Hypoxia-responsive protein degradation targeting chimera compound as well as preparation method and application thereof

Abstract

The invention provides a hypoxia-responsive protein degradation targeting chimera compound as well as a preparation method and application thereof. The structure of the compound is as shown in a formula (VII), and more specifically as shown in a formula (I). The compound disclosed by the invention takes an azo bond as a hypoxia response unit, can respond to a hypoxia tumor microenvironment and activate a protein degradation function of the hypoxia tumor microenvironment, and meanwhile, recovers a fluorescence signal of a photosensitizer so as to monitor drug release in real time. Under the irradiation of near-infrared light, the photosensitizer generates active oxygen to induce tumor cell apoptosis, so that photodynamic therapy and PROTAC combined therapy are realized. The compound provided by the invention can solve the problems of systemic toxicity after administration of an existing PROTACs drug system and poor effect in tumor treatment, and meanwhile, can monitor drug release in real time, so that the compound has good practical application value. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a hypoxia-responsive protein degradation targeting chimera compound and a composition and application thereof. Background Art

[0002] Protein degradation targeting chimeras (PROTACs) are an emerging drug development technology. Unlike the "occupancy-driven mechanism" of traditional small molecule inhibitors to regulate their activity, PROTAC can achieve direct chemical knockdown of target pathogenic proteins through an "event-driven mechanism". Specifically, PROTAC technology covalently links the binding ligand of the target protein to the binding ligand of the E3 ligase to obtain a class of bifunctional molecules. This type of molecule can bind to the target protein at one end and bind to the E3 ligase at the other end, eventually forming a ternary complex of the target protein-PROTAC-E3 ligase. Subsequently, the target protein is covalently labeled with ubiquitin (a 76-amino acid protein) and recognized and degraded by the ubiquitin-proteasome system. PROTAC can use low-affinity small molecule ligands (interacting transiently with possible binding sites of POI) or oligonucleotides as protein baits to relieve dependence on targetable pockets and provide a new method for degrading "undruggable" proteins. In addition, PROTACs also demonstrate multiple advantages over traditional small molecule inhibitors, such as their unique catalytic properties, lower dosage, and avoiding compensatory protein expression to overcome drug resistance. In the past few decades, a large number of studies have been devoted to designing new PROTACs to expand the range of target proteins, developing new E3 ligase ligands to broaden the application of PROTACs, and improving the performance of PROTACs from multiple angles.

[0003] Although PROTACs can effectively inhibit protein activity, due to their inherent lack of tissue selectivity, systemic toxicity caused by PROTACs after systemic administration remains an urgent problem to be solved. Selective activation of prodrugs is a promising molecular design strategy that can improve therapeutic efficacy and reduce systemic toxicity. Activatable PROTAC prodrugs remain inert in normal tissues and are only activated and restore their protein degradation activity under the action of specific triggers at the tumor site. This strategy provides a practical method for manipulating the degradation process. Currently, the means for PROTAC prodrug activation are mainly divided into two types: endogenous stimulation (mainly including active molecules overexpressed in tumor sites such as glutathione, hydrogen peroxide and enzymes) and external stimulation (ultraviolet light or visible light, etc.). These activatable PROTACs show great potential in controlling protein degradation.

[0004] Hypoxia is a common feature of most solid tumors, which is the result of uncontrolled cell proliferation, abnormal tumor vascularization and insufficient oxygen supply. Since healthy tissues usually do not contain hypoxic areas, tumor hypoxia can be used as a specific target for selective cancer therapy. In the hypoxic tumor microenvironment, endogenous reductases such as nitroreductase and azoreductase are overexpressed. The upregulation of physiological enzymes such as azoreductase and nitroreductase in hypoxic cancer cells can serve as a unique stimulus for the development of bioreductive prodrugs. To date, several nitroreductase-activated PROTAC prodrugs have been developed, mainly by introducing nitroimidazole groups into VHL E3 ligase ligands, which are inefficient in normal tissues and can be activated by overexpressed nitroreductase in tumor tissues. Although these PROTAC prodrugs show certain tumor inhibitory effects, the nitroimidazole masking group has limited hypoxia selectivity and may produce adverse toxicity in healthy tissues. In addition, due to the heterogeneity of hypoxic areas within solid tumors, the activation efficiency of hypoxia-activated prodrugs is limited, and poor effects have been observed in some clinical trials. Therefore, developing more intelligent hypoxia activation strategies to improve tissue specificity and promote effective prodrug activation is a major challenge in the current PROTAC prodrug design. In addition, most of the currently developed PROTAC prodrugs lack a reporting mechanism for drug activation and therefore cannot provide diagnostic feedback. Summary of the invention

[0005] The present invention designs a hypoxia-responsive PROTAC prodrug based on azobenzene and uses it for PROTAC and photodynamic therapy combined treatment, thereby achieving a more effective anti-tumor effect. The photosensitizer and PROTAC molecules are connected by the hypoxia-activated azobenzene structure, and the resulting PROTAC prodrug maintains low toxicity under normoxic conditions. The azo bond can be reduced and release the photosensitizer and PROTAC molecules in the hypoxic microenvironment, thereby respectively realizing the functions of monitoring the drug release process and inducing the degradation of the bromodomain exodomain (BET) protein. In addition, this degradation inhibits the transcriptional regulation function of BET protein and significantly promotes tumor cell apoptosis. Under the irradiation of near-infrared laser, the photosensitizer transfers energy to oxygen, produces cytotoxic reactive oxygen species, thereby inducing tumor cell damage. In in vivo treatment experiments, it was shown that the therapeutic effect of the prodrug was significantly enhanced when used in combination with photodynamic therapy. This method not only improves the safety of PROTAC, but also enhances the synergistic therapeutic effect on tumors.

[0006] In one aspect, the present invention provides a compound represented by formula (VII) or an isomer or a pharmaceutically acceptable salt or deuterated substance thereof:

[0007]

[0008] Wherein, X is an activatable photosensitizer.

[0009] The compounds of the present invention use azo bonds as hypoxia response units, which can respond to the hypoxic tumor microenvironment and activate its protein degradation function, while restoring the fluorescence signal of the photosensitizer to monitor drug release in real time. Under near-infrared light irradiation, the photosensitizer produces reactive oxygen to induce tumor cell apoptosis, thereby achieving photodynamic therapy and PROTAC combined therapy. The compounds of the present invention can solve the problems of systemic toxicity after administration of existing PROTACs drug systems and poor effects in tumor treatment, and can monitor drug release in real time, so they have good practical application value.

[0010] According to some specific embodiments of the present invention, DRUG is any PROTAC drug.

[0011] According to some specific embodiments of the present invention, DRUG is the following formula (VIII):

[0012] POI ligand-Linker-E3 ligand

[0013] (VIII)

[0014] According to some specific embodiments of the present invention, the compound represented by formula (VII) has a structure represented by formula (I), that is, the present invention provides a compound represented by formula (I) or an isomer or a pharmaceutically acceptable salt or deuterated substance thereof:

[0015]

[0016] Among them, POI ligand is selected from any target protein ligand of interest;

[0017] E3 ligand is the ligand of E3 ubiquitin ligase;

[0018] Linker is the connecting functional group between the target protein ligand and the E3 ubiquitin ligase ligand;

[0019] X is an activatable photosensitizer.

[0020] According to some specific embodiments of the present invention, the POI ligand structure (ie, the target protein ligand) can be derived from small molecules, nucleic acid aptamers, various forms of antibodies, and the like.

[0021] According to some specific embodiments of the present invention, the POI ligand is a BET family protein ligand structure. More specifically, the POI ligand is as shown in the following formulas (IV-1), (IV-2), (IV-3), (IV-4) and (IV-5):

[0022]

[0023]

[0024] According to some specific embodiments of the present invention, ring D is selected from any of the following structures:

[0025]

[0026] According to some specific embodiments of the present invention, ring E is selected from any of the following structures:

[0027]

[0028] According to some specific embodiments of the present invention, the POI ligand structure is selected from one of the following structures:

[0029] According to some specific embodiments of the present invention, the POI ligand structure is shown in the following formula (IV-1-1):

[0030]

[0031] According to some specific embodiments of the present invention, the structure of X is as shown in the following formula (II) or (III):

[0032]

[0033] Among them, R 1 , R 2 , R 3 and R 4 Each independently selected from C1-10 alkyl;

[0034] R 5 Selected from C1-10 alkyl or C1-10 alkyl sulfonate;

[0035] R 6 and R 7 Each independently selected from H or C1-2 alkyl;

[0036] R 8 is selected from F, Cl, Br, I or H;

[0037] Y 1 Selected from O or S;

[0038] Ring A is an aromatic ring selected from any of the following structures:

[0039]

[0040] The aromatic ring is optionally substituted by 0, 1, 2, 3, 4, 5 or 6 substituents R selected from F, Cl, Br, I, C1-6 alkyl, hydroxyl, amino, cyano or nitro. 9 When multiple R 9 When replaced by multiple R 9 Can be the same or different.

[0041] According to some specific embodiments of the present invention, the structure of X is as shown in the following formula (II-1) or formula (III-1):

[0042]

[0043] According to some specific embodiments of the present invention, the linker structure is selected from one of the following structures:

[0044]

[0045] wherein a is 1, 2 or 3; b is 1, 2 or 3; c is 0, 1, 2 or 3; d and e are each independently 0, 1, 2 or 3; f and g are each independently 0, 1, 2 or 3; h and i are each independently 0, 1, 2 or 3.

[0046] According to some specific embodiments of the present invention, the E3 ligand is selected from the E3 ligase ligands of VHL, CRBN, GID4, DCAF2, KLHL41, SPSB4, TRIM7, TRIM9, RNF43 and RNF182.

[0047] According to some specific embodiments of the present invention, the E3 ligand is selected from the E3 ligase ligand of VHL or CRBN.

[0048] According to some specific embodiments of the present invention, the E3 ligand structure is shown in the following formula (V-1) or formula (V-2):

[0049]

[0050] Among them, R 11 , R 12 and R 13 Each independently selected from H or C1-10 alkyl; optionally, the alkyl is further substituted by H, halogen, hydroxyl, carboxyl, amino, cyano, carbonyl or C1-5 alkyl;

[0051] Ring B is a C4-10 heterocycloalkyl group; the heterocycloalkyl group is optionally substituted by 0, 1, 2, 3 or 4 carbonyl groups; the heterocycloalkyl group contains 1, 2, 3 or 4 heteroatoms selected from N, O and S, and contains at least one N atom;

[0052] Ring C is a C6-12 aromatic ring; the aromatic ring is optionally substituted by 0, 1, 2, 3, 4, 5 or 6 substituents selected from F, Cl, Br, I, C1-6 alkyl, hydroxyl, amino, cyano or nitro;

[0053] R 14 is selected from -O- or -NH-;

[0054] R 15 is selected from hydrogen or carbonyl.

[0055] According to some specific embodiments of the present invention, X is connected to an azo bond.

[0056] According to some specific embodiments of the present invention, the compound structures are shown in formulas (VI-1) and (VI-2):

[0057]

[0058] According to some specific embodiments of the present invention, in formula (V-1) or formula (VI-1), R 11 , R 12 and R 13 each independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl or 3-ethylbutyl;

[0059] According to some specific embodiments of the present invention, in formula (V-2) or formula (VI-2), ring B is a heterocycloalkyl selected from the following structures:

[0060]

[0061] The heterocycloalkyl group is optionally substituted with 0, 1, 2, 3 or 4 carbonyl groups;

[0062] According to some specific embodiments of the present invention, ring C is an aromatic ring selected from the following structures (any position except two adjacent carbons on the same benzene ring marked can be connected to R14):

[0063]

[0064] The aromatic ring is optionally substituted by 0, 1, 2, 3, 4, 5 or 6 substituents R selected from F, Cl, Br, I, C1-6 alkyl, hydroxyl, amino, cyano or nitro. 9 When multiple R 9 When replaced by multiple R 9 Can be the same or different.

[0065] According to some specific embodiments of the present invention, ring B is selected from the following heterocycloalkyl groups:

[0066]

[0067] According to some specific embodiments of the present invention, ring C is selected from the following structural aromatic rings (preferably, two adjacent bonds on the same benzene ring are connected to the five-membered ring in V-2, and the remaining single bond is connected to R14):

[0068]

[0069] According to some specific embodiments of the present invention, the compound is selected from one of the structures shown below:

[0070]

[0071]

[0072] On the other hand, the present invention also provides a pharmaceutical composition containing any compound described in the present invention (including compounds represented by formula (I), formula (VI-1), (VI-2) and formula (VIII)) or its isomers or pharmaceutically acceptable salts or deuterated derivatives.

[0073] The pharmaceutical composition of the present invention can be used for drug treatment of patients in any feasible manner, including but not limited to: topical administration, oral administration, spray inhalation, rectal administration, nasal administration, vaginal administration, subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intraventricular injection, intracranial injection or infusion, or administration via an explanted reservoir, wherein oral administration, intramuscular injection or intravenous administration is preferred.

[0074] In another aspect, there is provided use of the above compounds in the preparation of drugs for preventing and / or treating (adjuvant therapy) tumor-related diseases.

[0075] Meanwhile, it should be noted that tumor is used in the present invention as known to those skilled in the art, and includes benign tumors and / or malignant tumors. Benign tumors are defined as excessive proliferation of cells that cannot form aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular abnormalities and biochemical abnormalities that can form systemic diseases (e.g., tumor metastasis in distant organs).

[0076] The compound of the present invention can be used alone or in combination with other drugs or carriers used in tumor treatment.

[0077] According to some specific embodiments of the present invention, there is provided a pharmaceutical preparation comprising the above-mentioned compound and a pharmaceutically acceptable carrier or excipient.

[0078] The above-mentioned pharmaceutical preparations can be any pharmaceutical dosage forms acceptable in pharmaceutical science, such as tablets (including dispersible tablets, sustained-release tablets, enteric-coated tablets, effervescent tablets, orally disintegrating tablets, chewable tablets, special-shaped tablets, etc.), hard capsules (including gastric-soluble, enteric-coated, and sustained-release capsules), soft capsules (including gastric-soluble and enteric-coated soft capsules), pellets, micropills, granules, dry suspensions, powders, oral liquids (including solutions, suspensions, and emulsions), injections (including powder injections and injection solutions for injection), etc. All of the above pharmaceutical dosage forms are recorded and described in the "Chinese Pharmacopoeia".

[0079] Depending on the dosage form of the drug, other suitable pharmaceutical carriers may be selectively contained as needed, such as diluents, fillers, disintegrants, surfactants, suspending agents, adhesives, lubricants, colorants, flavoring agents, etc. The "selectively contain" means that one or more of them may be selected, or none of them may be selected.

[0080] Suitable fillers or diluents include lactose, mannitol, sorbitol, microcrystalline cellulose, starch, modified starch, dextrin, cyclodextrin and its derivatives, calcium phosphate, sucrose, polyethylene glycol (polyethylene glycol of various molecular weights) pregelatinized starch, xylitol, fructose, maltitol, dextran, glucose, calcium sulfate, calcium hydrogen phosphate, etc.; the disintegrants include sodium carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, pregelatinized starch, corn starch, cross-linked sodium carboxymethyl starch, microcrystalline cellulose, calcium carboxymethyl cellulose, etc.; the surfactants include sodium lauryl sulfate , sodium tetradecyl sulfate, sodium hexadecyl sulfate, sodium octadecyl sulfate, polysorbate (generic name: Tween, including various types thereof), fatty acid sorbitan (generic name: Span, including various types thereof), etc.; the binder includes polyvinyl pyrrolidone, starch slurry, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, gelatin, guar gum, xanthan gum, and the like; the suspending agent includes but is not limited to hydroxypropyl methyl cellulose, ethyl cellulose, gum arabic, xanthan gum, sodium carboxymethyl cellulose, and the like; the lubricant includes magnesium stearate, stearic acid, talc, sodium stearyl fumarate, and the like. In addition, pH adjusters or buffers may be included, such as phosphate buffer, citric acid, sodium citrate, acetate buffer, dilute hydrochloric acid, sodium carbonate, sodium hydroxide, etc.; preservatives may be included, such as sodium benzoate, potassium sorbate, methyl paraben, propyl paraben, etc.; stabilizers and antioxidants may be included, such as calcium sodium edetate, sodium sulfite, vitamin C, vitamin E, etc.; taste adjusters may be included, such as maltitol, aspartame, stevioside, fructose, sucrose, saccharin sodium, flavors such as orange flavor, strawberry flavor, etc.

[0081] Of course, the pharmaceutical carrier is not limited to the above, and may include other conventional and appropriate additives or pharmaceutical excipients as needed, such as a wetting agent, which may be selected from water, ethanol, water-ethanol solution, etc., depending on the different dosage forms of the drug.

[0082] When the compound of the present invention is used in combination with other therapeutic drugs, the compound of the present invention and the pharmaceutical carrier or other drugs of suitable dose are usually made into suitable drug forms. Therefore, the present invention also provides a drug synergistic medication form, which comprises an effective dose of the compound of the present invention and at least one other type of pharmaceutically acceptable carrier or therapeutic drug.

[0083] In another aspect, the present invention provides a system, comprising:

[0084] a) the above-mentioned compound of the present invention or its isomer or pharmaceutically acceptable salt or deuterated substance, pharmaceutical composition or pharmaceutical preparation; and b) a light irradiation device.

[0085] The light source emitted by the illumination device is a near-infrared light source. Specifically, the wavelength of the near-infrared light source can be 660nm. It has been verified by experiments that after the above compound is released at the tumor site, the power density is 0.8W / cm 2 The 660nm laser irradiation effectively inhibited the growth of tumors and achieved a good combined treatment effect.

[0086] In yet another aspect, the present invention provides a method for preventing and / or treating tumors, comprising: administering a therapeutically effective dose of the above compound, the above pharmaceutical composition, the above pharmaceutical preparation or the above system to a subject.

[0087] The "optional" mentioned in the present invention should be understood as the event or situation may or may not occur. For example, the "optionally, the alkyl group is further substituted by H, halogen, hydroxyl, carboxyl, amino, cyano, carbonyl or C1-5 alkyl" should be understood as the alkyl group may or may not be substituted by the above-mentioned substituents such as H, halogen, hydroxyl, carboxyl, etc.

[0088] The “isomers” described in the present invention include geometric isomers (such as chiral isomers, cis-trans isomers, etc.) and tautomers.

[0089] Beneficial technical effects of the present invention:

[0090] The present invention provides a hypoxia-responsive protein-targeted degradation chimera prodrug, which blocks the active site of the PROTAC drug E3 ligase with an azobenzene structure and maintains a non-toxic state in normal tissues. In hypoxic tumors, the azo bond is cut by the reductase, thereby releasing the active PROTAC drug and inducing the degradation of the target protein. At the same time, byproducts such as carbon dioxide and 4-methylene-cyclohexadiene-imine with extremely low toxicity are produced, and no other substances that may be harmful to the body are produced, thereby reducing its toxic side effects on the whole body. This design effectively avoids the damage of PROTAC molecules to normal tissues while achieving selective activation of hypoxic tumors. At the same time, the synthesis method of the PROTAC prodrug is simple, no toxic reagents are introduced, and there is no problem of toxic reagent residues, and the biosafety is high, so it has good practical application value.

[0091] The present invention introduces an activatable photosensitizer into the prodrug, and the intramolecular charge transfer of the photosensitizer is inhibited under normoxic conditions, so that reactive oxygen cannot be generated. This design greatly reduces the phototoxicity of the prodrug and increases the flexibility and safety of the prodrug. The photosensitizer is activated at the hypoxic tumor site, and its fluorescence signal and photosensitivity are also restored, which can be used to monitor drug release in real time.

[0092] The prodrug of the present invention can release PROTAC drugs and photosensitizers simultaneously after being activated in the hypoxic environment of the tumor, wherein the PROTAC molecule can degrade the BET protein through the proteasome pathway, and the photosensitizer can generate reactive oxygen species under the irradiation of near-infrared light, significantly inducing tumor cell apoptosis. The activated prodrug combined with photodynamic therapy can effectively inhibit the growth of breast cancer cells (IC 50 =0.218 μM). BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 (a) The change of fluorescence spectrum with time and (b) liquid chromatography after the compound A1 of the present invention is reduced.

[0094] Figure 2 The toxicity curves of the compounds A1 / S1(a) and A2 / S2(b) of the present invention on tumor cells.

[0095] Figure 3 This is a diagram showing the effect of compound A1 of the present invention on inducing apoptosis of tumor cells.

[0096] Figure 4 This is a diagram showing the effects of compounds A1 / B1 and S1 of the present invention on BRD4 protein degradation.

[0097] Figure 5 This is the tumor growth curve after the combined action of the compound A1 of the present invention and photodynamic therapy. DETAILED DESCRIPTION

[0098] The implementation process of the present invention and the beneficial effects produced are described in detail below through specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0099] Experimental materials and instruments:

[0100] The organic solvents (such as dichloromethane, petroleum ether, methanol, acetone, ethyl acetate, tetrahydrofuran, etc.), inorganic salts (such as Na 2 CO 3 , NaCl, Na 2 S 2 O 4、 Na 2 SO 4 All organic solvents were of analytical grade and were not further processed upon receipt. 3, DMSO-D6), trifluoroacetic acid (CAS: 76-05-1), p-nitrobenzyl alcohol (CAS: 619-73-8), (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)acetate (CAS: 1268524-70-4), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (CAS: 1948273-02 -6), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (abbreviation: HATU, CAS: 148893-10-1), N,N-diisopropylethylamine (abbreviation: DIPEA, CAS: 7087-68-5), ultra-dry N,N-dimethylformamide (abbreviation: DMF, CAS: 68-12-2), bis(trichloromethyl) carbonate (CAS: 32315-10-9), ultra-dry dichloromethane (abbreviation: DCM, CAS: 75-09-2), 4-dimethylaminopyridine (CAS: 1122-58-3) and triethylamine (CAS: 121-44-8) were purchased from Anhui Zesheng Technology Co., Ltd. 3,7-Bis(dimethylamino)phenothiazine-5-ammonium chloride (CAS: 61-73-4), 4,4'-dihydroxymethylazobenzene (CAS: 37797-30-1), 5-iodo-2,3,3-trimethyl-3H-indole (CAS: 54136-25-3), 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde (CAS: 61010-04-6) and 5,8,11-trioxa-2-azatridecanedioic acid-1-tert-butyl ester (CAS: 462100-06-7) were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. p-Nitrophenyl chloroformate (CAS: 7693-46-1) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd. Tween 80 was purchased from MedChemExpress LLC. Fetal bovine serum (Gibco, catalog number: 10437010) and DMEM medium (Gibco, catalog number: 11995065) were purchased from Thermo Fisher Scientific. 3-color prestained protein marker (catalog number: 20351ES76), SDS-PAGE protein loading buffer (catalog number: 20315ES20), RIPA lysis buffer (catalog number: 20101ES60), 0.25% trypsin (catalog number: 40126ES60) and 1% penicillin-streptomycin double antibody (catalog number: 60162ES76) were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. CellTiter The kit was purchased from Promega (Cat. No. G3581). PBS solution (pH 7.2), ANNEXIN V-FITC / PI apoptosis detection kit (Cat. No. CA1020), 5% BSA blocking solution (Cat. No. SW3015) and cell culture grade DMSO (Cat. No. D8371) were purchased from Beijing Solebao Technology Co., Ltd. QuickBlock TM Blocking solution (Cat. No.: P0226), primary antibody diluent (Cat. No.: P0103), ECL chemiluminescence kit (Cat. No.: P0018FM; P0018A), Western washing solution (Cat. No.: P0023C6) and BCA protein concentration determination kit (Cat. No.: P0012) were purchased from Biotech. Bis-Tris SurePAGE gel was purchased from GenScript. PVDF membrane was purchased from Merck Millipore. PEG-300 (CAS: 25322-68-3, Cat. No.: T7022) and protease inhibitor cocktail (Cat. No.: C0001) were purchased from Target mol.USA.

[0101] Intelligent temperature-controlled digital display magnetic stirrer (MS-H-Pro+, Dalong), rotary evaporator (RE-2000, Shanghai Rongya), magnetic stirrer (85-2A, Zhengzhou Hengyan), one-tenth balance (BS124S, Sartorius), low-temperature coolant circulation pump (LTC-4 / 15, Beijing Xingde), low-temperature constant temperature stirring reaction bath (DHJK-4005, Zhengzhou Ketai), 400M liquid nuclear magnetic resonance spectrometer (JNM-ECZ400S, JEOL), eBlot fast wet transfer instrument (GenScript), and gel intelligent imaging workstation (GelView6000Plus, Bolu Teng).

[0102] Example 1 Preparation of hypoxia-responsive PROTAC prodrugs and analogs of the present invention

[0103] Ⅰ) Based on the above technical solution, the present invention first provides a method for preparing the above-mentioned compound A1 and its analogs A2, A3, A4, comprising the following steps:

[0104] a) First, a ligand targeting the target protein, compound P, is synthesized. Specifically:

[0105] Tert-butyl (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)acetate (1.0 g, 2.2 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (3 mL) was slowly added to the solution. The mixture was stirred at room temperature for 12 hours, and the reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated in vacuo and purified by silica gel column chromatography (dichloromethane: methanol = 100: 6) to obtain compound P as a white solid.

[0106] 1 H NMR (400MHz, DMSO-D6) δ9.31 (s, 1H), 7.50-7.48 (d, J = 8Hz, 2H), 7.44-7.42 (d, J = 8Hz, 2H), 4.48-4.44 (t, J = 8H z,1H),3.45-3.39(dd,J=16,8Hz,1H),3.34-3.28(dd,J=16,8Hz,1H),2.61(s,3H),2.41(s,3H),1.62(s,3H).

[0107] HRMS(ESI)m / z calcd for C 19 H 18 C1N 4 O 2 + [(M+H) + ]:401.0834,found:401.0857.

[0108]

[0109] b) Connecting the VHL ligand to the linker to synthesize compound L1. Specifically:

[0110] HATU (2.47 g, 6.6 mmol), DIPEA (1.13 mL, 6.6 mmol) and 1-tert-butyl 5,8,11-trioxa-2-azatridecanedioate (1.0 g, 3.25 mmol) were dissolved in DMF (12 mL). (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (1.47 g, 3.3 mmol) was slowly added to the solution. Subsequently, the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was diluted with a small amount of water, extracted with ethyl acetate (100 mL×3), and then washed with water (50 mL) and saturated sodium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain compound L1 as an oily liquid.

[0111] 1 H NMR (400MHz, DMSO-D6) δ8.97 (s, 1H), 8.43-8.41 (d, J = 8Hz, 1H), 7.44-7.42 (d, J = 8Hz, 2H), 7.40-7.35 (m ,2H),7.35(s,1H),6.76-6.73(m,1H),5.12-5.11(d,J=4Hz,1H),4.93-4.86(m,1H),4.55-4.52(d,J=12 Hz,1H),4.45-4.41(t,J=8Hz,1H),4.27(s,1H),3.95(s,2H),3.62-3.50(m,10H),3.38-3.35(t,J=6Hz, 2H),3.08-3.03(m,2H),2.44(s,3H),2.06-2.01(m,1H),1.79-1.73(m,1H),1.36(s,12H),0.93(s,9H).

[0112] HRMS(ESI)m / z calcd for C 36 H 55 N 5 O 9 S + [(M+H) + ]:734.3794,found:734.3760.

[0113]

[0114] Similarly, 5,8,11-trioxa-2-azatridecanedioic acid-1-tert-butyl ester in the above reaction is replaced by N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid or 11-((tert-butyloxycarbonyl)amino)undecanoic acid to obtain compounds L2 and L3, respectively, whose molecular structures are shown in Table 1:

[0115] Table 1

[0116]

[0117] c) Synthesis of PROTAC molecule compound S1. Specifically:

[0118] Compound L1 (1.0 g, 1.4 mmol) was slowly added to a mixed solution of dichloromethane (15 mL) and trifluoroacetic acid (3 mL), and the mixture was stirred at room temperature for 6 hours. The solvent in the mixture was then evaporated under reduced pressure. The above product, compound P (561.23 mg, 2.1 mmol), HATU (798.50 mg, 2.1 mmol) and DIPEA (365.80 μL, 2.1 mmol) were slowly added to a DMF (20 mL) solution, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was diluted with a small amount of water, extracted with ethyl acetate (50 mL × 3), and then washed with water (100 mL) and a saturated sodium chloride solution (50 mL × 3). The organic layer obtained after washing was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane: methanol = 15: 1) to obtain compound S1 as a white solid.

[0119] 1 H NMR (400MHz, DMSO-D6) δ8.97 (s, 1H), 8.43-8.42 (d, J = 4Hz, 1H), 8.29-8.26 (t, J = 6Hz, 1H), 7.48-7.45 (m, 2H), 7. 42(s,2H),7.41-7.38(m,2H),7.36-7.34(d,J=8Hz,2H),5.13-5.12(d,J=4Hz,1H),4.93-4.86(m,1H),4.55-4.42 (m,3H),4.27(s,1H),3.96(s,2H),3.63-3.58(m,4H),3.46-3.44(t,J=4Hz,2H),3.32(s,10H),2.58(s,3H),2.44 (s,3H),2.39(s,3H),2.06-2.01(m,1H),1.79-1.72(m,1H),1.61(s,3H),1.36-1.34(d,J=8Hz,3H),0.93(s,9H).

[0120] HRMS(ESI)m / z calcd for C 50 H 63 C1N 9 O 8 S 2 + [(M+H) + ]:1016.3925,found:1016.3833.

[0121]

[0122] Similarly, replacing L1 in the above reaction with L2 or L3, compounds S2 and S3 are obtained respectively, and the molecular structures are shown in Table 2:

[0123] Table 2

[0124]

[0125] d) Synthesis of compound P1. Specifically:

[0126] HATU (2.5 g, 6.6 mmol), DIPEA (0.85 g, 6.6 mmol) and P (1.0 g, 3.3 mmol) were dissolved in DMF (12 mL). 5-azidopentane-1-amine (423 mg, 3.3 mmol) was slowly added to the solution. Subsequently, the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was diluted with ethyl acetate (100 mL × 3), washed with water (50 mL) and saturated sodium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1: 1) to obtain a white solid, namely compound P1.

[0127] 1 H NMR (400MHz, DMSO-d6) δ8.29 (s, 1H), 7.49-7.47 (d, J = 8Hz, 2H), 7.42-7.40 (d, J = 8Hz, 2H), 5.53-5.50 (t, J = 6Hz, 1H), 3.62-3.59 (t,J=6Hz,2H),3.50-3.47(t,J=6Hz,2H),3.41-3.38(t,J=6Hz,2H),3.35–3.16(m,4H),2.59(s,3H),2.41(s,3H),1.62(s,3H). HRMS(ESI)m / z calcd for C 23 H 26 C1N 8 O 2 S + [(M+H)+ ]:511.1790,found:511.1778.

[0128]

[0129] e) Synthesis of compound L4. Specifically:

[0130] HATU (2.5 g, 6.6 mmol), DIPEA (0.85 g, 6.6 mmol) and 3-(prop-2-yn-1-oxy)propionic acid (422.8 mg, 3.3 mmol) were dissolved in DMF (12 mL). (2S, 4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (1.47 g, 3.3 mmol) was slowly added to the solution, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the mixture was diluted with ethyl acetate (100 mL×3), washed with water (50 mL) and saturated NaCl solution (50 mL). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain an oily liquid, which was compound L4.

[0131] 1 H NMR(400MHz, Methanol-d4)δ8.88(s,1H),7.51–7.38(m,4H),4.65(s,1H),4.60–4.48(m,4H),4.36-4.32(d,J=16Hz,1H),4.16-4.10 (m,2H),3.90-3.88(m,1H),3.84–3.72(m,3H),2.85-2.83(t,J=6Hz,1H),2.53–2.44(m,6H),2.08-2.04(d,J=16Hz,3H),1.04(s,9H).

[0132] HRMS(ESI)m / z calcd for C 29 H 39 N 4 O 5 S + [(M+H) + ]:555.2636,found:555.2580.

[0133]

[0134] f) Synthesis of compound S4. Specifically:

[0135] P1 (50 mM DMSO solution, 2.3 mL, 0.12 mmol) and L4 (50 mM DMSO solution, 2.3 mL, 0.12 mmol) were mixed and diluted with tert-butyl alcohol (4 mL), and then pre-mixed tris (3-hydroxypropyl triazolylmethyl) amine (THPTA, 50 mM aqueous solution, 4.7 mL, 0.23 mmol) and copper (II) sulfate (100 mM aqueous solution, 1.2 mL, 0.12 mmol) solution were added to the above solution. Finally, freshly prepared L-sodium ascorbate solution (200 mM in water, 3 mL, 0.59 mmol) was added thereto, and the reaction mixture was stirred at room temperature overnight. The solution was diluted with water (50 mL) and extracted twice with dichloromethane (50 mL) and ethyl acetate (50 mL). The organic layer was dried over anhydrous sodium sulfate, combined, concentrated under reduced pressure, and S4 was obtained as a white solid by silica gel column chromatography (dichloromethane: methanol = 10: 1).

[0136] 1 H NMR(400MHz,Chloroform-d)δ8.71(s,1H),7.98(s,1H),7.63–7.55(m,2H),7.46–7.29(m,9H),4.76-4.72 (t,J=8Hz,1H),4.70–4.47(m,7H),4.46–4.41(m,1H),4.35-4.33(d,J=16Hz,1H),4.15-4.12(d,J=12Hz,1H ),3.89–3.71(m,3H),3.70–3.63(m,1H),3.59-3.56(d,J=12Hz,1H),3.54–3.22(m,6H),2.65(s,3H),2.63 –2.60(m,7H),2.59–2.47(m,4H),2.46–2.32(m,4H),2.19-2.16(d,J=12Hz,1H),1.65(s,3H),0.96(s,9H).

[0137] HRMS(ESI)m / z calcd for C 53 H 66 C1N 12 O 6 S 2 + [(M+H) + ]:1065.4353,found:1065.4401.

[0138]

[0139] g) Synthesis of compound R12. Specifically:

[0140] Under argon atmosphere, methylene blue (640 mg, 2 mmol) was dissolved in water (50 mL), followed by addition of dichloromethane (25 mL) and sodium carbonate (847.92 mg, 8 mmol), and stirred at 40 ° C for 5 minutes. Sodium dithionite (1392.88 mg, 8 mmol) was dissolved in water (70 mL) and added directly to the solution with a syringe, followed by continued stirring until the solution turned yellow (usually within 15-30 minutes), and then the mixture was cooled with an ice-water bath. Di(trichloromethyl) carbonate (356.10 mg, 1.2 mmol) was dissolved in dichloromethane (20 mL) and added dropwise to the reaction system, and the stirring reaction was continued for 1 hour. Under stirring, the solution was slowly poured into ice water (200 mL), and the mixture was extracted with dichloromethane (100 mL × 3). The resulting mixture was washed with saturated brine and dried over anhydrous sodium sulfate. The product was filtered, concentrated under reduced pressure and purified by silica gel column chromatography (n-hexane:ethyl acetate=10:1) to obtain compound R12 as a white solid.

[0141] 1 H NMR (400MHz, Chloroform-d) δ7.38-7.36 (d, J = 8Hz, 2H), 6.68-6.67 (d, J = 4Hz, 2H), 6.62-6.59 (dd, J = 8, 4Hz, 2H), 2.94 (s, 12H).

[0142] HRMS(ESI)m / z calcd for C 17 H 19 C1N 3 OS + [(M+H) + ]:348.0932,found:348.0922.

[0143]

[0144] h) Synthesis of compound R11. Specifically:

[0145] 4,4'-dihydroxymethylazobenzene (72.68 mg, 0.3 mmol), sodium carbonate (31.80 mg, 0.3 mmol) and 4-dimethylaminopyridine (36.65 mg, 0.3 mmol) were dissolved in tetrahydrofuran (5 mL). Compound R12 (38.26 mg, 0.11 mmol) was then dissolved in anhydrous dichloromethane (3 mL) and added dropwise to the reaction mixture under stirring. After stirring the reaction at room temperature for 6 hours, the mixture was filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane: ethyl acetate = 10: 1) to obtain compound R11 as a yellow-green solid.

[0146] 1 H NMR (400MHz, DMSO-D6) δ8.13–8.00(m,2H),7.94-7.85(m,4H),7.54-7.52(d,J=8Hz,4H),7.36-7.34(d,J=8Hz,2H),6.69-6 .68(m,2H),5.38-5.36(t,J=4Hz,1H),5.26(s,1H),4.61-4.59(d,J=8Hz,2H),4.24-4.22(dd,J=8,4Hz,1H),2.88(s,12H).

[0147] HRMS(ESI)m / z calcd for C 31 H 32 N 5 O 3 S + [(M+H) + ]:554.2221,found:554.2230.

[0148]

[0149] i) Synthesis of compound R1. Specifically:

[0150] Compound R11 (276.84 mg, 0.5 mmol) and 4-dimethylaminopyridine (97.73 mg, 0.8 mmol) were dissolved in anhydrous dichloromethane (15 mL) and stirred for 10 minutes under an argon atmosphere. Then p-nitrophenyl chloroformate (161.25 mg, 0.8 mmol) was dissolved in anhydrous dichloromethane (5 mL) and slowly added dropwise to the reaction mixture, and stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane: ethyl acetate = 50: 1) to obtain compound R1 as an orange-yellow solid.

[0151] 1H NMR (400MHz, DMSO-D6) δ8.97 (s, 1H), 8.50-8.48 (d, J = 8Hz, 1H), 8.31-8.25 (m, 3H), 7.64-7.61 (m, 2H), 7.50-7.44 (m, 4H) ,7.42-7.38(m,4H),7.36(s,1H),5.27(s,1H),4.95-4.88(m,1H),4.54-4.47(m,2H),4.43-4.40(d,J=12Hz,1H),4.29-4 .26(d,J=12Hz,1H),3.98(s,2H),3.81-3.78(dd,J=12,4Hz,1H),3.60-3.54(m,8H),3.45-3.42(t,J=6Hz,2H),3.28-3.1 7(m,4H),2.58(s,3H),2.44(s,4H),2.39(s,3H),2.10-2.03(m,1H),1.60(s,3H),1.38-1.36(d,J=8Hz,3H),0.97(s,9H).

[0152] HRMS(ESI)m / z calcd for C 38 H 35 N 6 O 7 S + [(M+H) + ]:719.2283,found:719.2290.

[0153]

[0154] j) Synthesis of compound A1. Specifically:

[0155] Compound S1 (50.83 mg, 0.05 mmol), compound R1 (35.91 mg, 0.05 mmol) and 4-dimethylaminopyridine (7.33 mg, 0.06 mmol) were dissolved in anhydrous dichloromethane (10 mL), triethylamine (10.42 μL, 0.075 mmol) was added under stirring, and the mixture was reacted at 40° C. for 12 hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the obtained mixture was purified by column chromatography (dichloromethane: methanol = 20:1) to obtain compound A1 as a dark yellow solid.

[0156] 1H NMR(400MHz,Chloroform-d)δ8.65(s,1H),8.30-8.25(m,2H),8.17-8.13(m,2H ),8.02(s,1H),7.91-7.87(dd,J=8,4Hz,1H),7.52-7.45(m,4H),7.40(s,1H),7. 38(s,2H),7.35(s,4H),7.32(s,1H),7.29(s,1H),6.67-6.66(m,2H),6.63-6.60 (dd,J=8,4Hz,2H),5.30(s,2H),5.25-5.15(m,3H),5.10-5.06(t,J=8Hz,1H),4. 92-4.86(m,1H),4.66-4.58(m,2H),4.33-4.30(d,J=12Hz,1H),4.14-4.09(dd,J =16,4Hz,2H),4.02-3.98(d,J=16Hz,1H),3.87-3.84(d,J=12Hz,1H),3.70-3.68 (m,8H),3.44-3.40(m,2H),2.93(s,12H),2.62(s,3H),2.51(s,3H),2.37(s,3H) ,2.25-2.17(m,2H),2.02-1.99(m,3H),1.46-1.44(d,J=8Hz,4H),1.06(s,12H).

[0157] HRMS(ESI)m / z calcd for C 82 H 92 C1N 14 O 12 S 3 + [(M+H) + ]:1595.5865,found:1595.5787.

[0158]

[0159] Similarly, replacing S1 in the above reaction with S2, S3 or S4, compounds A2, A3 and A4 are obtained respectively, and the molecular structures are shown in Table 3:

[0160] Table 3

[0161]

[0162]

[0163] II) Synthesis of compounds B1-B4.

[0164] a) Synthesis of compound R23. Specifically:

[0165] 5-iodo-2,3,3-trimethyl-3H-indole (1.71 g, 6 mmol) and iodomethane (2.4 mL, 30 mmol) were dissolved in toluene (15 mL), and the mixture was then heated to reflux at 110 ° C for 24 hours. After the reactants were cooled to room temperature, the brown crystals were filtered and washed with ether (50 mL × 3) to obtain a light pink solid. The above product (854.14 mg, 2 mmol), 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (172.61 mg, 1 mmol) and anhydrous sodium acetate (164.07 mg, 2 mmol) were dissolved in anhydrous ethanol (10 mL), and the resulting mixture was heated to reflux for 4 hours under an argon atmosphere. Excess solvent was removed under reduced pressure, and compound R23 was purified by column chromatography (dichloromethane: methanol = 100: 1) as a green solid.

[0166] 1 H NMR (400MHz, DMSO-D6) δ8.97 (s, 1H), 7.88-7.84 (dd, J = 16Hz, 1H), 7.64-7.62 (dd, J =8Hz,1Hz,1H),7.39-7.32(m,2H),7.20-7.09(m,2H),6.96-6.92(m,2H),6.80-6.7 6(d,J=16Hz,1H),6.66-6.65(d,J=4Hz,1H),4.19-4.08(m,2H),3.71-3.66(m,2H), 2.77-2.46(m,4H),1.92-1.80(m,2H),1.63(s,7H),1.44(s,6H),1.46-1.33(m,4H).

[0167] HRMS(ESI)m / z calcd for C 34 H 38 C1I 2 N 2 + [(MI) + ]:763.0808,found:763.0910.

[0168]

[0169] b) Synthesis of compound R22. Specifically:

[0170] Compound R23 (325.17 mg, 0.5 mmol), m-nitrophenol (208.67 mg, 1.5 mmol) and potassium carbonate (207.32 mg, 1.5 mmol) were dissolved in acetonitrile (10 mL) and stirred at room temperature for 4 hours under an argon atmosphere. The solvent was then evaporated under reduced pressure and the obtained solid was dissolved in dichloromethane, washed with water and dried over anhydrous sodium sulfate, and the residue obtained after removing the solvent under reduced pressure was dissolved in methanol (30 mL). Stannous chloride dihydrate (1 g, 4.43 mmol) was dissolved in concentrated hydrochloric acid (1 mL) and then added to the above-mentioned methanol solution under an argon atmosphere. The reaction solution was heated to 70 ° C and stirred overnight, then the solution was neutralized with a saturated sodium carbonate solution, filtered to remove the precipitate, and washed with dichloromethane. The collected filtrate and washings were washed three times with water and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to give a green solid as compound R22.

[0171] 1 H NMR (400MHz, DMSO-D6) δ7.66 (s, 1H), 7.45-7.41 (d, J = 16Hz, 2H), 7.32-7.31 ( d,J=4Hz,1H),7.24-7.22(d,J=8Hz,1H),7.03-7.02(d,J=4Hz,1H),6.92-6.8 7(m,2H),6.62-6.59(d,J=12Hz,1H),3.78-3.73(m,2H),2.83-2.75(m,2H),2 .68-2.55(m,2H),1.98-1.92(m,2H),1.70(s,6H),1.49-1.45(t,J=8Hz,3H).

[0172] HRMS(ESI)m / z calcd for C 27 H 28 IN 2 O + [(MI) + ]:523.1241,found:523.1311.

[0173]

[0174] c) Synthesis of compound R21. Specifically:

[0175] Dissolve p-nitrobenzene alcohol (350mg, 2.1mmol), ammonium chloride (160mg, 2.94mmol) and zinc powder (410mg, 6.3mmol) in ethanol (5mL), and stir the reaction mixture at room temperature for 30 minutes. After the reaction is completed, filter out the insoluble material. Dissolve ferric chloride hexahydrate (680mg, 2.52mmol) in a mixed solution of ethanol (1mL) and water (2mL), and add the filtrate to the ferrous chloride hexahydrate solution at -5°C. The mixture is stirred and reacted for 30 minutes at -5°C. The resulting solution is diluted with brine and extracted with dichloromethane. The organic layer is then washed with water and dried over anhydrous magnesium sulfate. The solvent is removed under reduced pressure, and the resulting crude product does not need to be further purified.

[0176] The crude product (160 mg, 1.17 mmol) and compound R22 (487.7 mg, 0.75 mmol) from the previous step were dissolved in acetic acid (5 mL), and the mixture was stirred at room temperature overnight. The precipitate was separated by centrifugation, and the collected solid was dissolved in acetic acid again. Saturated sodium bicarbonate solution was slowly added to the solution to precipitate the product. The precipitate was separated by centrifugation, and the collected solid was washed with water to obtain compound R21.

[0177] 1 H NMR(400MHz, DMSO-D6)δ8.26-8.24(dd,J=8Hz,1H),7.48-7.47(d,J=4Hz,1H),7.39-7.32(m, 3H),7.30(s,1H),7.29(s,1H),7.27(s,1H),7.17-7.14(m,2H),7.10(s,1H),7.03-7.02(d,J= 4Hz,1H),6.52-6.48(d,J=16Hz,1H),4.28(s,2H),3.73-3.67(m,2H),2.80-2.72(m,2H),2.65 -2.59(m,1H),2.52-2.46(m,1H),2.12-1.96(m,2H),1.76(s,6H),1.52-1.48(t,J=16Hz,3H).

[0178] HRMS(ESI)m / z calcd for C 34 H 33 IN 3 O 2 + [(MI) + ]:642.1612,found:642.1630.

[0179]

[0180] d) Synthesis of compound R2. Specifically:

[0181] Compound R21 (384.73 mg, 0.5 mmol) and 4-dimethylaminopyridine (97.73 mg, 0.8 mmol) were dissolved in anhydrous dichloromethane (15 mL) and stirred for 10 minutes under an argon atmosphere. Then p-nitrophenyl chloroformate (161.25 mg, 0.8 mmol) was dissolved in anhydrous dichloromethane (5 mL) and slowly added dropwise to the reaction mixture, and stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane: ethyl acetate = 50: 1) to obtain compound R2.

[0182] 1 H NMR (400MHz, DMSO-D6) δ8.26-8.24(dd,J=8Hz,1H),8.18-8.15(dd,J=12Hz,2H),7.48-7.47(d,J=4H z,1H),7.39-7.36(m,3H),7.35(s,2H),7.32(s,1H),7.30(s,1H),7.17-7.14(m,2H),7.10(s,1H),7. 03-7.02(d,J=4Hz,1H),6.52-6.48(d,J=16Hz,1H),5.01(s,2H),3.72-3.68(m,2H),2.80-2.72(m,2H ),2.65-2.59(m,1H),2.52-2.46(m,1H),2.12-1.96(m,2H),1.73(s,6H),1.51-1.47(t,J=16Hz,3H).

[0183] HRMS(ESI)m / z calcd for C 41 H 36 IN 4 O 6 + [(MI) + ]:807.1675,found:807.1700.

[0184]

[0185] e) Synthesis of compounds B1-B4. Specifically:

[0186] The synthesis operation of B1, B2, B3, and B4 is consistent with that of A1, A2, A3, and A4. Specifically, slowly dissolve compound S1 (or S2, S3, and S4), compound R2, and 4-dimethylaminopyridine in anhydrous DMF, and stir the mixture at 50° C. for 12 hours. Evaporate the solvent under reduced pressure to obtain a crude product, which is purified by silica gel column chromatography (dichloromethane: methanol = 20: 1) to obtain a yellow solid, which is compound B1 (or B2, B3, and B4).

[0187] Table 4

[0188]

[0189]

[0190] III) Synthesis of compounds C1 and C2.

[0191] a) Synthesis of compound PS1. Specifically:

[0192] Compound P (1.0 g, 2.5 mmol), 13-amino-5,8,11-trioxa-2-azatridecanoic acid 1,1-dimethylethyl ester (470 mg, 2.5 mmol), HATU (1.14 g, 3.0 mmol) and DIPEA (388 mg, 3.0 mmol) were slowly added to a DMF (20 mL) solution, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reaction mixture was diluted with ethyl acetate (50 mL × 3) and washed with water (100 mL) and saturated sodium chloride solution (50 mL × 3). The organic layer obtained after washing was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane: methanol = 100: 3) to obtain compound PS1.

[0193] 1 H NMR(400MHz,DMSO-d6)δ7.55-7.53(m,4H),5.52-5.48(t,J=8Hz,1H),3.65(s,8H),3.50–3 .42(m,8H),3.18-3.16(d,J=8Hz,2H),2.36-2.34(d,J=8Hz,6H),2.21(s,3H),1.43(s,9H).

[0194] HRMS(ESI)m / z calcd for C 32 H 44 C1N 6 O 6 S + [(M+H) + ]:675.2727,found:675.2680.

[0195]

[0196] Similarly, by replacing 1,1-dimethylethyl 13-amino-5,8,11-trioxa-2-azatridecanoate with 1-BOC-1,10-diaminodecane in the above reaction, compound PS2 can be obtained.

[0197] Table 5:

[0198]

[0199] b) Synthesis of compound RS1. Specifically:

[0200] The compound 2-(2,6-dioxacyclic piperidine-3-yl)-4-fluoroisoindoline-1,3-dione (100 mg, 0.36 mmol) was slowly added to dichloromethane (15 mL), cooled to 0 ° C, NaH (10.3 mg, 0.43 mmol) was added, reacted at 0 ° C for 1 hour, and then compound R1 (308.8 mg, 0.43 mmol) was added and the mixture was stirred at room temperature for 6 hours. The solvent in the mixture was then evaporated under reduced pressure. After the reaction was completed, the reacted mixture was diluted with ethyl acetate (30 mL × 3) and washed with water (50 mL) and saturated NaCl solution (20 mL × 3). The organic layer obtained after washing was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane: petroleum ether = 1: 3) to obtain compound RS1.

[0201] 1 H NMR (400MHz, DMSO-D6) δ8.03-8.00(m,1H),7.61(s,1H),7.60(s,1H),7.52-7.46(m,4H),7.44(s,3H),7.42(s,1H),7.19-7.15(t,J=8Hz,2H),7 .08-7.07(d,J=4Hz,2H),6.80-6.75(m,2H),5.09(s,2H),5.07-5.05(m ,1H),5.04(s,2H),2.80(s,12H),2.42-2.39(m,2H),2.09-1.90(m,2H).

[0202] HRMS(ESI)m / z calcd for C 45 H 39 FN 7 O 8 S + [(M+H) + ]:856.2560,found:856.2573.

[0203]

[0204] c) Synthesis of compound C1. Specifically:

[0205] Compound PS1 (121.4 mg, 0.18 mmol) was slowly added to a mixed solution of dichloromethane (5 mL) and trifluoroacetic acid (1 mL), and the mixture was stirred at room temperature for 6 hours. The solvent in the mixture was then evaporated under reduced pressure. The above product, compound RS1 (154.06 mg, 0.18 mmol) and DIPEA (93.1 mg, 0.72 mmol) were slowly added to a DMF (10 mL) solution, and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reacted mixture was diluted with ethyl acetate (20 mL × 3) and washed with water (30 mL) and saturated NaCl solution (20 mL × 3). The organic layer obtained after washing was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane: methanol = 20: 1) to obtain compound C1.

[0206] 1 H NMR (400MHz, DMSO-D6) δ8.03-8.00(m,1H),7.61(s,1H),7.60(s,1H),7.52-7.46(m,4H),7.44(s,3H),7.42(s,1H),7.39- 7.37(m,2H),7.29-7.27(t,J=8Hz,2H),7.23-7.22(d,J=4Hz,2H),7.18-7.15(m,2H),7.00-6.98(m,1H),6.09-6.05(t,J=8 Hz,1H),5.09(s,2H),5.07-5.05(m,1H),5.04(s,2H),4.21(s,1H),4.20(s,1H),4.18(s,8H),4.17-4.14(m,2H),4.03-3. 94(m,6H),3.52(s,12H),2.94(s,3H),2.91(s,3H),2.79(s,3H),2.78-2.61(m,1H),2.44-2.38(m,1H),2.25-2.18(m,2H).

[0207] HRMS(ESI)m / z calcd for C 72 H 73 C1N 13 O 12 S 2 + [(M+H) +]:1410.4627,found:1410.4580.

[0208]

[0209] Similarly, by replacing PS1 in the above reaction with PS2, compound C2 can be obtained.

[0210] Table 6:

[0211]

[0212] Example 2 Characterization of the release capacity of hypoxia-responsive PROTAC prodrugs

[0213] Experimental procedure: PBS was bubbled with argon for 1 hour to remove oxygen. 50 μM of compound A1 was mixed with 50 mM sodium disulfite (Na 2 S 2 O 4 , a chemical mimetic of azoreductase) and studied its fluorescence spectra at different time points. Figure 1 The image (a) shows that the prodrug itself has almost no fluorescence in the range of 400-800nm. As the reaction time increases, the photosensitizer methylene blue (MB) is released, resulting in a gradual recovery of fluorescence. The above changes can be attributed to the breaking of the azo bond in the prodrug and the occurrence of intramolecular charge transfer. Figure 1 As shown in the picture (b), this reduction process can also be confirmed by high performance liquid chromatography (HPLC) analysis. HPLC experimental conditions: Welch Ultisil XB-C18, 5μm, 4.6×250mm chromatographic column, flow rate of 0.5mL / min, detection wavelength: 254nm; analysis method: gradient elution with 30% acetonitrile-water solution (containing 0.1% trifluoroacetic acid) from 0 to 3 minutes, then gradient elution with 30%-95% acetonitrile-water solution (containing 0.1% trifluoroacetic acid) for 3-13 minutes, then elution with 95% acetonitrile-water solution for 5 minutes, and then gradient elution with 95%-30% acetonitrile-water solution (containing 0.1% trifluoroacetic acid), and stop detection at 30 minutes. The solvents used in this experiment are all HPLC grade solvents.

[0214] Example 3 In vitro antitumor effect of hypoxia-responsive PROTAC prodrug

[0215] Experimental procedure: Logarithmic phase MCF-7 cells were used for the experiment. The cells were seeded in a 96-well plate at a density of 5000 cells per well and adhered to the wall at 37°C for 24 hours. The cells were divided into different control groups: compound S1 / S2, compound A1 / A2, compound A1 / A2 + hypoxia, and compound A1 / A2 + hypoxia + laser. Different concentrations of compound S1 / S2 and compound A1 / A2 were added to the well plate, and the compound S1 / S2 group and compound A1 / A2 group were incubated under normoxic conditions for 72 hours; the compound A1 / A2 + hypoxia group was incubated in a hypoxic bag for 24 hours, and then incubated under normoxic conditions for 48 hours; the compound A1 / A2 + hypoxia + laser group was incubated in a hypoxic bag for 24 hours, and then irradiated with a 660nm laser at 0.8W / cm 2 The cells were irradiated at a power density of 500 nm for 5 minutes and incubated under normoxic conditions for 48 hours. Subsequently, 20 μL of CellTiter The solution was added and the well plate was incubated in an incubator for 2-4 hours, and then the absorbance was measured using a microplate reader.

[0216] The experimental results are as follows Figure 2 As shown, the IC50 of compound S1 in MCF-7 cells is 0.264 μM, and the corresponding IC50 of compound A1 under normoxic conditions is 2.89 μM. It is inferred that when the hydroxyl group on the VHL ligand is masked by the azobenzene structure, compound A1 cannot effectively degrade the BET protein, so the tumor killing effect is weak. However, when the cells to which the drug compound A1 is added are treated with hypoxia, the hypoxic environment causes the azo group in the prodrug to break, releasing the PROTAC molecule, thereby producing significant cytotoxicity. Subsequently, under the irradiation of near-infrared light, the reactive oxygen species produced by the photosensitizer enhances the tumor therapeutic effect of the drug, and its corresponding IC50 is 0.218 μM. Similarly, the toxicity of the compound A2+hypoxia+laser group is also significantly increased, proving the safety of the drug under normoxic conditions, and photodynamic therapy increases the drug toxicity of the prodrug after activation.

[0217] Example 4: Detection of apoptosis by Annexin V / PI double staining

[0218] The surface of normal cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflets of the plasma membrane. Among them, phosphatidylserine (PS) is usually only distributed in the inner leaflet of the plasma membrane and is therefore only exposed in the cytoplasm. However, when apoptosis occurs, PS is exposed on the outer leaflet of the plasma membrane. Annexin V is a phospholipid-binding protein that can bind to PS. Therefore, fluorescently labeled Annexin V can be used to detect PS exposed to the outside of early apoptotic cells. By co-staining with propidium iodide (PI), Annexin V-PI can distinguish between normal living cells, early apoptotic cells, and late apoptotic cells.

[0219] Cell treatment: MCF-7 cells in logarithmic growth phase were used for the experiment. The cells were cultured at 2×10 5 Cells were seeded at a density of 10 cells per well in a 12-well plate and allowed to adhere to the wall at 37°C for 24 hours. The cells were treated with DMSO, compound S1, or compound A1 (400 nM). The normoxic group was cultured in an incubator for 48 hours, and the hypoxic group was incubated in a hypoxic bag for 24 hours and then incubated under normoxic conditions for another 24 hours. After 24 hours of hypoxia treatment, the laser group was treated with a 660 nm laser at 0.8 W / cm 2 The irradiation was carried out at a power density of 500 nm for 5 minutes.

[0220] Cell collection: Collect the culture medium in a 1.5 mL centrifuge tube for later use. Wash the cells three times with pre-cooled PBS, then digest with 0.25% trypsin (without EDTA) at 37°C, add the previously collected culture medium to terminate the digestion, and prepare a cell suspension solution. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. Resuspend the cells with 4°C pre-cooled PBS, centrifuge again under the same conditions, and discard the supernatant.

[0221] Cell staining: Staining was performed according to the instructions of ANNEXIN V-FITC / PI apoptosis detection kit. 6 The cells were resuspended in 100 μL Binding Buffer, 5 μL Annexin V-FITC solution was added, mixed and incubated for 5 minutes, then 5 μL propidium iodide solution and PBS (200 μL) were added, mixed and fluorescence detection was immediately performed using a flow cytometer.

[0222] According to the working principle of Annexin V / PI double staining reagent, the scatter plot is divided into 4 quadrants. The lower left quadrant is FITC / PI negative, representing living cells; the lower right quadrant is FITC positive and PI negative, representing early apoptotic cells; the upper right quadrant is FITC / PI positive, representing necrotic cells or late apoptotic cells. Figure 3 It can be seen that the ability of compound A1 to induce apoptosis in MCF-7 cells under hypoxia and normoxic conditions is significantly different. Under normoxic conditions, the ability of compound A1 to induce cell apoptosis is comparable to that of the control group DMSO, and there is no obvious cell killing ability. However, under hypoxia, after compound A1 treated MCF-7 cells, a significant increase in apoptotic cells was detected in flow cytometry. After hypoxia treatment, the apoptotic cells were further detected using a 660nm laser at 0.8W / cm -2 The results show that the number of apoptotic cells increased further, proving that the combined treatment of compound A1 and photodynamic therapy can further promote cell apoptosis.

[0223] Example 5 Protein degradation activity test of hypoxia-responsive PROTAC prodrug

[0224] Cell treatment: Logarithmic phase MCF-7 cells were cultured at 5×10 5 Cells were seeded at a density of 10 cells / well in a six-well plate and adhered to the wall at 37°C for 24 hours. The cells were divided into different control groups: compound S1, compound A1 / B1, compound A1 / B1+hypoxia, and compound A1 / B1+hypoxia+laser. Different concentrations (0nM, 10nM, 30nM, 100nM, 300nM, 1000nM) of compound S1, compound A1, and compound B1 were added to the well plate, and the compound S1 group, compound A1, and compound B1 group were incubated under normoxic conditions for 24 hours; the compound A1 / B1+hypoxia group was incubated in a hypoxic bag for 12 hours, and then incubated under normoxic conditions for another 12 hours; the compound A1 / B1+hypoxia+laser group was incubated in a hypoxic bag for 12 hours, and then irradiated with a 660nm laser at 0.8W / cm 2 Irradiate at a power density of 5 minutes, and then continue incubation under normoxic conditions for 12 hours. Extraction of total cell protein: After drug treatment, discard the culture medium and wash the cells three times with cold PBS. Scrape the cells, carefully aspirate the resulting solution, and centrifuge at 1000 rpm for 5 minutes at 4°C to obtain the cells. Lyse the cells washed with pre-cooled PBS in a pre-lysis buffer (containing 1% protease and phosphatase inhibitors) for 30 minutes, vortexing the cells vigorously several times during the lysis process. After the lysis is completed, centrifuge at 14000 rpm for 15 minutes at 4°C, and collect the supernatant as the total cell protein.

[0225] Protein denaturation: Measure protein concentration using the BCA protein concentration assay kit. After quantification, dilute the protein with protein loading buffer and heat the sample at 100°C for 15 minutes to obtain denatured protein.

[0226] Western blotting (WB):

[0227] 1) Protein electrophoresis: Proteins were separated in 4-12% or 4-20% Bis-Tris SDS-PAGE gel. An appropriate volume of cell whole protein extract was loaded into the lane of the SDS-PAGE gel. After 20 minutes of electrophoresis at 80V, the voltage was adjusted to 120V and the electrophoresis was stopped at the bottom of the gel.

[0228] 2) Transfer: After electrophoresis, remove the gel, cut the PVDF membrane (0.45 μm) of appropriate size and activate the membrane with methanol, then balance it with PVDF membrane balance solution. Install the transfer device in the following order from the negative electrode to the positive electrode: sponge pad, gel, PVDF membrane, sponge pad. After installing the transfer device, place it in the transfer tank, check the liquid level of the solution required for transfer, turn on the power, and complete the transfer after 3 transfer cycles.

[0229] 3) Blocking: Place the membrane in QuickBlock TM Western blocking buffer was added and shaken at room temperature for 30 minutes.

[0230] 4) Primary antibody incubation: After blocking, discard the blocking solution, add the primary antibody diluted to an appropriate concentration, and gently shake on a shaker at 4°C overnight. After incubation, wash the membrane 4 times with TBST, 5 minutes each time.

[0231] 5) Secondary antibody incubation: Add secondary antibody diluted to an appropriate concentration and gently shake the PVDF membrane on a shaker for 1 hour at room temperature. After incubation, wash the membrane 4 times with TBST, 5 minutes each time.

[0232] 6) Development: Prepare ECL developer before development. Spread the PVDF membrane on a clean plastic wrap, add developer to the membrane and leave it in the dark for 1 minute, then image the PVDF membrane on the gel system.

[0233] The degradation activity of PROTAC drug S1 of this example on BRD4 is as follows: In the breast cancer cell line MCF-7, it can be observed in the WB results that PROTAC drug S1 effectively degrades BRD4 at the test concentration (0-1000nM), and the DC50 is 90.23nM. Similarly, the degradation activity of hypoxia-responsive protein degradation chimera prodrug compound A1 / B1 on BRD4 is as follows: In the breast cancer cell line MCF-7, it can be observed in the WB results that PROTAC prodrug A1 / B1 has significantly inhibited BRD4 degradation activity at the test concentration before activation. On the contrary, when MCF-7 cells are treated with hypoxia and near-infrared light, BRD4 protein is significantly degraded, and 100nM of the prodrug can degrade more than half of the BRD4 protein. The results are as follows. Figure 4 shown.

[0234] Example 6 Study on the antitumor activity of compound A1 in vivo

[0235] In order to study the in vivo activation and anti-tumor activity of hypoxia-responsive PROTAC prodrug compound A1, the present invention conducted an in vivo anti-tumor activity study on it. MCF-7 cell line was selected and the experiment was carried out in BALB / c nude mice.

[0236] Experimental animals: BALB / c nude mice (female; 6-8 weeks old) were purchased from Beijing Weitongli Huashi Biotechnology Co., Ltd. and housed in the Experimental Animal Center of Tsinghua University. All animal experiments were approved by the Animal Ethics Committee of Tsinghua University.

[0237] Tumor model establishment: Take MCF-7 cells in the logarithmic growth phase, add pre-cooled PBS buffer to wash the cells three times, and then digest the cells with 0.25% trypsin. The digested cells are centrifuged at 1000rpm / min for 5 minutes, the precipitate is discarded, and the cells are resuspended with pre-cooled PBS and carefully blown to make a single cell solution. After the cells are counted by a counter, the cell density is adjusted to 1×10 7 cells / mL and put into a centrifuge tube and store on ice. Before inoculating cancer cells, gently wipe the subcutaneous tissue on the right back of the nude mouse with a paper towel sprayed with 75% alcohol. Draw 100μL of cell suspension into a 1mL disposable syringe, exhaust the air, puncture about 1cm forward from the injection site, and perform a single subcutaneous injection. After the injection, slowly withdraw the needle to avoid leakage. Observe the tumor growth daily.

[0238] Dosing: When the tumor volume reaches 60mm 3 The tumor model mice were randomly divided into five groups, namely PBS group, laser group, compound A1 group, PROTAC drug compound S1 group and compound A1 + laser irradiation group. Various drugs were dispersed in the following solution: 1% DMSO + 20% PEG + 5% Tween 80 + 74% dd H 2 O; Dosage: 10 mg / kg; Injection into tail vein, 24 hours after drug injection, 660 nm laser at 0.8 W / cm 2 The irradiation was carried out at a power density of 100 for 10 minutes.

[0239] Experimental Log:

[0240] Tumor size was measured and body weight was monitored every other day. The formula for calculating tumor volume was: tumor volume = length × width × width / 2. After 16 days of monitoring, mice in each group were euthanized and tumors and major organs were collected. The experimental results showed that the mice were in good health during the experiment, and their movements, eating, and excretion were normal.

[0241] The curve of tumor volume change over time in mice in the compound A1 group is shown in Figure 5. From the results of administration, compared with the PBS group, although compound S1 can inhibit tumor growth, the anti-tumor effect is not good, which is mainly due to the lower dosage and frequency of administration. On the contrary, when compound A1 is used in combination with near-infrared light, tumor growth is significantly inhibited. This result shows that the combination of compound A1 and photodynamic therapy can enhance the killing of tumor cells ( Figure 5 The p values ​​used were calculated by t-test, ***p<0.001, **p<0.01. Data are expressed as mean±standard deviation, n=4).

[0242] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A compound represented by formula (VII) or an isomer or a pharmaceutically acceptable salt or deuterated substance thereof: in, X is an activatable photosensitizer.

2. The compound according to claim 1 or its isomer or pharmaceutically acceptable salt or deuterated substance, wherein: Drug is any PROTAC drug.

3. The compound according to claim 1 or 2, or an isomer or a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: DRUG is represented by the following formula (VIII): POIligand-Linker-E3ligand (VIII).

4. The compound according to claim 3 or its isomer or pharmaceutically acceptable salt or deuterated substance, which has a structure as shown in formula (I): in, POI ligand is selected from any target protein ligand of interest; E3 ligand is the E3 ubiquitin ligase ligand; linker is the connecting functional group between the target protein ligand and the E3 ubiquitin ligase ligand.

5. The compound according to any one of claims 1 to 4, or an isomer or a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The structure of X is shown in the following formula (II) or (III): wherein R1, R2, R3 and R4 are selected from C1-10 alkyl; R5 is selected from C1-10 alkyl or C1-10 alkyl sulfonate; R6 and R7 are selected from H or C1-2 alkyl; Y1 is selected from O or S; R8 is selected from F, Cl, Br, I or H; Ring A is an aromatic ring selected from the following structures: The aromatic ring is optionally substituted by 0, 1, 2, 3, 4, 5 or 6 substituents R9 selected from F, Cl, Br, I, C1-6 alkyl, hydroxy, amino, cyano or nitro.

6. The compound according to any one of claims 1 to 5, or an isomer or a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The structure of X is shown in the following formula (II-1) or formula (III-1):

7. The compound according to any one of claims 3 to 6, or an isomer or a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The POI ligand is derived from a small molecule, a nucleic acid aptamer or an antibody; preferably, the POI ligand is a BET family protein targeting ligand JQ1 or a derivative thereof.

8. The compound according to any one of claims 3 to 7, or an isomer, a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The POI ligand structure is shown in the following formula (IV-1), (IV-2), (IV-3), (IV-4) or (IV-5): Ring D is selected from any of the following structures: Ring E is selected from any of the following structures: Preferably, the POI ligand structure is selected from one of the following structures:

9. The compound according to any one of claims 3 to 8, or an isomer, a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The Linker structure is selected from one of the following structures: wherein a and b are each independently 1, 2 or 3, and c, d, e, f, g, h, i are each independently 0, 1, 2 or 3.

10. The compound according to any one of claims 3 to 9, or an isomer, a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The E3 ligand is selected from the E3 ligase ligand of VHL, CRBN, GID4, DCAF2, KLHL41, SPSB4, TRIM7, TRIM9, RNF43 or RNF182; Preferably, the E3 ligand is selected from the E3 ligase ligands of VHL or CRBN.

11. The compound according to any one of claims 3 to 10, or an isomer or a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The E3 ligand structure is shown in the following formula (V-1) or formula (V-2): Among them, R 11 , R 12 and R 13 Each independently selected from H or C1-10 alkyl; optionally, the alkyl is further substituted by H, halogen, hydroxyl, carboxyl, amino, cyano, carbonyl or C1-5 alkyl; Ring B is a C4-10 heterocycloalkyl group; the heterocycloalkyl group is optionally substituted by 0, 1, 2, 3 or 4 carbonyl groups; the heterocycloalkyl group contains 1, 2, 3 or 4 heteroatoms selected from N, O and S, and contains at least one N atom; Ring C is a C6-12 aromatic ring; the aromatic ring is optionally substituted by 0, 1, 2, 3, 4, 5 or 6 substituents selected from F, Cl, Br, I, C1-6 alkyl, hydroxyl, amino, cyano or nitro; R14 is selected from -O- or -NH-; R15 is selected from hydrogen or carbonyl.

12. The compound according to any one of claims 1 to 11, or an isomer, a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: The compound structures are shown in the following formulas (VI-1) and (VI-2):

13. The compound according to any one of claims 11 to 12, or an isomer or a pharmaceutically acceptable salt or a deuterated substance thereof, wherein: R 11 , R 12 and R 13 each independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl or 3-ethylbutyl; Ring B is a heterocycloalkyl group selected from the following structures: The heterocycloalkyl group is optionally substituted with 0, 1, 2, 3 or 4 carbonyl groups; Ring C is an aromatic ring selected from the following structures: The aromatic ring is optionally substituted with 0, 1, 2, 3, 4, 5 or 6 substituents R9 selected from F, Cl, Br, I, C1-6 alkyl, hydroxyl, amino, cyano or nitro; Preferably, ring B is selected from the following heterocycloalkyl groups: Preferably, ring C is selected from the following aromatic rings:

14. The compound according to claim 1 or its isomer, pharmaceutically acceptable salt or deuterated substance, wherein: The compound is selected from one of the structures shown below:

15. A pharmaceutical composition comprising the compound according to any one of claims 1 to 14 or an isomer or a pharmaceutically acceptable salt or deuterated substance thereof.

16. Use of the compound according to any one of claims 1 to 14 or its isomer or pharmaceutically acceptable salt or deuterated substance or the pharmaceutical composition according to claim 15 in preventing and / or treating tumor-related diseases.

17. A pharmaceutical preparation comprising: the compound according to any one of claims 1 to 14 or its isomer or pharmaceutically acceptable salt or deuterated substance, and pharmaceutically acceptable excipients and / or carriers.

18. A device, comprising: a) the compound according to any one of claims 1 to 14 or its isomer or pharmaceutically acceptable salt or deuterated substance, the pharmaceutical composition according to claim 15 or the pharmaceutical preparation according to claim 17; b) Lighting device.

19. The device of claim 18, wherein: The light source emitted by the illumination device is a near-infrared light source.

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