Light-controlled heat shock protein 90 degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a light-controlled heat shock protein 90 degradation agent as well as a preparation method and application thereof, and belongs to the technical field of medicines. The degradation agent is synthesized by coupling a BIIB021 derivative of targeted Hsp90 and a dihydroporphyrin photosensitizer. The compound can degrade Hsp90 protein in tumor cells, client protein HIF-1alpha of the Hsp90 and Hsp70 protein capable of forming a compound with the Hsp90 in a light-controlled manner. The compound is expected to solve the problems of tumor drug resistance and the like of the traditional Hsp90 inhibitor and Hsp90 PROTACs, and the hypoxic microenvironment in tumors is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a light-controlled heat shock protein 90 degradation agent, a preparation method and application thereof. BACKGROUND

[0002] The classical approach in drug discovery is to use high-affinity small molecules to modulate the activity of target proteins, which is a space-occupying driven way, that is, the inhibitor binds to its target and occupies the binding site, thereby inhibiting the function of the target. However, 80-85% of proteins cannot be druggable due to the lack of available binding pockets and suitable chemicals. Targeted protein degradation technology (TPD) provides a new treatment option by using the endogenous protein degradation system to induce the degradation of the target protein. Compared with traditional small molecule inhibitors, TPD only needs to be combined with the target protein as an adhesive to induce protein degradation, and does not require high affinity with the protein, thus having the potential to target the undruggable proteome. Targeted protein degradation technology includes hydrophobic tags, molecular glue and PROTACs. However, traditional protein degradation technology has off-target effects, that is, it can degrade proteins throughout the body, thereby causing toxic side effects, and thus a method is needed to controllably degrade the target protein.

[0003] The biological function of a protein can be affected by photochemical reactions. Many researchers have reported that direct or indirect light-induced processes can cause enzyme inactivation. The primary photochemical reaction of a protein can cause modification of the side chain of a specific residue, thereby affecting the conformation and structure of the protein. Proteins oxidized by free radical-radical reactions or nucleophilic reactions can aggregate to form inclusion bodies, which have a direct impact on cells. The formation of aggregates is usually irreversible (except for those formed entirely by disulfide bonds), and can exceed the protein degradation capacity of cellular systems (such as proteasomes and lysosomes) that remove damaged proteins. Protein photooxidation initially mediates the oxidation of susceptible amino acids, leading to changes in the molecular mass or conformation of the protein, thereby affecting the function, thermal stability or proteasome function of the protein.

[0004] Photodynamic therapy (PDT) has become a treatment for tumors and non-malignant lesions. Photosensitizer (PS), light and ROS are important components of PDT. Under light irradiation, photosensitizer is activated and generates highly active singlet oxygen from oxygen molecules. Due to the short half-life (about 3 μs), the damage caused by singlet oxygen is highly localized. In cancer treatment, PDT can directly destroy tumor cells and surrounding vasculature, and activate immune response against tumors. The commonly used photosensitizers are 5-aminolevulinic acid (5-ALA), 2-[1-hexyloxyethyl]-2-diethyl vinyl pyropheophorbide-a (HPPH), mono-aspartyl chlorin e6 (NPe6), pyropheophorbide-a (Ppa) and chlorin e6 (Ce6) and the like.

[0005] Heat shock proteins are molecular chaperones that are ubiquitously expressed and evolutionarily conserved in organisms, and are essential for maintaining cellular protein homeostasis. Under cellular stress, members of the 90 kDa heat shock protein (Hsp90) family maintain cellular homeostasis by promoting the folding, activation, and protection of client proteins that are essential for cellular functions such as growth, survival, and differentiation. Therefore, Hsp90 plays a key role in tumorigenesis by maturing or activating client proteins associated with cancer. Studies have shown that Hsp90 is overexpressed in the serum or tissues of many malignant tumor patients, such as breast cancer (non-patent literature 1), lung cancer (non-patent literature 2), melanoma (non-patent literature 3), prostate cancer (non-patent literature 4), cervical cancer (non-patent literature 5), gastric cancer (non-patent literature 6), colon cancer (non-patent literature 7), liver cancer (non-patent literature 8), and pancreatic cancer (non-patent literature 9). In addition, correlation analysis of some tumor-related clinical studies found that Hsp90 was positively correlated with metastasis and malignancy (non-patent literature 10), and can be used as a biomarker for diagnosing tumors (non-patent literature 11). However, so far, most Hsp90 inhibitors can regulate the degradation of Hsp90 downstream clients by inhibiting Hsp90 function, but few inhibitors can regulate the level of Hsp90.

[0006] (Non-patent literature 1) LEE Y C, CHANG W W, CHEN Y Y, et al. Hsp90a Mediates BMI1 Expression in Breast Cancer Stem / Progenitor Cells through Facilitating Nuclear Translocation of c-Myc and EZH2 [J]. International Journal of Molecular Sciences, 2017, 18(9): 1986.

[0007] (Non-patent literature 2) SHI Y, LIU X, LOU J, et al. Plasma Levels of Heat Shock Protein 90 Alpha Associated with Lung Cancer Development and Treatment Responses [J]. Clinical Cancer Research, 2014, 20(23): 6016-6022.

[0008] (Non-patent literature 3) MCCARTHY M M, PICK E, KLUGER Y, et al. HSP90 as a marker of progression in melanoma [J]. Annals of Oncology, 2008, 19(3): 590-594.

[0009] (Non-patent literature 4) BURGESS E F, HAM A L, TABB D L, et al. Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes [J]. PROTEOMICS - Clinical Applications, 2008, 2(9): 1223-1233.

[0010] (NPL 5) ZENG J, HE S L, LI L J, et al. Hsp90 up-regulates PD-L1 to promote HPV-positive cervical cancer via HER2 / PI3K / AKT pathway [J]. Molecular Medicine, 2021, 27(1).

[0011] (NPL 6) WANG H, DENG G, AI M, et al. Hsp90ab1 stabilizes LRP5 to promote epithelial-mesenchymal transition via activating of AKT and Wnt / β-catenin signaling pathways in gastric cancer progression [J]. Oncogene, 2018, 38(9): 1489-1507.

[0012] (NPL 7) QIU C, SHEN X, LU H, et al. Combination therapy with HSP90 inhibitors and piperlongumine promotes ROS-mediated ER stress in colon cancer cells [J]. Cell death discovery, 2023, 9(1): 375.

[0013] (NPL 8) FU Y, XU X, HUANG D, et al. Plasma Heat Shock Protein 90alpha as a Biomarker for the Diagnosis of Liver Cancer: An Official, Large-scale, and Multicenter Clinical Trial [J]. EBioMedicine, 2017, 24: 56-63.

[0014] (Non-patent document 9) JIANG H, DUAN B, HE C, et al. Cytoplasmic HSP90α expression is associated with perineural invasion in pancreatic cancer [J]. International journal of clinical and experimental pathology, 2014, 7(6): 3305-3311.

[0015] (Non-patent document 10) WANG X, SONG X, ZHUO W, et al. The regulatory mechanism of Hsp90α secretion and its function in tumor malignancy [J]. Proceedings of the National Academy of Sciences, 2009, 106(50): 21288-21293.

[0016] (Non-patent document 11) WEI W, LIU M, NING S, et al. Diagnostic value of plasma HSP90α levels for detection of hepatocellular carcinoma [J]. BMC Cancer, 2020, 20(1). SUMMARY

[0017] To solve the problems existing in the prior art, the present application provides a kind of light-controlled heat shock protein 90 degradation agent and preparation method and application.The degradation agent is combined with the BIIB021 derivative of target Hsp90 and dihydrogen porphyrin photosensitizer.The compound can photodegrade Hsp90 protein, Hsp90 client protein HIF-1 alpha and Hsp70 protein that can form complex with Hsp90 in tumor cells.The compound is expected to solve the problems of tumor drug resistance and other problems existing in traditional Hsp90 inhibitors and Hsp90 PROTACs, and improve the hypoxic microenvironment in tumor.

[0018] The technical scheme of the present application: a kind of compound, the structure of the compound is as follows:

[0019] ;

[0020] Wherein, R0 is H、 ;

[0021] R1 is H, ;

[0022] and R0 and R1 are not simultaneously H;

[0023] R2 is , , , CH2;

[0024] R3 is , ;

[0025] wherein x is an integer from 1 to 10, and y is an integer from 1 to 5;

[0026] R4 is a pyropheophorbide-a dihydrohalocyanine e6 derivative;

[0027] the pyropheophorbide-a is ;

[0028] the dihydrohalocyanine e6 derivative is , , ;

[0029] wherein R5, R6 and R7 are each independently H, ;

[0030] wherein m is an integer from 0 to 10.

[0031] Further, R0 is ; R1 is H.

[0032] Further, R2 is .

[0033] Further, R3 is .

[0034] Further, x is an integer from 2 to 5.

[0035] Further, the structure of the compound is selected from:

[0036] , , .

[0037] Use of the above compound or pharmaceutically acceptable carboxylate salt thereof in the preparation of an antitumor drug.

[0038] Further, the tumor comprises breast cancer, lung cancer, melanoma, prostate cancer, cervical cancer, gastric cancer, colon cancer, liver cancer and pancreatic cancer.

[0039] A pharmaceutical composition comprising the compound described above or a pharmaceutically acceptable carboxylate salt thereof.

[0040] The present application has the beneficial effect that the BIIB021 derivative targeting Hsp90 is combined with a dihydrogen porphyrin photosensitizer. The compound can photodegrade Hsp90 protein, Hsp90 client protein HIF-1α and Hsp70 protein which can form a complex with Hsp90 in tumor cells, and is expected to solve the problems of tumor drug resistance and the like existing in traditional Hsp90 inhibitors and Hsp90 PROTACs, and improve the hypoxic microenvironment in tumors. Moreover, compared with traditional PROTAC and other targeted protein degradation technologies, the light-controlled protein degradation technology of the present application has lower toxicity in the absence of light, and only plays a role in degrading proteins after light irradiation. In addition, the photosensitizer parent nucleus has certain tumor targeting ability, and is expected to avoid the systemic toxicity caused by off-target effects of traditional degrading agents. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a high-performance liquid chromatogram of compound Y-1.

[0042] Figure 2 is a fluorescence quenching spectrum of compound Y-1 and recombinant Hsp90 protein incubated at different concentrations (b) F0 / F curve at different temperatures.

[0043] Figure 3 is a protein degradation SDS-PAGE result chart in solution (a) time-dependent degradation result chart (b) concentration-dependent degradation result chart.

[0044] Figure 4 is the compound toxicity of BIIB021, CHPpa and Y-1 after 24h administration in different cell lines. (a) Dark toxicity of the compound in 4T1 cell line; (b) light toxicity of the compound in 4T1 cell line; (c) dark toxicity of the compound in MCF-7 cell line; (d) light toxicity of the compound in MCF-7 cell line.

[0045] Figure 5 is the uptake of CHPpa and Y-1 in 4T1 cell line at different administration times. (a) Flow cytometry uptake chart of CHPpa; (b) flow cytometry uptake column chart of CHPpa; (c) flow cytometry uptake chart of Y-1; (d) flow cytometry uptake column chart of Y-1.

[0046] Figure 6Figure 1 shows the results of Western Blot analysis of the expression of Hsp90a and HIF-1a in cells treated with compound Y-1. Figure 2 shows the results of Western Blot analysis of the expression of Hsp90a and HIF-1a in cells treated with compound BIIB021. Figure 3 shows the results of Western Blot analysis of the expression of Hsp90a and HIF-1a in cells treated with compound CHPpa.

[0047] Figure 7 Figure 1 shows the results of Western Blot analysis of the expression of Hsp90a and HIF-1a in cells treated with compound Y-1. Figure 2 shows the results of Western Blot analysis of the expression of Hsp90a and HIF-1a in cells treated with compound BIIB021. Figure 3 shows the results of Western Blot analysis of the expression of Hsp90a and HIF-1a in cells treated with compound CHPpa. DETAILED DESCRIPTION

[0048] The present application is described in detail below by way of examples. It is necessary to point out that the following examples are only used to further illustrate the present application, but not limited thereto, and unless otherwise stated.

[0049] A class of light-controlled Hsp90 protein degradation agents targeting Hsp90 protein with BIIB021 derivatives, the structure is as follows:

[0050] .

[0051] wherein R0, R1are independently selected from H, ; and both are not H at the same time.

[0052] R2is , , , CH2.

[0053] R3is , .

[0054] wherein x is an integer from 1 to 10, and y is an integer from 1 to 5.

[0055] R4is a self-made compound Chenghai Pyropheophorbide a (CHPpa, equivalent to commercially available Pyropheophorbide a), a self-made compound Chenghai Chlorin e6 (CHC, equivalent to commercially available Chlorin e6) derivative;

[0056] The pyrophylloxera-a described in CHPpaCHPpa is... ;

[0057] The dihydroporphyrin E6 derivative is , , ;

[0058] Among them, R5, R6, and R7 are each independently H, ;

[0059] Where m is an integer between 0 and 10.

[0060] In some specific compounds, R0 is R1 is H.

[0061] In some specific compounds, R2 is .

[0062] In some specific compounds, R3 is .

[0063] In some specific compounds, x is an integer between 2 and 5.

[0064] Some specific compounds, the structures of which are selected from:

[0065] , , .

[0066] This invention also provides a method for preparing a compound, comprising the following steps:

[0067]

[0068] Example I: Compound a reacts with compound A in a solvent to give compound b. Compound b reacts in ethyl hydrochloride solution to remove the tert-butyloxycarbonyl group to give compound c. Compound c undergoes a condensation reaction with compound B to give the target compound.

[0069] The definitions of R2, R3, and R4 are the same as those in the above structural formula.

[0070] Y1 and Y2 are independent of each other. H, -NH2, -OH, -COOH, F, Cl, Br, or I.

[0071] Z is H, -NH2, -OH, -COOH, F, Cl, Br, or I.

[0072] A method for preparing a compound, comprising the following steps:

[0073] A specific preparation method of a light-controlled Hsp90 protein degradation agent, comprising the following steps:

[0074] Compound a is used as raw material, dissolved in N, N-dimethylformamide, tetraphenylphosphonium palladium (0.01-0.1 equiv.), compound A (1-2 equiv.), cuprous iodide (0.1-1 equiv.) and triethylamine (2-10 equiv.) are added, and compound b is obtained after 1-10 h. Compound b is dissolved in hydrochloric acid ethyl acetate solution, and compound c is obtained after 10 min-1.5 h. Compound c is added with compound B (1-4 equiv.), 1-hydroxybenzotriazole (1-3 equiv.), 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride (1-4 equiv.), triethylamine (1-10 equiv.) and N, N-dimethylformamide, and compound Y-1 is obtained after 2-10 h.

[0075] The light-controlled Hsp90 protein degradation agent described above is used to prepare a targeted antitumor drug by targeting the degradation of Hsp90 as an active part. The tumor includes breast cancer, lung cancer, melanoma, prostate cancer, cervical cancer, gastric cancer, colon cancer, liver cancer and pancreatic cancer.

[0076] The following detailed description of the technical solutions will describe the specific embodiments of the present application:

[0077] Example 1

[0078]

[0079] (1) Synthesis of compound 2

[0080] Accurately weigh compound 1 (165.5 mg, 1 equiv.), add tetraphenylphosphonium palladium (0.05 equiv.), N-Boc-butyn-4-amine (2 equiv.), cuprous iodide (0.3 equiv.) and triethylamine (5 equiv.), then add DMF (9.5 mL), set the reaction temperature to 70°C, and the reaction is complete after 3 h. A large amount of ice water is added to the reaction solution, and a large amount of yellow solid is precipitated. After suction filtration, column chromatography separation, vacuum concentration, the target compound 2 is obtained as a white solid with a yield of 70%. 1H NMR (400 MHz, Methanol-d4) δ 8.49 (s, 1H), 7.62 (s, 1H), 5.67 (s, 2H), 4.13 (s, 3H), 3.22 (s, 2H), 2.96 - 2.87 (m, 2H), 2.49 (s, 3H), 2.41 (d, J = 10.7 Hz, 3H). HR-MS for C 24 H 29 Cl2IN6O: (calculated) 420.1232; (found) 384.2224 [M+H-Cl] + .

[0081] (2) Synthesis of compound 3

[0082] Compound 2 (20.0 mg, 1 equiv.) was accurately weighed in a vial, and the pre-prepared hydrochloric acid / ethyl acetate reaction solution (1 mL) was added. The reaction was complete after 30 min of reaction at 0°C. The reaction solution was concentrated under reduced pressure, and the reaction solution was dissolved in water, and the aqueous phase was washed with ethyl acetate three times, and then the aqueous phase was concentrated under reduced pressure to obtain the target compound 3 as a yellow oil (18.6 mg), with a yield of 99%. 1 H NMR (400 MHz, Methanol-d4) δ 8.49 (s, 1H), 7.62 (s, 1H), 5.67 (s, 2H), 4.13 (s, 3H), 3.22 (s, 2H), 2.96 - 2.87 (m, 2H), 2.49 (s, 3H), 2.41 (d, J = 10.7 Hz, 3H). HR-MS for C 19 H 22 Cl2IN6O: (calculated) 420.1232; (found) 384.2224 [M+H-Cl] + .

[0083] (3) Synthesis of compound Y-1

[0084] Compound CHPpa (30.5 mg, 1 equiv.) was accurately weighed in a pear-shaped flask, 1-hydroxybenzotriazole (2 equiv.), l-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (2 equiv.), triethylamine (3 equiv.), compound 3 (2 equiv.) and DMF (5 mL) were added, and the reaction was complete after 12 h at room temperature. Ethyl acetate was added to the reaction, and the ethyl acetate phase was washed three times with water, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and then column chromatography was used to isolate the target compound Y-1 (12.8 mg) as a dark green solid, with a yield of 25%. The high-performance liquid chromatography test of Y-1 used reverse-phase chromatography with a condition of 95% methanol / 5% 1% acetic acid in water, and a detection wavelength of 410 nm. The final test result was: purity 97%. 1 H NMR (400 MHz, Chloroform-d) δ 9.29 (s, 1H), 9.23 (s, 1H), 8.55 (s, 1H), 8.06 (s, 1H), 7.93 (s, 1H), 6.78 (s, 1H), 6.24 (d, J = 17.8 Hz, 1H), 6.12 (d, J = 11.3 Hz, 1H), 5.87 (s, 1H), 5.22 (s, 1H), 5.07 (s, 2H), 4.54 (s, 1H), 4.31 (s, 1H), 3.65 (s, 3H), 3.57 (s, 2H), 3.37 (s, 3H), 3.35 (s, 3H), 3.19 (s, 3H), 2.74 (d, J = 13.9 Hz, 2H), 2.41 (s, 6H), 2.13 (s, 3H), 1.96 (s, 3H), 1.79 (s, 3H), 1.61 (s, 3H), -1.75 (s, 1H). HR-MS (ESI) m / z Calcd for C 52 H 53 ClIN 10 O3: (calculated) 900.3991; (found) 901.4068 [M+H] + 。

[0085] Example 2

[0086]

[0087] (1) Synthesis of compound 4

[0088] Compound 1 (165.5 mg, 1 equiv.) was weighed accurately in a flask, and then tetraphenylphosphonium palladium (0.06 equiv.), N-Boc-pent-5-yn-l-amine (2 equiv.), cuprous iodide (0.3 equiv.) and triethylamine (5 equiv.) were added, followed by the addition of DMF (10 mL), and the reaction temperature was set to 70 °C. After 4 h, the reaction was complete. A large amount of ice water was added to the reaction solution, and a large amount of yellow solid was precipitated. After suction filtration, the target compound 4 was separated by column chromatography and obtained as a white solid with a yield of 75%. HR-MS for C 24 H 29 ClIN6O3: (calculated) 498.2146; (found) 499.2152 [M+H] + .

[0089] (2) Synthesis of compound 5

[0090] Compound 4 (15.0 mg, 1 equiv.) was weighed accurately in a flask, and then hydrochloric acid / ethyl acetate solution (1 mL) prepared in advance was added at 0 °C. After 15 min, the reaction was complete. The reaction solution was concentrated under reduced pressure, and then the reaction solution was dissolved in water and washed with ethyl acetate three times. The aqueous phase was then concentrated under reduced pressure to obtain the target compound 5 as a yellow oil with a yield of 97%. HR-MS for C 19 H 22 Cl2IN6O: (calculated) 434.1389; (found) 399.1793 [M+H-Cl] + .

[0091] (3) Synthesis of compound Y-2

[0092] Compound CHPpa (50 mg, 1 equiv.) was weighed accurately in a flask, and then 1-hydroxybenzotriazole (2 equiv.), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (2 equiv.), triethylamine (2 equiv.), compound 5 (1.5 equiv.) and DMF (5 mL) were added, and the reaction was complete after 12 h at room temperature. Ethyl acetate was added to the reaction solution, and then the ethyl acetate phase was washed with water three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The target compound Y-2 was separated by column chromatography and obtained as a dark green solid with a yield of 30%. HR-MS (ESI) m / z Calcd for C 52 H 53 ClIN 10 O3:(calculated) 914.4147; (found) 915.4205 [M+H]+ .

[0093] Example 3

[0094]

[0095] Compound CHC (45.5 mg, 1 equiv.) was weighed accurately in a flask, 1-ethyl-(3- dimethylaminopropyl)carbodiimide hydrochloride (2 equiv.), triethylamine (5 equiv.), compound 3 (2 equiv., synthesized as described in Example 1) and DMF (5 mL) were added, and the reaction was complete after 12 h at room temperature. Ethyl acetate was added to the reaction, and the ethyl acetate phase was washed three times with water, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The target compound Y-3 was isolated as a dark green solid by column chromatography, with a yield of 33%. HR-MS (ESI) m / z Calcd for C 52 H 53 ClIN 10 O3:(calculated) 962.3995; (found) 963.3991 [M+H] + .

[0096] Example 4

[0097] Performance test experiment: The purity of compound Y-1 was characterized by high performance liquid chromatography, with a purity of more than 97% (HPLC). Figure 1 .

[0098] Example 5

[0099] The compound Y-1 prepared in Example 1 was subjected to a solution binding experiment with Hsp90 protein.

[0100] Recombinant human Hsp90a protein was added to a 96-well plate to a final concentration of 0.1 μM, then compound Y-1 was added to a final concentration of 25 μM, the same volume of DMSO as the drug was added to the negative control group, and no compound or DMSO was added to the blank group. After incubation at room temperature for 30 min, the fluorescence was measured using a microplate reader. The excitation wavelength was fixed at 262 nm, and the emission wavelength was tested from 310 nm to 360 nm, as shown in Figure 2 The results showed that the fluorescence quenching of the protein could be detected after incubation of compound Y-1 with recombinant human Hsp90a protein, and the quenching degree was concentration-dependent. The fluorescence quenching constant was measured at three temperatures of 298 K, 303 K and 308 K, and it was found that the quenching constant decreased with increasing temperature, indicating that the fluorescence quenching of the protein was due to the binding of the compound to the protein.

[0101] Example 6

[0102] The compound Y-1 prepared in Example 1 was subjected to Hsp90 protein degradation experiment in solution.

[0103] (1) Time gradient degradation

[0104] The recombinant human Hsp90a protein was diluted and added with compound Y-1 to have a final concentration of 0.78 μM, and the negative control group was added with DMSO, and incubated at room temperature for 30 min, and then irradiated under red light for 0 min, 2 min, 5 min, 10 min and 15 min, respectively, and the control group was not irradiated, and then subjected to SDS-PAGE experiment.

[0105] (2) Concentration gradient degradation

[0106] The recombinant human Hsp90a protein was diluted and added with compound Y-1 to have a final concentration of 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM and 25 μM, and the negative control group was added with DMSO, and incubated at room temperature for 30 min, and then irradiated under red light for 5 min, and the control group was not irradiated, and then subjected to SDS-PAGE experiment.

[0107] The experimental results (Figure 3) show that 0.78 μM of Y-1 can cause obvious degradation of recombinant human Hsp90a protein after irradiation for 2 min, and the protein is almost completely degraded after irradiation for 5 min, while the protein in the non-drug group and the non-irradiation group is not degraded, proving that Y-1 can bind to recombinant human Hsp90a protein and degrade the protein under light in PBS.

[0108] Example 7

[0109] The 4T1 and MCF-7 cell toxicity experiments were performed on compound Y-1 and CHPpa prepared in Example 1 and BIIB021 to explore the dosing amount. Among them, the structure of BIIB021 is as follows: .

[0110] The two cells were inoculated in a 96-well plate and cultured. After the cells adhered, different concentrations of compound solutions prepared with culture medium were added to the 96-well plate, and placed in the incubator for 24 h and irradiated under red light for 5 min. When measuring the dark toxicity, no operation was required, and after a period of time, 20 μL of MTT solution was added to each well under dark conditions, and placed back in the incubator for incubation for 4 h. After incubation, the culture solution was aspirated, DMSO was added to each well, and the absorbance of each well at 570 nm was measured and the survival rate was calculated. The experimental results show that Figure 4) Compound Y-1 has the lowest dark toxicity in two cell lines (no toxicity below 3.13 μM); the cytotoxicity of compound Y-1 is greatly improved compared with non-irradiation, and has higher phototoxicity to 4T1 cells above 12 nM and no toxicity below 6 nM; and has higher phototoxicity to MCF-7 cell line above 390 nM and no toxicity below 195 nM.

[0111] Example 8

[0112] A 4T1 cell uptake experiment was performed on compound Y-1 prepared in Example 1 and CHPpa and BIIB021 to explore the administration time.

[0113] The digested cells were inoculated in a 24-well plate and cultured at 37°C until adherent. The drug prepared with the culture medium was added to the well plate, and the final concentration was 2 μM. The drug was administered at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, and 24 h, respectively. After the drug was removed, trypsin digestion was added to each well, and centrifugation was performed at 4°C. The supernatant was discarded, and 1% paraformaldehyde was added for fixation before flow detection. The experimental results (Figure 5) show that Y-1 is slowly taken up by 4T1 cells within 12 h, and the uptake amount increases sharply at 12 h-18 h, and reaches a plateau around 18 h-24 h, i.e., the maximum uptake.

[0114] Example 9

[0115] A 4T1 cell experiment was performed on compound Y-1 prepared in Example 1 and CHPpa and BIIB021 to degrade Hsp90 protein and other related proteins in the cells.

[0116] The cells were inoculated in a 12-well plate and cultured. After the cells adhered, they were treated with different concentrations of drugs for 24 h, then irradiated with red light for 5 min, and then placed in a low-oxygen incubator for low-oxygen culture. 5xLoading Buffer was diluted to 2xwith RIPA containing PMSF, and the cells were lysed with the solution, followed by gel electrophoresis. After electrophoresis, PVDF membrane was used for transmembrane under the condition of 300 mA, and after completion, the prepared blocking solution was blocked for 2 h. After blocking, the corresponding primary antibody was incubated at 4°C overnight, and the next day, the secondary antibody was added and incubated for 2 h. After incubation, the developing solution was prepared for development.

[0117] The experimental results (Figure 6) show that compound Y-1 has a better effect on degrading Hsp90 protein and other related proteins in 4T1 cells than CHPpa and BIIB021. Figure 6) indicated that Y-1 could significantly degrade Hsp90 under the condition of higher than 1.5 nM and light for 5 min, and could significantly degrade HIF-1a under the condition of higher than 1.5 nM and light for 5 min, while the control compounds CHPpa and BIIB021 had no effect on the expression level of the two proteins under the condition of lower than 12 nM, whether light or not, proving that the compound Y-1 can selectively bind to Hsp90 protein and degrade Hsp90 protein under the condition of light, thereby down-regulating the expression level of its client protein HIF-1a.

[0118] The effect of the compound on the expression level of Hsp70 protein in 4T1 cell line after light was evaluated by Western Blot experiment, and it was found that 12 nM of the control compound BIIB021 could up-regulate the expression level of Hsp70, 12 nM of CHPpa also had a certain up-regulation effect on Hsp70 after light for 5 min, while 12 nM and 6 nM of the compound Y-1 not only had no up-regulation effect on Hsp70 after light for 5 min, but also had a significant down-regulation effect on Hsp70. Figure 7 This effect is expected to improve the drug resistance of traditional Hsp90 inhibitors and Hsp90 PROTACs.

[0119] The above embodiments are only used to illustrate the present application, and any equivalent transformation and improvement on the basis of the technical scheme of the present application should not be excluded from the protection scope of the present application.

Claims

1. A class of compounds, characterized by: The structure of the compound is as follows: ； wherein R0is H, ; R1is H, ; and R0 and R1 are not simultaneously H; R2 is , , , CH2; R3 is , ; wherein x is an integer from 1 to 10, and y is an integer from 1 to 5; R4 is pyropheophorbide-a, a chlorin e6 derivative; The pyro-magnesiofieroxin-a is ; The chlorin e6 derivative is , , ; wherein R5, R6and R7are each independently H, ; wherein m is an integer from 0 to 10.

2. The compound of claim 1, wherein R0 is ; R1 is H.

3. The compound of claim 2, wherein R2 is .

4. The compound of claim 3, wherein R3 is .

5. The compound of claim 4, wherein x is an integer from 2 to 5.

6. The compound of claim 5, wherein The structure of the compound is selected from the group consisting of: 、 、 。 7. Use of a compound according to any one of claims 1-6 or a pharmaceutically acceptable carboxylate salt thereof in the manufacture of an antitumor medicament.

8. Use according to claim 7, characterized in that, The tumor comprises breast cancer, lung cancer, melanoma, prostate cancer, cervical cancer, gastric cancer, colon cancer, liver cancer and pancreatic cancer.

9. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises a compound according to any one of claims 1-6 or a pharmaceutically acceptable carboxylate salt thereof.

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