, utilizes its downregulation of HIF-1α and its downstream effects to reduce tumor hypoxia and antioxidant resistance to PDT therapy, thus achieving a synergistic effect between PDT and PROTAC.

[0036] This invention designs and synthesizes a series of photo-PROTAC molecules ZnPc-O targeting BRD4. n-JQ1 (n = 0, 1, 2, 3, 4, 6). Utilizing the BRD4 expression characteristic of bladder cancer tumor tissue, combined with selective tumor site phototherapy, this invention achieves highly efficient PROTAC degradation independent of E3 ubiquitinase. Simultaneously, this invention can disrupt the antioxidant and hypoxia inhibition barriers of PDT by degrading BRD4, achieving a synergistic effect between PDT and PROTAC. This invention utilizes a photoactivated photo-PROTAC strategy to precisely degrade BRD4 protein in tumor tissue. BRD4 degradation disrupts the antioxidant and hypoxia inhibition barriers of PDT, resulting in a synergistic effect between photodynamic therapy and PROTAC. The series of photo-PROTAC drugs prepared in this invention are all prepared for the first time. This invention specifically binds the photosensitizer ZnPc in PDT to the BRD4 small molecule inhibitor JQ1, which is activated by photoirradiation, generating reactive oxygen species near the BRD4 protein, thereby degrading it. This degradation method does not require the participation of E3 ligase, achieving selective degradation of the protein through ROS in both normal and tumor tissues.

[0037] Compared to dBET1, a commercially available PROTAC degrader for BRD4, ZnPc-O3-JQ1 exhibits superior in vivo antitumor activity. This invention compares ZnPc-JQ1 and ZnPc-O3-JQ1... n The photosensitive tumor cell killing activity of JQ1 was derived, and the photosensitive tumor killing IC50 was obtained. 50 Concentration from 1351 nM (IC50) 50 The concentration of ZnPc-JQ1 was significantly reduced to 41 nM (IC). 50 Below the ZnPc-O1-JQ1 level, the activity is increased by more than 33 times. With the extension of the linker, ZnPc-O... n -JQ1's IC 50 The concentration gradually decreased and reached a basic stability at ZnPc-O3-JQ1, with its IC50 value decreasing. 50 The concentration was 22 nM. Furthermore, ZnPc-O3-JQ1 exhibited ideal photosensitizing activity against tumor cells and a high PI (phototoxicity index: the IC50 value of a drug under light irradiation). 50 IC under dark conditions 50 The ratio of PDT to PROTAC is a characteristic that allows it to bind to BRD4 and, after photodynamic therapy, generate ROS to effectively degrade BRD4. PDT and PROTAC mutually promote each other, thereby damaging cancer cells. In vivo, ZnPc-O3-JQ1 exhibits the best inhibitory effect on tumor growth.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0039] (1) The present invention uses a photo-PROTAC strategy system to effectively combine photodynamic therapy (PDT) with targeted protein degradation to improve the treatment effect of bladder cancer.

[0040] (2) This invention selectively irradiates tumor sites to stimulate photosensitizers to generate reactive oxygen species that degrade target proteins. This strategy does not depend on the expression level and activity of E3 ubiquitinase, opening up a new path for the development of safe and efficient PROTAC drugs.

[0041] (3) The series of photo-PROTAC drugs prepared in this invention specifically bind the photosensitizer ZnPc in PDT to the BRD4 small molecule inhibitor JQ1. Upon activation by light irradiation, reactive oxygen species are generated near the BRD4 protein, thereby degrading it. This degradation method does not require the participation of E3 ligase and achieves selective degradation of the protein through ROS in both normal and tumor tissues. At the same time, BRD4 degradation disrupts the antioxidant and hypoxia inhibition barrier of PDT, achieving synergistic effects between PDT and PROTAC.

[0042] (4) The drug prepared by this invention, such as ZnPc-O3-JQ1, has a PI value greater than 9091, which is much higher than the approved phthalocyanine photosensitizing drug Photosens® (PI = 725), and has excellent photosensitizing and tumor cell killing activity and high PI value characteristics.

[0043] (5) The drug prepared by the present invention has a significant tumor growth inhibition effect in vivo. Compared with the commercially available PROTAC degrader dBET1 that degrades BRD4, such as ZnPc-O3-JQ1, it has better in vivo antitumor activity.

[0044] (6) The preparation method of the present invention is simple and efficient, the raw materials are readily available, and it has good industrial production value. Attached Figure Description

[0045] Figure 1 The UV-Vis absorption spectra of the six photo-PROTAC drugs of this invention in aqueous solution;

[0046] Figure 2 Phototoxic IC50 of six photo-PROTAC drugs in T24 cells 50 picture;

[0047] Figure 3 Dark toxicity IC50 of six photo-PROTAC drugs in T24 cells 50 picture;

[0048] Figure 4 PI plots of six photo-PROTAC drugs in T24 cells;

[0049] Figure 5 The relative expression levels of BRD4 in T24 cells after ZnPc-JQ1, ZnPc-O3-JQ1, and dBET1 were treated without light.

[0050] Figure 6 The relative expression levels of BRD4 in T24 cells after treatment with ZnPc-JQ1, ZnPc-O3-JQ1, and dBET1 following light exposure are shown.

[0051] Figure 7 Figure 1 shows the reactive oxygen species production of ZnPc-JQ1 and ZnPc-O3-JQ1 in T24 cells;

[0052] Figure 8 The image shows the Western blotting and quantitative analysis of HIF-1α level changes induced by ZnPc-JQ1 and ZnPc-O3-JQ1 treatments after light irradiation under hypoxic conditions.

[0053] Figure 9 Flow cytometry analysis and intensity comparison of GSH level changes after treatment with ZnPc-JQ1 and ZnPc-O3-JQ1.

[0054] Figure 10 WB and quantitative analysis graphs of GLC level changes after treatment with ZnPc-JQ1 and ZnPc-O3-JQ1.

[0055] Figure 11 This is a schematic diagram comparing the ability of PBS, dBET1, Cis-platin, ZnPc-JQ1+L, and ZnPc-O3-JQ1+L groups to inhibit tumor growth in subcutaneous xenograft tumor models.

[0056] In each figure, ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0057] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0058] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0059] (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (JQ1), CAS No. 202592-23-2.

[0060] 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid, CAS No. 1260092-44-1.

[0061] N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid, CAS No. 1365655-91-9.

[0062] 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecane-17-acid, CAS No. 1347750-75-7.

[0063] 5,8,11,14-Tetraoxa-2-azaheptadecanoic acid 1-tert-butyl ester, CAS No. 756525-91-4.

[0064] 21-(Boc-amino)-4,7,10,13,16,19-hexaoxadocaoconic acid, CAS No. 882847-13-4.

[0065] The commercially available PROTAC drug dBET1 targets BRD4 as its protein and CRBN as its E3 ubiquitinase. Bidex Pharmaceuticals, CAS No.: 1799711-21-9.

[0066] Cis-platin, Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 15663-27-1.

[0067] Commercial phthalocyanine photosensitizer Photosens ® CAS number 122170-90-5.

[0068] Reference for GSH probe synthesis: Q. Zhang, D. Yu, S. Ding, G. Feng, A low dose, highly selective and sensitive colorimetric and fluorescent probe for biothiols and its application in bioimaging, Chem. Commun. 50 (2014) 14002-14005. https: / / doi.org / 10.1039 / c4cc04978k.

[0069] Example 1

[0070] 4-Nitrophthalonitrile (0.5 g, 2.89 mmol), 4-(boc-amino)phenol (0.78 g, 3.73 mmol), and potassium carbonate (0.62 g, 4.49 mmol) were dissolved in 25 mL of N,N-dimethylformamide. Under nitrogen protection, the mixture was heated to 50 °C and stirred for 6 h. After the reaction was complete, the solution was poured into 200 mL of ice water and allowed to stand until a solid precipitated. The solution was filtered, and the filter cake was washed with distilled water until the filtrate was colorless. The filter cake was then dried in a vacuum drying oven to obtain a white solid intermediate 1.

[0071] Intermediate 1 (0.4 g, 1.19 mmol), phthalonitrile (1.3 g, 10.15 mmol), and anhydrous zinc acetate (0.7 g, 3.82 mmol) were added to a three-necked flask, along with 25 mL of n-pentanol as solvent. Under nitrogen protection, the mixture was stirred and heated to 80 °C, refluxed for 1 h, and then 400 μL of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) was added via syringe. The mixture was then heated to 140 °C and refluxed for 9 h. After the reaction was complete, the reaction solution was cooled to room temperature, and n-pentanol was removed by vacuum distillation. The remaining solid was dissolved in 40 mL of dichloromethane (DCM) by sonication, filtered, and the filtrate was collected, mixed, and separated by silica gel column chromatography (petroleum ether / ethyl acetate (v / v, 1:1)) to obtain the crude product. Then, 10 mL of dichloromethane and 4 mL of trifluoroacetic acid (TFA) were added, and the mixture was stirred for 8 h to remove ammonia and BOC. The solvent was then removed by rotary evaporation under reduced pressure. Next, 30 mL of methanol and 5 mL of 10% sodium hydroxide were added, and the mixture was heated to 130 °C and refluxed for 4 h. After centrifugation, the lower solid layer was collected. Finally, 40 mL of methanol and 2 mL of concentrated hydrochloric acid (36%–38%) were added, and the mixture was heated to 100 °C and refluxed for 4 h (the methanol and concentrated hydrochloric acid reflux process was repeated twice). After centrifugation, the lower solid layer was collected and dried in a vacuum drying oven to obtain the blue product compound 2 (ZnPc) (150 mg, yield 18.36%). MALDI-TOP (m / z) calculated for C 38 H 21 N9OZn:685.12, found[M+H] + : 685.03. 1 H NMR (400 MHz, DMSO) δ 9.40 – 9.09 (m, 7H), 8.72 (s, 1H), 8.28 – 8.09 (m, 6H), 7.92 – 7.81 (m, 2H), 7.68 – 7.52 (m, 4H).

[0072] Example 2

[0073] Compound 2 (380 mg, 0.12 mmol), (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetic acid (JQ1 (52 mg, 0.13 mmol)) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (90 mg, 0.23 mmol) were weighed into a round-bottom flask, dissolved in 15 mL of dimethyl sulfoxide (DMSO), stirred for 10 min, and then 80 μL of N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at room temperature for 4 h, and the reaction process was monitored by TLC. After the reaction was complete, the reaction solution was poured into 100 mL of distilled water and extracted three times with ethyl acetate (30 mL × 3) and 10 mL of saturated brine. The extracted organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (dichloromethane / methanol (v / v, 20:1)) to give the blue product compound 4 (ZnPc-JQ1) (30 mg, yield 23.96%). MALDI-TOP(m / z) calculated for C 57 H 36 ClN 13 O2SZn: 1067.89, found[M+H] + : 1067.18. 1 H NMR(400 MHz, DMSO) δ 10.62 (s, 1H), 9.19 – 8.80 (m, 7H), 8.38 (s, 1H), 8.24 –7.90 (m, 8H), 7.71 (d, J = 7.7 Hz, 1H), 7.54 (dd, J = 15.0, 9.8 Hz, 6H), 4.73(s, 1H), 3.65 (d, J = 6.8 Hz, 2H), 2.66 (s, 3H), 2.44 (s, 3H), 1.69 (s, 3H).

[0074] Example 3

[0075] Compound 2 (150 mg, 0.22 mmol), 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid (61.3 mg, 0.26 mmol), and HATU (166 mg, 0.44 mmol) were dissolved in 20 mL of DMSO. After stirring for 10 min, 150 μL of DIPEA was added, and the mixture was stirred at room temperature for 6 h. The reaction process was monitored by TLC. After the reaction was completed, the reaction solution was poured into 100 mL of distilled water and extracted three times with ethyl acetate (50 mL × 3) and 10 mL of saturated brine. The extracted organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (dichloromethane / methanol (v / v, 100:3)). The purified compound was dissolved in DCM (5 mL) and TFA (2 mL) and stirred for 6 h to remove ammonia and BOC. After removing the solvent by evaporation under reduced pressure, the compound was washed with water and methanol, centrifuged, and the lower solid layer was dried in a vacuum drying oven to obtain the blue solid product compound 3a.

[0076] Using the same synthesis and purification methods as compound 4, compound 3a (80 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), 80 μL of DIPEA, and JQ1 (44 mg, 0.11 mmol) were reacted to give the blue solid product compound 5a (ZnPc-O1-JQ1) (28 mg, yield 23.67%). MALDI-TOP (m / z) calculated for C 62 H 45 ClN 14 O4SZn: 1183.03, found[M+H] + : 1183.24. 1H NMR (400 MHz, DMSO) δ 9.22 – 9.03 (m, 5H), 8.95 (d, J = 6.9 Hz, 2H), 8.39 (d, J = 17.1 Hz, 2H), 8.22 – 8.03 (m, 6H), 7.95 (d, J =8.8 Hz, 2H), 7.75 – 7.68 (m, 1H), 7.58 – 7.39 (m, 6H), 4.60 – 4.53 (m, 1H), 3.85 (t, J = 6.2 Hz, 2H), 3.57 (t, J = 5.7 Hz, 2H), 3.17 (d, J = 5.2 Hz, 2H),2.72 (t, J = 6.0 Hz, 2H), 2.57 (s, 3H), 2.33 (s, 3H), 1.59 (s, 3H).

[0077] Example 4

[0078] Following the synthesis and purification method of compound 3a, compound 2 (150 mg, 0.22 mmol), HATU (380 mg, 0.44 mmol), 100 μL of DIPEA and N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid (72.87 mg, 0.26 mmol) were reacted to give the blue solid product compound 3b.

[0079] Using the same synthesis and purification methods as compound 4, compound 3b (80 mg, 0.095 mmol), HATU (72 mg, 0.19 mmol), 80 μL of DIPEA, and JQ1 (41.79 mg, 0.1 mmol) were reacted to give the blue solid product compound 5b (ZnPc-O2-JQ1) (27 mg, yield 23.22%). MALDI-TOP (m / z) calculated for C 64 H 49 ClN 14 O5SZn:1227.08, found[M+H] + : 1227.27. 1H NMR (400 MHz, DMSO) δ 9.13 (ddd, J = 31.9,17.1, 4.8 Hz, 5H), 8.94 (d, J = 7.7 Hz, 2H), 8.37 (dd, J = 18.6, 13.3 Hz,2H), 8.24 – 8.03 (m, 3.64 (s, 4H), 3.53 (t, J = 5.8 Hz, 2H), 3.17 (d, J = 5.3 Hz, 3H), 2.69 (t, J= 6.1 Hz, 2H), 2.55 (s, 2H), 2.29 (s, 3H), 1.57 (s, 3H).

[0080] Example 5

[0081] Following the synthesis and purification method of compound 3a, compound 2 (150 mg, 0.22 mmol), HATU (380 mg, 0.44 mmol), 100 μL of DIPEA, and 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecane-17-acid (84.44 mg, 0.26 mmol) were reacted to give the blue solid product compound 3c.

[0082] Using the same synthesis and purification methods as compound 4, compound 3c (80 mg, 0.09 mmol), HATU (69 mg, 0.18 mmol), 80 μL of DIPEA, and JQ1 (52 mg, 0.1 mmol) were reacted to give the blue solid product compound 5c (ZnPc-O3-JQ1) (25 mg, yield 21.84%). MALDI-TOP (m / z) calculated for C 66 H 53 ClN 14 O6SZn:1271.13, found[M+H] + : 1271.30. 1H NMR (400 MHz, DMSO) δ 9.26 – 9.07 (m, 4H),9.02 (t, J = 6.9 Hz, 2H), 8.50 (s, 1H), 8.31 (s, 1H), 8.26 – 8.05 (m, 5H),7.92 (d, J = 8.9 Hz, 2H), 7.75 (dd, J = 8.2, 2.1 Hz, 1H), 7.62 – 7.33 (m,6H), 4.56 – 4.49 (m, 1H), 3.81 (t, J = 6.2 Hz, 2H), 3.59 (dd, J = 10.0, 7.3Hz, 7H), 3.50 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 6.2 Hz, 2H), 2.52 (s, 2H), 2.29 (s, 3H), 1.56 (s, 3H).

[0083] Example 6

[0084] Following the synthesis and purification method of compound 3a, compound 2 (160 mg, 0.23 mmol), HATU (175 mg, 0.46 mmol), 100 μL of DIPEA and 1-tert-butyl 5,8,11,14-tetraoxa-2-azaheptadecanoic acid (102.4 mg, 0.28 mmol) were reacted to give the blue solid product compound 3d.

[0085] Using the same synthesis and purification methods as compound 4, compound 3d (80 mg, 0.085 mmol), HATU (65 mg, 0.17 mmol), 80 μL of DIPEA, and JQ1 (37.84 mg, 0.094 mmol) were reacted to give the blue solid product compound 5d (ZnPc-O4-JQ1) (25 mg, yield 22.15%). MALDI-TOP (m / z) calculated for C 68 H 57 ClN 14 O7SZn:1315.19, found[M+H] +: 1315.32. 1H NMR (400 MHz, DMSO) δ 9.26 – 9.07 (m, 4H), 9.05 – 8.94 (m, 2H), 8.47 (s, 1H), 8.31 (t, J = 5.4 Hz, 1H), 8.25 – 8.05 (m, 5H), 7.93 (d, J = 8.8 Hz, 2H), 7.79 – 7.71 (m, 1H), 7.58 – 7.35 (m, 6H), 4.54– 4.47 (m, 1H), 3.80 (t, J = 6.2 Hz, 2H), 3.58 (d, J = 14.4 Hz, 10H), 3.47(t, J = 5.8 Hz, 2H), 3.17 (d, J = 4.9 Hz, 5H), 2.67 (t, J = 6.0 Hz, 2H), 2.53(s, 2H), 2.31 (s, 3H), 1.56 (s, 2H).

[0086] Example 7

[0087] Following the synthesis and purification method of compound 3a, compound 2 (160 mg, 0.23 mmol), HATU (175 mg, 0.46 mmol), 100 μL of DIPEA and 21-(Boc-amino)-4,7,10,13,16,19-hexaoxadocosuccinic acid (127 mg, 0.28 mmol) were reacted to give the blue solid product compound 3e.

[0088] Using the same synthesis and purification methods as compound 4, compound 3e (80 mg, 0.078 mmol), HATU (59 mg, 0.16 mmol), 80 μL of DIPEA, and JQ1 (34.57 mg, 0.086 mmol) were reacted to give the blue solid product compound 5e (ZnPc-O6-JQ1) (20 mg, yield 18.18%). MALDI-TOP (m / z) calculated for C 72 H 65 ClN 14 O9SZn:1403.29, found[M+H] + : 1403.38. 1H NMR (400 MHz, DMSO) δ 9.28 – 9.06 (m, 4H), 9.07 – 8.91 (m, 2H), 8.47 (s, 1H), 8.29 (t, J = 5.2 Hz, 1H), 8.25 – 8.03 (m,5H), 7.93 (d, J = 8.8 Hz, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.60 – 7.34 (m, 6H), 4.54 – 4.45 (m, 1H), 3.80 (t, J = 6.2 Hz, 2H), 3.63 – 3.41 (m, 18H), 3.18(dd, J = 11.5, 5.7 Hz, 4H), 2.67 (t, J = 6.1 Hz, 2H), 2.54 (s, 2H), 2.33 (s, 3H), 1.57 (s, 3H).

[0089] Example 8

[0090] UV-Vis absorption curve

[0091] The UV absorption spectra of six phthalocyanines, 5a-5e (photo-PROTAC, 10 μM), in water were determined using a UV-Vis spectrophotometer. UV standard curves were plotted based on the absorbance at the maximum absorption wavelengths of 633 nm and 683 nm.

[0092] like Figure 1 As shown, the UV-Vis absorption spectra of the six molecules are basically the same, with the maximum absorption peaks all located near 680 nm.

[0093] Example 9

[0094] In vitro cell phototoxicity detection

[0095] In vitro cytotoxicity was evaluated using the CCK-8 assay. T24 cells were cultured at 1.12 × 10⁻⁶ cells / cells. 4 One sample per well was seeded into a 96-well plate and incubated in DMEM medium at 37 °C with 5% CO2 for 24 h in the dark. Afterwards, the medium was replaced with DMEM medium containing different concentrations of the drug, and incubated in the dark for 9 h. Then, the sample was incubated at 680 nm (20.4 J / cm²). 2 Irradiate with light for 10 min, then incubate in the dark for 15 hours. Subsequently, add CCK-8 solution and measure the absorbance of each well using a microplate reader.

[0096] like Figure 2As shown, in bladder cancer cells T24, molecules with Linker have a significantly stronger ability to generate total reactive oxygen species than ZnPc-JQ1 (compound 4), and their photosensitizing tumor-killing IC50 is significantly enhanced. 50 Concentration from 1351 nM (IC50 of ZnPc-JQ1) 50 The IC value of ZnPc-O1-JQ1 was significantly reduced to 41 nM. 50 Below this level, activity is increased by more than 33 times. With the extension of the linker, the IC of ZnPc-On-JQ1... 50 The concentration gradually decreased and reached a basic stability at ZnPc-O3-JQ1, with its IC50 value decreasing. 50 At a concentration of 22 nM, it exhibits ideal photosensitive activity in killing tumor cells.

[0097] Example 10

[0098] In vitro cell toxicity detection

[0099] T24 cells were fed at a dose of 1.12 × 10⁻⁶. 4 Each well was seeded with one sample of the drug and incubated in DMEM medium at 37 °C for 24 h in the dark. Afterward, the medium was replaced with DMEM medium containing different concentrations of the drug. After incubation in the dark for 9 h, CCK-8 solution was added, and the absorbance of each well was measured using a microplate reader.

[0100] like Figure 3 As shown, under dark conditions, the IC50 of ZnPc-O3-JQ1 in killing T24 cells is [data missing]. 50 Greater than 2×10 5 nM. Combination Figure 2 The phototoxicity index (PI: the ratio of the IC50 of a drug under dark conditions to the IC50 of a drug under light conditions) of ZnPc-O3-JQ1 is greater than 9091, which is much higher than that of ZnPc-JQ1 (PI greater than 148) and the approved phthalocyanine photosensitizer Photosens® (PI = 725). See ACS Med. Chem. Lett. 2021, 12, 502−507 for details. ZnPc-O3-JQ1 exhibits a high PI value. Figure 4 High-PI photosensitizers typically exhibit better solubility, facilitating more effective delivery to target tissues or cells. They also exhibit lower toxicity to non-target tissues. Furthermore, high-PI photosensitizers show low cellular toxicity in the dark, thus minimizing their impact on normal tissues and cells under non-light conditions, thereby reducing side effects and improving treatment safety. When used with selective illumination of tumor tissue, high-PI photosensitizers demonstrate a stronger selective killing effect on cancer cells.

[0101] Example 11

[0102] Detection of relative expression level of BRD4 in T24 cells under no-light treatment

[0103] T24 cells were used at a rate of 5 × 10 5 Cells were seeded per well in 6-well plates and incubated in DMEM medium at 37 °C for 24 h in the dark. Then, ZnPc-JQ1 (30 nM), ZnPc-O3-JQ1 (30 nM), and dBET1 (1 μM) were added and incubated in the dark for 9 h. The level of BRD4 in the cells was detected using Western blotting.

[0104] like Figure 5 As shown, neither ZnPc-JQ1 (30 nM) nor ZnPc-O3-JQ1 (30 nM) exhibited BRD4 degradation activity when not exposed to light.

[0105] Example 12

[0106] Detection of relative expression level of BRD4 in T24 cells under light treatment

[0107] T24 cells were used at a rate of 5 × 10 5 One sample per well was seeded into a 6-well plate and incubated in DMEM medium at 37 °C with 5% CO2 for 24 h in the dark. Then, ZnPc-JQ1 (30 nM), ZnPc-O3-JQ1 (30 nM), and dBET1 (1 μM) were added and incubated in the dark for 9 h at 680 nm (20.4 J / cm²). 2 Irradiate with light for 10 min, then incubate in the dark for 15 hours. Detect BRD4 levels in cells using Western blotting analysis.

[0108] like Figure 6 As shown, after light irradiation, ZnPc-O3-JQ1 exhibits a significant degradation effect on BRD4, combined with... Figure 5 The drug does not produce ROS when not exposed to light; it is only after light exposure that ROS is generated and damages cells. This demonstrates that the degradation of BRD4 by ZnPc-O3-JQ1 does not originate from indiscriminate protein damage by ROS, but rather from the precise disruption of ROS after JQ1 binds to BRD4. Moreover, the degradation rate of BRD4 by ZnPc-O3-JQ1 at 30 nM can reach 87%, far exceeding the degradation effect of dBET1 at 10000 nM (35% degradation rate).

[0109] Example 13

[0110] Detection of reactive oxygen species in cells

[0111] T24 cells were used at a rate of 5 × 10 5Cells were seeded per well in 6-well plates and incubated in DMEM medium at 37 °C with 5% CO2 for 24 h in the dark. The cells were then divided into three groups: blank, ZnPc-JQ1 (1 μM), and ZnPc-O3-JQ1 (1 μM). After treatment with ZnPc-JQ1 (1 μM) and ZnPc-O3-JQ1 (1 μM) for 9 h, the cells were incubated with 5 μM of the probe fluorescent dye DCFH-DA (Beyotime, S0034S) for 4 h in the dark; subsequently, they were incubated at 680 nm (20.4 J / cm²). 2 Cells were exposed to light for 10 min, incubated in an incubator for 30 min, and then washed twice with PBS. The fluorescence intensity of the probe was detected using a fluorescence inverted microscope. The relative ROS content in T24 cells was measured using the fluorescent dye DCFH-DA. The fluorescence intensity of the probe was directly proportional to its reactive oxygen species production capacity. Figure 7 As shown, in T24 cells, ZnPc-O3-JQ1 has a significantly stronger reactive oxygen species production capacity than ZnPc-JQ1, indicating that ZnPc-O3-JQ1 has higher photosensitive killing activity against tumor cells.

[0112] Example 14

[0113] HIF-1α protein inhibition assay

[0114] T24 cells were used at a rate of 5 × 10 5 The cells were seeded at a density of 1 cell / well in 6-well plates and incubated in DMEM medium in the dark at 37 °C with 5% CO2 for 24 h. After incubation in the dark (O2 concentration 1%) with ZnPc-JQ1 (30 nM) and ZnPc-O3-JQ1 (30 nM) respectively for 9 h, the solution was then applied at 680 nm (20.4 J / cm²). 2 Cells were exposed to light for 10 min and then cultured in the dark for 15 min. Protein was extracted and Western blot was performed to detect the level of HIF-1α protein in each group of cells (β-tubulin was used as the internal reference).

[0115] like Figure 8 As shown, ZnPc-O3-JQ1-induced BRD4 degradation affects HIF-1α transcription and significantly reduces intracellular HIF-1α levels in tumor cells compared to the control group. This indicates that ZnPc-O3-JQ1 not only degrades BRD4 but also downregulates HIF-1α, which can disrupt treatment resistance caused by aggravated hypoxia, thereby achieving synergistic effects of PDT and PROTAC.

[0116] Example 15

[0117] Intracellular GSH level detection:

[0118] T24 cells were used at a rate of 5 × 10 5Cells / well were seeded in six-well plates and incubated in DMEM medium at 37 °C with 5% CO2 for 24 h in the dark. After incubation with ZnPc-JQ1 and ZnPc-O3-JQ1 in the dark for 9 h, the cells were then incubated at 680 nm (20.4 J / cm²). 2 Irradiate for 10 min. Then, add GSH (5 μM) probe to the cells and incubate in the dark for 2 h. Flow cytometry and fluorescence inverted microscope are used to detect the fluorescence intensity of the probe in the cells.

[0119] like Figure 9 As shown, GSH in the ZnPc-O3-JQ1 group increased by only 1.37 times compared to the control group, indicating that ZnPc-O3-JQ1 can negatively regulate GSH synthesis by downregulating GCL, thereby inhibiting GSH synthesis.

[0120] Example 16

[0121] Research on the impact of PDT on GCL

[0122] T24 cells were used at a rate of 5 × 10 5 The cells were seeded at a density of cells / well in 6-well plates and incubated in DMEM medium at 37 °C with 5% CO2 for 24 h in the dark. After incubation in the dark for 9 h with ZnPc-JQ1 (30 nM) and ZnPc-O3-JQ1 (30 nM), respectively, at 680 nm (20.4 J / cm²). 2 Cells were exposed to light for 10 min and then cultured in the dark for 15 min. Protein was extracted and Western blot was performed to detect the level of GCL protein in each group of cells (internal control was GAPDH).

[0123] like Figure 10 As shown, GCL protein was significantly degraded, indicating that ZnPc-O3-JQ1 can downregulate GCL, thereby affecting GSH synthesis.

[0124] As can be seen from Examples 15 and 16, the ZnPc-O3-JQ1 of the present invention downregulates GCL and inhibits GSH synthesis, indicating that it can disrupt the cellular antioxidant defense barrier and has superior antitumor activity.

[0125] Example 17

[0126] In vivo anti-tumor detection

[0127] A subcutaneous xenograft tumor model was constructed by injecting MB49 cells into the lateral hind limbs of female C57BL / 6 mice (6–7 weeks old, 20–22 g). The length and width of the tumor were measured using calipers and calculated according to the formula (Volume = 0.5 × Length × Width). 2The tumor volume was calculated by using Length and Width to represent the length and width of the tumor, respectively, until the tumor grew to 100 mm on one side. 3 Treatment experiments were conducted on both sides.

[0128] Mice were divided into PBS, dBET1, Cis-platin, ZnPc-JQ1+L, and ZnPc-O3-JQ1+L groups. Mice were injected with the drug (3 mg / kg) via the tail vein every other day for 10 consecutive days and then treated with a 665 nm laser (25 mW / cm²). 2 Irradiate the tumor site for 5 minutes and monitor the tumor volume.

[0129] like Figure 11 As shown, in terms of tumor inhibition, all drug-treated groups had varying degrees of anti-tumor effects compared to the PBS group. Among them, the ZnPc-O3-JQ1+L group showed the best tumor growth inhibition effect and had a significant difference compared to the ZnPc-JQ1+L group and other groups.

Claims

1. A phthalocyanine-based photo-PROTAC drug characterized in that, The structure of the drug is shown in the following formula I or formula II: ， Wherein, n = any integer from 1 to 10.

2. The phthalocyanine-based photo-PROTAC drug of claim 1, wherein The n is 1, 2, 3, 4 or 6.

3. A method of preparing a phthalocyanine-based photo-PROTAC drug of claim 2, characterized in that, Comprising the following steps: (1) synthesis of intermediate 1: 4-nitrophthalonitrile, 4-(boc-amino) phenol and potassium carbonate are uniformly mixed in an organic solvent to obtain intermediate 1; (2) synthesis of compound 2 ZnPc: intermediate 1, phthalonitrile and anhydrous zinc acetate are dissolved in an organic solvent, DBU is added under the protection of inert gas, and reaction is carried out to obtain ZnPc; (3) synthesis of compound 4 ZnPc-JQ1: compound 2, (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid JQ1 and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) are dissolved in an organic solvent, stirred and reacted, then N,N-diisopropylethylamine (DIPEA) is added, and reaction is carried out at room temperature to obtain compound 4; (4) synthesis of compound 5a ZnPc-O1-JQ1: compound 2, 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoic acid and HATU are dissolved in an organic solvent, stirred and reacted, then DIPEA is added, reaction is carried out at room temperature, after the reaction is completed, an organic solvent and trifluoroacetic acid are added, and the BOC is removed by stirring to obtain compound 3a; compound 3a is reacted with JQ1 at room temperature to obtain compound 5a; (5) synthesis of compound 5b ZnPc-O2-JQ1: compound 2, N-Boc-3-[2-(2-aminoethoxy)ethoxy]propanoic acid and HATU are dissolved in an organic solvent, stirred and reacted, then DIPEA is added, reaction is carried out at room temperature, after the reaction is completed, an organic solvent and trifluoroacetic acid are added, and the BOC is removed by stirring to obtain compound 3b; Compound 3b is reacted with JQ1 to obtain compound 5b; (6) synthesis of compound 5c ZnPc-O3-JQ1: compound 2, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecanoic acid and HATU are dissolved in an organic solvent, stirred and reacted, then DIPEA is added, reaction is carried out at room temperature, after the reaction is completed, an organic solvent and trifluoroacetic acid are added, and the BOC is removed by stirring to obtain compound 3c; 3c is reacted with JQ1 to obtain compound 5c; (7) synthesis of compound 5d ZnPc-O4-JQ1: compound 2, 5,8,11,14-tetraoxa-2-azahexadecanedioic acid 1-tert-butyl ester and HATU are dissolved in an organic solvent, stirred and reacted, then DIPEA is added, reaction is carried out at room temperature, after the reaction is completed, an organic solvent and trifluoroacetic acid are added, and the BOC is removed by stirring to obtain compound 3d; 3d is reacted with JQ1 to obtain compound 5d; (8) Synthesis of compound 5e ZnPc-O6-JQ1: compound 2, 21- (Boc-amino) -4, 7, 10, 13, 16, 19-hexaoxahenicosanoic acid and HATU were dissolved in an organic solvent, stirred and reacted, DIPEA was added, reacted at room temperature, after the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the amino BOC was removed by stirring to obtain compound 3e; 3e was reacted with JQ1 to obtain compound 5e; The reaction formula is as follows: 。 4. The production method according to claim 3, characterized by, In step (1), the molar ratio of 4-nitrophthalonitrile, 4- (boc-amino) phenol and potassium carbonate is 1: 1.2 ~ 1.5: 1.55 ~ 2, the reaction temperature is 50 ~ 60 ℃, and the stirring reaction time is 6 ~ 7 h.

5. The preparation method according to claim 3, characterized in that, In step (2), the molar ratio of intermediate 1, phthalonitrile and anhydrous zinc acetate is 1: 8.5 ~ 9: 3 ~ 4, the reaction temperature is 130 ~ 140 ℃, and the stirring reaction time is 9 ~ 10 h.

6. The preparation method according to claim 3, characterized in that, In step (3), the molar ratio of compound 2, JQ1 and HATU is 1: 1.0 ~ 1.3: 1.8 ~ 3, the reaction temperature is room temperature, and the stirring reaction time is 4 ~ 5 h.

7. The preparation method according to claim 3, characterized in that, In step (4), the molar ratio of compound 2, 3- (2- ( (tert-butoxycarbonyl) amino) ethoxy) propionic acid and HATU is 1: 1.0 ~ 1.3: 2 ~ 3, the reaction temperature is room temperature, and the stirring reaction time is 6 ~ 7 h.

8. The preparation method according to claim 3, characterized in that, The reaction conditions of steps (5), (6), (7) and (8) are the same as those of step (4).

9. Use of the phthalocyanine-based photo-PROTAC drug of claim 1 or 2 in the preparation of an anticancer drug.

10. Use according to claim 9, characterized in that, Use of the phthalocyanine-based photo-PROTAC drug as a photosensitizer in the preparation of an anticancer drug.

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