Phthalocyanine-based photo-PROTAC medicine as well as preparation method and application thereof

Abstract

The invention discloses a photo-PROTAC drug based on phthalocyanine as well as a preparation method and application of the photo-PROTAC drug. The photo-PROTAC drug taking BRD4 as a target spot is constructed by mainly adopting a chemical synthesis method of coupling amino and carboxyl, taking photosensitizers ZnPc and BRD4 (BRD4 inhibitor) as structural main bodies and selecting polyethylene glycol (PEG) chains with different lengths as linkers of ZnPc and JQ1. The raw materials are wide in source, and the preparation method is simple. According to the drug, efficient PROTAC degradation independent of E3 ubiquitin enzyme is achieved by utilizing the characteristic of BRD4 expression of bladder cancer tumor tissue and combining selective tumor site illumination. Meanwhile, the BRD4 degrades and destroys the antioxidation and hypoxia inhibition barrier of the PDT, so that the synergistic interaction of the PDT and the PROTAC is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a phthalocyanine-based photo-PROTAC drug and a preparation method and application thereof. Background Art

[0002] It is well known that the occurrence and development of cancer depends 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, the proteolysis targeting chimera (PROTAC) technology has become a promising and attractive method in cancer targeted therapy because of its advantage of selectively degrading proteins rather than directly inhibiting protein activity. The PROTAC molecule is a heterobifunctional molecule consisting of a ligand that recruits the target protein and another ligand that recruits the E3 ubiquitin ligase. The two ligands are connected to each other through a connector. This chemically induced connection between POI and E3 ligase leads to ubiquitination and POI degradation by the ubiquitin-proteasome system (UPS). Compared with traditional small molecule inhibitors driven by "occupancy", 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 extra-terminal domain (BET) family. It can recognize acetylated histones and plays an important role in transcription, replication and DNA repair. Overexpression or abnormal activation of BRD4 is closely related to the occurrence and development of many tumors. In normal tissues, the strict regulation of BRD4 expression is essential for maintaining cell cycle progression. Currently, a commercial PROTAC degrader for degrading BRD4 is dBET1. This degrader uses thalidomide derivatives as E3 ligase recruitment ligands to hijack CRBN to degrade BRD4 protein. This degradation mechanism targets all BRD4 in the body and has no selectivity for tumors and adjacent tissues.

[0004] PROTAC technology offers many potential advantages, but also faces significant challenges in cancer therapy. The activity of PROTAC depends on its associated E3, whose expression may vary in different cell types, tissues or species, and there may be off-target effects with its E3 ligand. In addition, it remains a challenge to precisely control the function of PROTAC in the tumor area in vivo. Summary of the invention

[0005] Purpose of the invention: To solve the off-target problem that may occur when PROTAC drugs are used to treat tumors, the present invention designs a phthalocyanine-based photo-PROTAC drug, which directly degrades the target protein by generating reactive oxygen species through photoactivation, independent of E3 ubiquitinase. On the basis that the BRD4 level in most tumor tissues is higher than that in adjacent adjacent tissues, the photo-assisted targeted activation strategy of selectively irradiating the tumor site is used to further improve the selective degradation of the target protein in the tumor tissue by the Photo-PROTAC drug, which is expected to achieve the goal of safe and efficient tumor treatment.

[0006] The present invention also provides a preparation method and application of the phthalocyanine-based photo-PROTAC drug.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the photo-PROTAC drug based on phthalocyanine of the present invention has a structural formula as shown in Formula I or Formula II below:

[0008]

[0009] Wherein, n=any integer from 1 to 10.

[0010] Preferably, n is any integer of 1, 2, 3, 4, 6, and its structure is:

[0011]

[0012] The method for preparing the phthalocyanine-based photo-PROTAC compound of the present invention comprises the following steps:

[0013] (1) Synthesis of Intermediate 1:

[0014] 4-nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate are uniformly mixed in an organic solvent to react to obtain intermediate 1;

[0015] (2) Synthesis of compound 2 (ZnPc): Compound 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;

[0016] (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) were dissolved in an organic solvent, stirred for reaction, and then N,N-diisopropylethylamine (DIPEA) was added and reacted at room temperature to obtain compound 4;

[0017] (4) Compound 5a (ZnPc-O 1 -JQ1): Compound 2, 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid and HATU are dissolved in an organic solvent, stirred for reaction, and then DIPEA is added to react at room temperature. After the reaction is completed, an organic solvent and trifluoroacetic acid are added, and the ammonia BOC is removed by stirring to obtain compound 3a; Compound 3a is reacted with JQ1 at room temperature to obtain compound 5a;

[0018] (5) Compound 5b (ZnPc-O 2 -JQ1): Compound 2, N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid and HATU are dissolved in an organic solvent, stirred for reaction, and then DIPEA is added to react at room temperature. After the reaction is completed, an organic solvent and trifluoroacetic acid are added, and the ammonia BOC is removed by stirring to obtain compound 3b; compound 3b is reacted with JQ1 to obtain compound 5b;

[0019] (6) Compound 5c (ZnPc-O 3 -JQ1) synthesis: Compound 2, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3c; 3c was reacted with JQ1 to obtain compound 5c;

[0020] (7) Compound 5d (ZnPc-O 4 -JQ1) synthesis: Compound 2, 1-tert-butyl 5,8,11,14-tetraoxa-2-azaheptadecanedioate and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3d; 3d was reacted with JQ1 to obtain compound 5d;

[0021] (8) Compound 5e (ZnPc-O 6 -JQ1) synthesis: Compound 2, 21-(Boc-amino)-4,7,10,13,16,19-hexaoxahene-11-carbonic acid and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3e; 3e was reacted with JQ1 to obtain compound 5e;

[0022] The reaction formula is as follows:

[0023]

[0025] Wherein, the molar ratio of 4-nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate in step (1) is 1:1.2-1.5:1.55-2, the volume of the solvent is 25-30 mL, the reaction temperature is 50-60° C., and the stirring reaction time is 6-7 h.

[0026] Preferably, in step (1), the molar ratio of 4-nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate is 1:1.3:1.55, the volume of the solvent is 25 mL, the reaction temperature is 50° C., and the stirring reaction time is 6 h.

[0027] Wherein, the molar ratio of the compound 1 to phthalonitrile and anhydrous zinc acetate in step (2) 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° C., and the stirring reaction time is 9-10 h.

[0028] 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° C., and the stirring reaction time is 9 h.

[0029] Wherein, in step (3), the molar ratio of compound 2, JQ1 and HATU is 1:1.0-1.3:1.8-3, the volume of the 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.

[0030] Preferably, in step (3), the molar ratio of compound 2, JQ1 and HATU is 1:1.1:1.92-2, the volume of the 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.

[0031] Wherein, the molar ratio of the compound 2, 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid and HATU in step (4) is 1:1.0-1.3:2-3, the volume of the 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.

[0032] 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.

[0033] The reaction conditions in steps (5), (6), (7) and (8) are the same as those in step (4).

[0034] Application of the phthalocyanine-based photo-PROTAC drug of the present invention in the preparation of anticancer drugs.

[0035] Among them, the phthalocyanine-based photo-PROTAC drug is used as a photosensitizer in the preparation of anticancer drugs.

[0036] Furthermore, the photo-PROTAC compound (ZnPc-O 3 -JQ1) has excellent photosensitizing activity, good physiological compatibility and selectivity, and is suitable as a photosensitizer for photodynamic therapy of tumors.

[0037] The phthalocyanine-based photo-PROTAC compound of the present invention mainly adopts a chemical synthesis method of amino and carboxyl coupling, has a wide range of raw materials and a simple preparation method. The present invention uses photosensitizer ZnPc and BRD4 ligand JQ1 (BRD4 inhibitor) as the main structure, selects polyethylene glycol (PEG) chains of different lengths as linkers of ZnPc and JQ1, constructs photo-PROTAC compounds targeting BRD4, and screens out the best photosensitizer ZnPc-O 3 -JQ1. ZnPc-O 3 -JQ1 has excellent BRD4 binding and light-triggered 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 the level of HIF-1α in cells, thereby reducing the tumor's resistance to treatment induced by PDT-induced hypoxia. In response to the oxidative stress caused by reactive oxygen species produced by photodynamic therapy (PDT), cells will synthesize more glutathione to enhance the antioxidant capacity of cells. HIF-1α can negatively regulate glutathione (GSH) synthesis by downregulating GCL (a key enzyme for the de novo synthesis of glutathione), thereby destroying the antioxidant defense capacity of tumor cells. Therefore, ZnPc-O 3 -JQ1 not only achieves efficient photo-PROTAC degradation of BRD4, but also utilizes its downregulation of HIF-1α and its downstream effects to reduce the hypoxia and antioxidant resistance of tumors to PDT treatment, thereby achieving synergistic enhancement of PDT and PROTAC.

[0038] The present invention designs and synthesizes a series of photo-PROTAC molecules ZnPc-O with BRD4 as the target protein. n-JQ1 (n=0, 1, 2, 3, 4, 6). By utilizing the BRD4 expression characteristic of bladder cancer tumor tissue and combining it with selective illumination of the tumor site, efficient PROTAC degradation independent of E3 ubiquitinase is achieved. At the same time, the present invention can destroy the antioxidant and hypoxic inhibition barrier of PDT by degrading BRD4, thereby achieving synergistic enhancement of PDT and PROTAC. The present invention uses a light-activated photo-PROTAC strategy to accurately degrade BRD4 protein in tumor tissue. BRD4 degradation destroys the antioxidant and hypoxic inhibition barrier of PDT, and photodynamic therapy and PROTAC synergize. The series of photo-PROTAC drugs prepared by the present invention are all prepared for the first time. The present invention specifically combines the photosensitizer ZnPc in PDT with the BRD4 small molecule inhibitor JQ1, which is activated by light irradiation to generate reactive oxygen near the BRD4 protein, thereby degrading it. This degradation method does not require the participation of E3 ligases, and accurately degrades proteins through ROS, achieving selectivity in normal and tumor tissues.

[0039] Compared with the commercial PROTAC degrader dBET1 for BRD4 degradation, ZnPc-O 3 -JQ1 has better in vivo anti-tumor activity. The present invention compares ZnPc-JQ1 and ZnPc- n -JQ1 photosensitized killing of tumor cells activity, photosensitized killing of tumor IC 50 The concentration ranged from 1351nM (IC 50 of ZnPc-JQ1) was significantly reduced to 41nM (IC 50 of ZnPc-O 1 -JQ1), the activity increased by more than 33 times. n -JQ1 IC 50 The concentration gradually decreased and 3 -JQ1 reaches basic stability, and its IC 50 The concentration was 22 nM. In addition, ZnPc-O 3 -JQ1 has ideal photosensitizing tumor cell killing activity and high PI (phototoxicity index: refers to the IC value of a drug under light conditions). 50 With IC in dark conditions 50 The ratio of the value of ZnPc-O can bind to BRD4 and generate ROS after photodynamic therapy to effectively degrade BRD4. PDT and PROTAC promote each other to achieve cancer cell damage. 3 -JQ1 had the best effect in inhibiting tumor growth.

[0040] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0041] (1) The present invention adopts the photo-PROTAC strategy system to effectively combine photodynamic therapy (PDT) with targeted protein degradation to improve the treatment effect of bladder cancer.

[0042] (2) The present invention selectively illuminates the tumor site to stimulate the photosensitizer to produce reactive oxygen species to degrade the target protein. This strategy is independent of the expression level and activity of E3 ubiquitinase, opening up a new path for the development of safe and efficient PROTAC drugs.

[0043] (3) A series of photo-PROTAC drugs prepared by the present invention specifically bind the photosensitizer ZnPc in PDT to the BRD4 small molecule inhibitor JQ1, which is activated by light irradiation to generate reactive oxygen species near the BRD4 protein, thereby degrading it. This degradation method does not require the participation of E3 ligases, and accurately degrades proteins through ROS, achieving selectivity in normal and tumor tissues. At the same time, BRD4 degradation destroys the antioxidant and hypoxic inhibition barriers of PDT, achieving synergistic enhancement of PDT and PROTAC.

[0044] (4) The drug prepared by the present invention, such as ZnPc-O 3 - The PI value of JQ1 is greater than 9091, which is much higher than the approved phthalocyanine photosensitizers (PI=725), with excellent photosensitizing tumor cell killing activity and high PI value characteristics.

[0045] (5) The drug prepared by the present invention has a significant tumor growth inhibitory effect in vivo, compared with the commercial PROTAC degrader dBET1 for degrading BRD4, such as ZnPc-O 3 -JQ1 has superior antitumor activity in vivo.

[0046] (6) The preparation method of the present invention is simple and efficient, the raw materials are easily available, and it has good industrial production value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 are the UV-visible absorption spectra of the six photo-PROTAC drugs of the present invention in aqueous solution;

[0048] Figure 2 The phototoxic IC values ​​of six photo-PROTAC drugs in T24 cells 50 picture;

[0049] Figure 3 Dark toxic IC of six photo-PROTAC drugs in T24 cells 50 picture;

[0050] Figure 4PI graphs of six photo-PROTAC drugs in T24 cells;

[0051] Figure 5 For ZnPc-JQ1, ZnPc-O 3 - Relative expression level of BRD4 in T24 cells after JQ1 and dBET1 treatment without light;

[0052] Figure 6 After illumination, ZnPc-JQ1 and ZnPc-O 3 - Relative expression of BRD4 in T24 cells after treatment with JQ1 and dBET1;

[0053] Figure 7 For ZnPc-JQ1, ZnPc-O 3 -JQ1 reactive oxygen species production in T24 cells;

[0054] Figure 8 Under hypoxic conditions, ZnPc-JQ1 and ZnPc-O 3 -WB and quantitative analysis of the changes in HIF-1α levels induced by JQ1 treatment.

[0055] Fig. 9 For ZnPc-JQ1 and ZnPc-O 3 -Flow cytometric detection and intensity comparison of GSH level changes after JQ1 treatment.

[0056] Fig.10 For ZnPc-JQ1 and ZnPc-O 3 - Western blot and quantitative analysis of the changes in GLC levels after JQ1 treatment.

[0057] Fig.11 In the subcutaneous transplanted tumor model animal, PBS, dBET1, Cis-platin, ZnPc-JQ1+L and ZnPc-O 3 -Schematic diagram comparing the ability of the JQ1+L group to inhibit tumor growth.

[0058] In each figure, ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. DETAILED DESCRIPTION

[0059] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0060] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0061] (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), CAS No. 202592-23-2.

[0062] 3-(2-((tert-Butyloxycarbonyl)amino)ethoxy)propanoic acid, CAS No. 1260092-44-1.

[0063] N-Boc-3-[2-(2-aminoethoxy)ethoxy]propanoic acid, CAS No. 1365655-91-9.

[0064] 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid, CAS No. 1347750-75-7.

[0065] 1-tert-Butyl 5,8,11,14-tetraoxa-2-azaheptadecanedioate, CAS No. 756525-91-4.

[0066] 21-(Boc-amino)-4,7,10,13,16,19-hexaoxaheneicosanoic acid, CAS No. 882847-13-4.

[0067] Commercial PROTAC drug dBET1, whose target protein is BRD4 and E3 ubiquitinase is CRBN. Bidex Pharmaceuticals, CAS number: 1799711-21-9.

[0068] Cis-platin, Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 15663-27-1.

[0069] Commercialized phthalocyanine photosensitizers CAS No. 122170-90-5.

[0070] GSH probe synthesis reference: 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.

[0071] Example 1

[0072] Dissolve 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) in 25 mL of N,N-dimethylformamide. Under nitrogen protection, heat to 50 ° C and stir to react for 6 hours. After the reaction is completed, pour into 200 mL of ice water and let stand until solid precipitates. Filter by suction, wash the filter cake with distilled water until the filtrate is colorless, take the filter cake and put it into a vacuum drying oven to dry, and obtain white solid compound 1.

[0073] Compound 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, and 25 mL of n-pentanol was added 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 with a syringe, and then the mixture was heated to 140 °C and refluxed for 9 h. After the reaction was completed, the reaction solution was cooled to room temperature and n-pentanol was removed by vacuum distillation. The remaining solid was ultrasonically dissolved with 40 mL of dichloromethane (DCM), filtered, the filtrate was taken, the sample was mixed, and the crude product was separated by silica gel column chromatography (petroleum ether / ethyl acetate (v / v, 1:1)). Then 10 mL of dichloromethane and 4 mL of trifluoroacetic acid (TFA) were added and stirred for 8 h to remove ammonia BOC, and the solvent was removed by vacuum rotary evaporation. Then, 30 mL of methanol and 5 mL of 10% sodium hydroxide were added, the temperature was raised to 130°C and refluxed for 4 h, centrifuged, and the lower layer of solid was removed. Finally, 40 mL of methanol and 2 mL of concentrated hydrochloric acid (36% to 38%) were added, the temperature was raised to 100°C and refluxed for 4 h (the process of methanol and concentrated hydrochloric acid reflux was repeated twice), centrifuged, the lower layer of solid was removed, and placed in a vacuum drying oven for drying to obtain a blue product compound 2 (ZnPc) (150 mg, yield 18.36%). MALDI-TOP (m / z) calculated for C 38 H 21 N9 OZn:685.12,found[M+H] + :685.03. 1 H NMR (400MHz, DMSO) δ9.40-9.09(m,7H),8.72(s,1H),8.28-8.09(m,6H),7.92-7.81(m,2H),7.68-7.52(m,4H).

[0074] Example 2

[0075] 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]diazepin-6-yl)acetic acid (JQ1 (52 mg, 0.13 mmol)) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium 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 reaction was stirred at room temperature for 4 h, and the reaction progress of the mixture was monitored by TLC. After the reaction, 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 organic layer after extraction was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column chromatography (dichloromethane / methanol (v / v, 20:1)) to obtain a blue product compound 4 (ZnPc-JQ1) (30 mg, yield 23.96%). MALDI-TOP (m / z) calculated for C 57 H 36 C1N 13 O 2 SZn:1067.89,found[M+H] + :1067.18. 1 H NMR (400MHz, DMSO) δ10.62(s,1H),9.19-8.80(m,7H),8.38(s,1H),8.24-7.90(m,8H),7.71(d,J=7.7Hz,1H ),7.54(dd,J=15.0,9.8Hz,6H),4.73(s,1H),3.65(d,J=6.8Hz,2H),2.66(s,3H),2.44(s,3H),1.69(s,3H).

[0076] Example 3

[0077] 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, stirred for 10 min, and then 150 μL of DIPEA was added. The mixture was stirred at room temperature for 6 h, and the reaction process of the mixture 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 organic layer after extraction was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain a crude product, which was separated and 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 BOC. After evaporating the solvent under reduced pressure, the mixture was washed with water and methanol and centrifuged. The lower solid layer was taken and dried in a vacuum drying oven to obtain a blue solid product compound 3a.

[0078] Using the synthesis and purification method of compound 4, compound 3a (80 mg, 0.1 mmol), HATU (76 mg, 0.2 mmol), 80 μL DIPEA and JQ1 (44 mg, 0.11 mmol) were reacted to obtain a blue solid product compound 5a (ZnPc-O 1 -JQ1) (28 mg, yield 23.67%). MALDI-TOP (m / z) calculated for C 62 H 45 C1N 14 O 4 SZn:1183.03,found[M+H] + :1183.24. 1 HNMR(400MHz,DMSO)δ9.22-9.03(m,5H),8.95(d,J=6.9Hz,2H),8.39(d,J=17.1 Hz,2H),8.22-8.03(m,6H),7.95(d,J=8.8Hz,2H),7.75-7.68(m,1H),7.58-7.3 9(m,6H),4.60-4.53(m,1H),3.85(t,J=6.2Hz,2H),3.57(t,J=5.7Hz,2H),3.17 (d,J=5.2Hz,2H),2.72(t,J=6.0Hz,2H),2.57(s,3H),2.33(s,3H),1.59(s,3H).

[0079] Example 4

[0080] According to the method for synthesis, separation and purification of compound 3a, compound 2 (150 mg, 0.22 mmol), HATU (380 mg, 0.44 mmol), 100 μL DIPEA and N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid (72.87 mg, 0.26 mmol) were reacted to obtain a blue solid product, compound 3b.

[0081] Using the synthesis and purification method of compound 4, compound 3b (80 mg, 0.095 mmol), HATU (72 mg, 0.19 mmol), 80 μL DIPEA and JQ1 (41.79 mg, 0.1 mmol) were reacted to obtain a blue solid product compound 5b (ZnPc-O 2 -JQ1) (27 mg, yield 23.22%). MALDI-TOP (m / z) calculated for C 64 H 49 C1N 14 O 5 SZn:1227.08,found[M+H] + :1227.27. 1 H NMR (400MHz, DMSO) δ9.13(ddd,J=31.9,17.1,4.8Hz,5H),8.94(d,J=7.7Hz,2H),8.37(dd,J =18.6,13.3Hz,2H),8.24-8.03(m,5H),7.94(d,J=8.9Hz,2H),7.71(dd,J=8.2,2.0Hz,1H), 7.57-7.36(m,6H),4.59-4.51(m,1H),3.83(t,J=6.2Hz,2H),3.64(s,4H),3.53(t,J=5.8Hz ,2H),3.17(d,J=5.3Hz,3H),2.69(t,J=6.1Hz,2H),2.55(s,2H),2.29(s,3H),1.57(s,3H).

[0082] Example 5

[0083] According to the method for synthesis, separation and purification of compound 3a, compound 2 (150 mg, 0.22 mmol), HATU (380 mg, 0.44 mmol), 100 μL DIPEA and 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid (84.44 mg, 0.26 mmol) were reacted to obtain a blue solid product, compound 3c.

[0084] Using the synthesis and purification method of compound 4, compound 3c (80 mg, 0.09 mmol), HATU (69 mg, 0.18 mmol), 80 μL DIPEA and JQ1 (52 mg, 0.1 mmol) were reacted to obtain a blue solid product compound 5c (ZnPc-O 3 -JQ1) (25 mg, yield 21.84%). MALDI-TOP (m / z) calculated for C 66 H 53 C1N 14 O 6 SZn:1271.13,found[M+H] + :1271.30. 1 HNMR (400MHz, DMSO) δ9.26-9.07 (m, 4H), 9.02 (t, J = 6.9Hz, 2H), 8.50 (s, 1H), 8.31 (s ,1H),8.26-8.05(m,5H),7.92(d,J=8.9Hz,2H),7.75(dd,J=8.2,2.1Hz,1H),7.62-7 .33(m,6H),4.56-4.49(m,1H),3.81(t,J=6.2Hz,2H),3.59(dd,J=10.0,7.3Hz,7H), 3.50(t,J=5.9Hz,2H),2.68(t,J=6.2Hz,2H),2.52(s,2H),2.29(s,3H),1.56(s,3H).

[0085] Example 6

[0086] According to the method for synthesis, separation and purification of compound 3a, compound 2 (160 mg, 0.23 mmol), HATU (175 mg, 0.46 mmol), 100 μL DIPEA and 1-tert-butyl 5,8,11,14-tetraoxa-2-azaheptadecanedioate (102.4 mg, 0.28 mmol) were reacted to obtain a blue solid product compound 3d.

[0087] Using the synthesis and purification method of compound 4, compound 3d (80 mg, 0.085 mmol), HATU (65 mg, 0.17 mmol), 80 μL DIPEA and JQ1 (37.84 mg, 0.094 mmol) were reacted to obtain a blue solid product compound 5d (ZnPc-O 4 -JQ1) (25 mg, yield 22.15%). MALDI-TOP (m / z) calculated for C 68 H 57 C1N14 O 7 SZn:1315.19,found[M+H] + :1315.32.1H NMR (400MHz, DMSO) δ9.26-9.07(m,4H),9.05-8.94(m,2H),8.47(s,1H),8.31(t,J=5.4H z,1H),8.25-8.05(m,5H),7.93(d,J=8.8Hz,2H),7.79-7.71(m,1H),7.58-7.35(m,6H), 4.54-4.47(m,1H),3.80(t,J=6.2Hz,2H),3.58(d,J=14.4Hz,10H),3.47(t,J=5.8Hz,2H ),3.17(d,J=4.9Hz,5H),2.67(t,J=6.0Hz,2H),2.53(s,2H),2.31(s,3H),1.56(s,2H).

[0088] Example 7

[0089] According to the method for synthesis, separation and purification of compound 3a, compound 2 (160 mg, 0.23 mmol), HATU (175 mg, 0.46 mmol), 100 μL DIPEA and 21-(Boc-amino)-4,7,10,13,16,19-hexaoxaheneicosanoic acid (127 mg, 0.28 mmol) were reacted to obtain a blue solid product compound 3e.

[0090] Using the synthesis and purification method of compound 4, compound 3e (80 mg, 0.078 mmol), HATU (59 mg, 0.16 mmol), 80 μL DIPEA and JQ1 (34.57 mg, 0.086 mmol) were reacted to obtain a blue solid product compound 5e (ZnPc-O 6 -JQ1) (20 mg, yield 18.18%). MALDI-TOP (m / z) calculated for C 72 H 65 C1N 14 O 9 SZn:1403.29,found[M+H] + :1403.38. 1H NMR (400MHz, DMSO) δ9.28-9.06(m,4H),9.07-8.91(m,2H),8.47(s,1H),8.29(t,J= 5.2Hz,1H),8.25-8.03(m,5H),7.93(d,J=8.8Hz,2H),7.74(d,J=8.1Hz,1H),7.60-7 .34(m,6H),4.54-4.45(m,1H),3.80(t,J=6.2Hz,2H),3.63-3.41(m,18H),3.18(dd ,J=11.5,5.7Hz,4H),2.67(t,J=6.1Hz,2H),2.54(s,2H),2.33(s,3H),1.57(s,3H).

[0091] Example 8

[0092] UV-Vis absorption curve

[0093] The UV absorption spectra of six phthalocyanine photo-PROTAC compounds 5a-5e (10 μM) in water were measured using a UV-visible spectrophotometer. The corresponding UV standard curves were drawn based on the absorbance at the maximum absorption wavelengths of 633 nm and 683 nm.

[0094] like Figure 1 As shown, the UV-visible absorption spectra of the six molecules are basically consistent, and the maximum absorption peaks are all located near 680nm.

[0095] Example 9

[0096] In vitro cellular phototoxicity assay

[0097] CCK-8 assay was used to evaluate in vitro cytotoxicity. T24 cells were cultured at 1.12×10 4 Each well of a 96-well plate was inoculated with 5% CO 2 , incubated in DMEM medium in the dark for 24 h at 37 °C. Then, replaced with DMEM medium containing different concentrations of drugs, incubated in the dark for 9 h, 680 nm (20.4 J / cm 2 ) for 10 min, and then incubate in the dark for 15 h. Then, CCK-8 solution was added, and the absorbance of each well was measured using an enzyme-labeled instrument.

[0098] like Figure 2 As shown, in bladder cancer cells T24, the total reactive oxygen species production capacity of the Linker molecule was significantly stronger than that of ZnPc-JQ1 (compound 4), and the photosensitive tumor killing IC 50 The concentration ranged from 1351 nM (IC of ZnPc-JQ1) 50 ) was significantly reduced to 41nM (ZnPc-O1 -JQ1 IC 50 ) below, the activity increased by more than 33 times. As the linker lengthened, the IC 50 The concentration gradually decreased and 3 -JQ1 reaches basic stability, and its IC 50 The concentration is 22nM, which has ideal photosensitizing activity in killing tumor cells.

[0099] Example 10

[0100] In vitro cytotoxicity assay

[0101] T24 cells were cultured at 1.12 × 10 4 Each well of a 96-well plate was inoculated with 5% CO 2 Incubate the cells in DMEM medium in the dark for 24 hours at 37°C. Then replace with DMEM medium containing different concentrations of drugs. After incubation in the dark for 9 hours, add CCK-8 solution and measure the absorbance of each well with an enzyme-labeled instrument.

[0102] like Figure 3 As shown, under dark conditions, ZnPc-O 3 -IC of JQ1 killing T24 cells 50 Greater than 2×10 5 nM. Binding Figure 2 , ZnPc-O 3 - The PI (phototoxicity index: the ratio of IC50 under dark conditions to IC50 of the drug under light conditions) value of JQ1 is greater than 9091, which is much higher than ZnPc-JQ1 (PI greater than 148) and approved phthalocyanine photosensitizers (PI=725), see ACS Med. Chem. Lett. 2021, 12, 502-507, ZnPc-O 3 -JQ1 has high PI value characteristics, ( Figure 4 ), drugs with high PI values ​​usually have better solubility, which helps the drugs reach the target tissue or target cells more effectively and has lower toxicity to non-target tissues. High PI value photosensitizers have low toxicity to cells in the dark, so under non-illumination conditions, they have less impact on normal tissues and cells, thereby reducing side effects during treatment and improving the safety of treatment. High PI value photosensitizers have a stronger selective killing effect on cancer cells when selectively irradiating tumor tissues.

[0103] Embodiment 11

[0104] Detection of relative expression level of BRD4 in T24 cells without light treatment

[0105] T24 cells were cultured at 5 × 10 5 Each well of a 6-well plate was inoculated with 5% CO 2 , and incubate in DMEM medium in the dark for 24 h at 37°C. Then, ZnPc-JQ1 (30 nM), ZnPc-O 3 -JQ1 (30 nM) and dBET1 (1 μM) were incubated in the dark for 9 h, and the level of BRD4 in the cells was detected by immunoblotting analysis.

[0106] like Figure 5 As shown, when not irradiated, ZnPc-JQ1 (30 nM) and ZnPc-O 3 -JQ1 (30 nM) had no BRD4 degradation activity.

[0107] Example 12

[0108] Detection of relative expression level of BRD4 in T24 cells treated with light

[0109] T24 cells were cultured at 5 × 10 5 Each well of a 6-well plate was inoculated with 5% CO 2 , and incubate in DMEM medium in the dark for 24 h at 37°C. Then, ZnPc-JQ1 (30 nM), ZnPc-O 3 -JQ1 (30 nM) and dBET1 (1 μM) were incubated in the dark for 9 h, and the concentration was 680 nm (20.4 J / cm 2 ) for 10 min, and then incubated in the dark for 15 h. The level of BRD4 in cells was detected by immunoblotting analysis.

[0110] like Figure 6 As shown, after illumination, ZnPc-O 3 -JQ1 has a significant effect on the degradation of BRD4. Figure 5 The drug does not generate ROS when not exposed to light, and only when it is exposed to light will it damage cells. 3 -The degradation of BRD4 by JQ1 does not originate from the indiscriminate protein damage of ROS, but from the precise destruction of ROS after JQ1 binds to BRD4. 3 -The degradation rate of BRD4 by JQ1 can reach 87%, far exceeding the degradation effect of dBET1 under 10000nM conditions (degradation rate is 35%).

[0111] Example 13

[0112] Detection of reactive oxygen species in cells

[0113] T24 cells were collected at 5×10 5 Each well of a 6-well plate was inoculated with 5% CO2 , and incubated in DMEM medium in the dark for 24 h at 37 °C. 3 -JQ1 (1 μM). 3 -JQ1 (1 μM) was treated for 9 h, and then incubated with 5 μM probe fluorescent dye DCFH-DA (Biyuntian, S0034S) for 4 h in the dark; then, 680 nm (20.4 J / cm 2 ) The cells were illuminated for 10 min, placed in an incubator and incubated for 30 min, then washed twice with PBS and the fluorescence intensity of the probe was detected using an inverted fluorescence microscope. The relative content of ROS in T24 cells was determined using the fluorescent dye DCFH-DA. The fluorescence intensity of the probe was proportional to the ability to produce reactive oxygen species. Figure 7 As shown, in T24 cells, ZnPc-O 3 -JQ1 has a significantly stronger ability to produce reactive oxygen than ZnPc-JQ1, indicating that ZnPc- 3 -JQ1 has higher photosensitizing activity in killing tumor cells.

[0114] Embodiment 14

[0115] HIF-1α protein inhibition experiment

[0116] T24 cells were cultured at 5×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and incubated in 5% CO 2 Incubate the cells in DMEM medium at 37°C in the dark for 24 h. 3 -JQ1 (30 nM) and incubated in the dark (O 2 concentration of 1%) after 9h; 680nm (20.4J / cm 2 ) The cells were illuminated for 10 min and then cultured in the dark for 15 min. Protein was extracted and WB experiment was performed to detect the level of HIF-1α protein in each group of cells (the internal reference was β-tubulin).

[0117] like Figure 8 As shown, ZnPc-O 3 -JQ1-induced degradation of BRD4 affects the transcription of HIF-1α and significantly reduces the level of HIF-1α in tumor cells compared with the control group, indicating that ZnPc-O 3 -JQ1 not only degrades BRD4, but also downregulates HIF-1α, which can destroy the therapeutic resistance caused by increased hypoxia, thereby achieving synergistic enhancement of PDT and PROTAC.

[0118] Embodiment 15

[0119] Intracellular GSH level detection:

[0120] T24 cells were cultured at 5×10 5 Cells were seeded per well in a six-well plate and incubated in 5% CO 2 Incubate in DMEM medium at 37°C for 24 h in the dark. 3 -JQ1 was incubated in the dark for 9 hours, 680nm (20.4J / cm 2 ) for 10 min. Subsequently, GSH (5 μM) probes were added to the cells, and after incubation in the dark for 2 h, the fluorescence intensity of the probes in the cells was detected using a flow cytometer and a fluorescence inverted microscope.

[0121] like Fig. 9 As shown, ZnPc-O 3 -JQ1 group GSH increased only 1.37 times compared with the control group, indicating that ZnPc-O 3 -JQ1 can negatively regulate GSH synthesis by downregulating GCL and inhibiting the synthesis of GSH.

[0122] Example 16

[0123] Study on the influence of PDT on GCL

[0124] T24 cells were collected at 5×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate and incubated in 5% CO 2 At 37°C, the cells were incubated in DMEM medium in the dark for 24 h and then incubated with ZnPc-JQ1 (30 nM) and ZnPc-O 3 -JQ1 (30 nM) was incubated in the dark for 9 h; 680 nm (20.4 J / cm 2 ) The cells were illuminated for 10 minutes and then cultured in the dark for 15 minutes; proteins were extracted and WB experiments were performed to detect the GCL protein levels in each group of cells (the internal reference was GAPDH).

[0125] like Fig.10 As shown in the figure, GCL protein was significantly degraded, indicating that ZnPc-O 3 -JQ1 can downregulate GCL, thereby affecting the synthesis of GSH.

[0126] It can be seen from Examples 15 and 16 that the ZnPc-O 3 -JQ1 downregulates GCL and inhibits the synthesis of GSH, indicating that it can destroy the antioxidant defense barrier of cells and has better anti-tumor activity.

[0127] Embodiment 17

[0128] In vivo anti-tumor assay

[0129] MB49 cells were injected into the lateral hind limbs of C57BL / 6 female mice (6-7 weeks, 20-22 g) to construct a subcutaneous transplant tumor model. The length and width of the tumor were measured with a vernier caliper and calculated according to the formula (Volume = 0.5 × Length × Width 2 , where Length and Width represent the length and width of the tumor, respectively) to calculate the tumor volume. When the tumor grows to 100 mm on one side, 3 Then conduct treatment experiments.

[0130] The mice were divided into PBS, dBET1, Cis-platin, ZnPc-JQ1+L and ZnPc-O 3 -JQ1+L group. The mice were injected with drug (3 mg / kg) via tail vein every other day and irradiated with 665 nm laser (25 mW / cm 2 ) irradiated the tumor site for 5 min and monitored the tumor volume.

[0131] like Fig.11 As shown in the figure, in terms of tumor inhibition, compared with the PBS group, the drug-treated groups had different degrees of anti-tumor effects, among which ZnPc-O 3 The -JQ1+L group had the best effect in inhibiting tumor growth, which was significantly different from the ZnPc-JQ1+L group and other groups.

Claims

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

2. The phthalocyanine-based photo-PROTAC drug according to claim 1, characterized in that The n is preferably any integer of 1, 2, 3, 4, 6, and its structure is:

3. A method for preparing a phthalocyanine-based photo-PROTAC drug according to claim 2, characterized in that: The steps include: (1) Synthesis of Intermediate 1: 4-nitrophthalonitrile, 4-(boc-amino)phenol and potassium carbonate are uniformly mixed in an organic solvent to react to obtain intermediate 1; (2) Synthesis of compound 2 (ZnPc): Compound 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; (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) were dissolved in an organic solvent, stirred for reaction, and then N,N-diisopropylethylamine (DIPEA) was added and reacted at room temperature to obtain compound 4; (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, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3a. Compound 3a was 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]propionic acid and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3b; Compound 3b reacts 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-azaheptadecan-17-oic acid and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3c; 3c was reacted with JQ1 to obtain compound 5c; (7) Synthesis of compound 5d (ZnPc-O4-JQ1): Compound 2, 1-tert-butyl 5,8,11,14-tetraoxa-2-azaheptadecanedioate and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia BOC was removed by stirring to obtain compound 3d. 3d reacts with JQ1 to give compound 5d; (8) Synthesis of compound 5e (ZnPc-O6-JQ1): Compound 2, 21-(Boc-amino)-4,7,10,13,16,19-hexaoxahene-11-carbonic acid and HATU were dissolved in an organic solvent, stirred for reaction, and then DIPEA was added to react at room temperature. After the reaction was completed, an organic solvent and trifluoroacetic acid were added, and the ammonia 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 preparation method according to claim 3, characterized in that: 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° C., and the stirring reaction time is 6-7 hours.

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

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 hours.

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 hours.

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 according to claim 1 or 2 in the preparation of anticancer drugs.

10. The use according to claim 9, characterized in that: The phthalocyanine-based photo-PROTAC drug is used as a photosensitizer in the preparation of anticancer drugs.

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