CAIX targeted degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a CAIX targeted degradation agent. The CAIX targeted degradation agent comprises a CAIX target protein ligand, an integrin ligand and a connector, the CAIX target protein ligand and the integrin ligand are connected through the connecting body. As the carbonic anhydrase IX is enriched in the tumor region, the CAIX targeted degradation agent can specifically target the tumor region; the compound can effectively induce degradation of CAIX and inhibit survival of tumor cells under the hypoxia condition, has the functions of targeting a tumor area and degrading carbonic anhydrase IX by utilizing integrin and lysosome, further changes hypoxia and subacid environments of the internal environment of a tumor patient, and plays a role in inhibiting survival of hypoxia tumor cells; the tumor targeting property is high, the toxic and side effects on normal cells are relatively small, and a relatively good effect can be obtained by using a relatively small dosage.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a CAIX targeted degrader, a preparation method of the CAIX targeted degrader, and an application thereof in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is currently one of the major health problems. Hypoxia and acidic microenvironment are typical features of solid tumors. The two will promote the processes of tumor cell proliferation, angiogenesis and metastasis, glycolytic metabolism, etc., and will also have a negative impact on the expression of genes and proteins. In addition, hypoxia and acidic microenvironment are recognized as key factors leading to treatment resistance and poor prognosis in oncology. The bio-reducing environment caused by hypoxia and low pH will affect the stability, penetrability and biological processes of most chemotherapeutic drugs, thereby reducing their anti-cancer effects. Therefore, regulating tumor hypoxia and acidic microenvironment may improve the efficacy of anti-cancer drugs and has profound clinical significance in cancer treatment.

[0003] Carbonic anhydrase IX (CAIX / CA9) is a transmembrane protein overexpressed in developing and invasive tumors, which is only associated with hypoxic tumors. Its expression is regulated in a hypoxia-dependent manner by hypoxia-inducible factor (HIF-1α), and is generally considered as an indicator of poor prognosis. In addition, CAIX can catalyze the conversion of carbon dioxide hydrate into bicarbonate and protons, thereby participating in the pH regulation of hypoxic solid tumors. CAIX plays an important role in promoting the proliferation, invasion and metastasis of tumor cells and is a therapeutic target worthy of research. Therefore, inhibiting CAIX can effectively improve the characteristics of hypoxia and acidic microenvironment and provide a more effective means for the clinical treatment of solid tumors.

[0004] Since the processes of development, angiogenesis, metastasis and drug resistance characteristics of various cancers are directly or indirectly related to the abnormal activity of CAIX, CAIX has become a promising drug target. Inhibiting the activity of CAIX enzyme can improve hypoxia and acidic microenvironment and provide a more effective means for the clinical treatment of solid tumors. However, there is currently a lack of compounds that can specifically target and degrade CAIX, and there is a certain gap in the clinical evaluation of this part of the research content in cancer treatment.

[0005] At present, there are mainly two strategies for inhibiting CAIX: monoclonal antibodies and small molecule inhibitors of CAIX. The first monoclonal antibody-based therapeutic strategy is to inhibit CAIX through CAIX-mediated immunotherapy using specific antibodies with induced cytotoxicity, thereby treating cancer clinically. Among them, the chimeric monoclonal antibody Girentuximab targeting CAIX is used to treat metastatic renal cell carcinoma. However, in the adjuvant therapy in the phase III clinical study ARISER, the drug-related adverse reactions in RCC patients with a high risk of recurrence after nephrectomy reached 21.6%, and the expected clinical benefit was not achieved.

[0006] The second small molecule inhibitor strategy of CAIX is based on the theoretical basis that the catalytic activity of CAIX is crucial for the survival and invasiveness of tumor cells. However, simply reducing CAIX-expressing cells or inhibiting CAIX is not sufficient to have a good therapeutic response because tumors are heterogeneous and the drug targets have stage-specific differential expression characteristics. The main problem in designing CA inhibitors is that the similar structures of the active sites of CA isomers will interfere with the selectivity of small molecules, resulting in low tumor specificity of existing small molecule inhibitors of CAIX and causing damage to normal cells. Therefore, there is an urgent need to develop small molecule compounds with high specificity for targeted degradation of CAIX. Summary of the Invention

[0007] The object of the present invention is to provide a CAIX targeted degrader, which solves the deficiencies of existing CAIX degrading agents that are prone to drug resistance and have low tumor specificity.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A CAIX targeted degrader, comprising a CAIX target protein ligand, an integrin ligand and a linker; the CAIX target protein ligand and the integrin ligand are connected through the linker.

[0010] In the present invention, the linker in the CAIX-targeted degrader connects the ligand that specifically recognizes carbonic anhydrase IX and the ligand that specifically binds to the integrin receptor. Since carbonic anhydrase IX is enriched in the tumor region, the CAIX-targeted degrader can specifically target the tumor region; integrin, as a heterodimeric transmembrane protein receptor that can connect the intracellular and extracellular matrix (ECM), is expressed on the cell surface, mainly acts as a signaling protein in mammals, and is widely expressed on the surfaces of various tumor cells. When integrin binds to its ligand, a receptor-ligand-mediated transmission system can be formed, thereby guiding secreted proteins or membrane proteins to the lysosome for degradation. Therefore, the CAIX-targeted degrader of the present invention is a new CAIX degrader that can effectively degrade highly expressed CAIX and integrin on tumor cells. This degrader can effectively induce the degradation of CAIX, inhibit the survival of tumor cells under hypoxic conditions, has the functions of targeting the tumor region and using integrin and lysosome to degrade carbonic anhydrase IX, and further changes the hypoxic and mildly acidic environment of the internal environment of tumor patients, playing a role in inhibiting the survival of hypoxic tumor cells. It has high tumor targeting, low toxic and side effects on normal cells, and can achieve better effects with a smaller dose.

[0011] In the present invention, the CAIX target protein ligand has a sulfonamide group. For the ligand with a sulfonamide group, the sulfonamide group can target Zn at the CA active center 2+ , form a coordination bond, and interfere with the function of carbonic anhydrase.

[0012] Furthermore, the CAIX target protein ligand is 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutanoic acid.

[0013] In the present invention, the integrin ligand includes one or more of a polypeptide with an RGD sequence, a cyclic RGD sequence, a cyclopentapeptide sequence containing RGD, a cyclic peptide cilengitide sequence, an antibody containing an RGD sequence, and a protein containing an RGD sequence.

[0014] Preferably, the integrin ligand is a cyclic RGD sequence. The integrin αvβ3 receptor specifically recognized by RGD is highly expressed in tumor cells, and the specifically bound CAIX is highly expressed in highly invasive tumor cells rather than normal tissues, showing high selectivity for tumor cells. The cyclic RGD sequence enables the degrader to degrade the entire pathogenic protein via lysosomes, which can overcome the drug resistance of tumors, thereby reducing the dosage of drugs and avoiding the off-target effect caused by high doses, making it have low toxicity and high safety. Compared with the above-mentioned other antibodies and proteins targeting integrin αvβ3, the cyclic small molecule containing the RGD (Arg-Gly-Asp) sequence also has the following advantages: 1. Small molecular weight, small volume, and simple synthesis; 2. High stability and good hydrophilicity; 3. Easier to conjugate with fluorescent labels, chelating agents or carriers.

[0015] In the present invention, the structure of the linker includes an alkyl chain containing one or more of an aromatic ring, a heterocyclic ring, a heteroatom, and a functional group.

[0016] Further, the structural formula of the linker is as follows:

[0017]

[0018] Further, the structural formula of the linker is as follows:

[0019] This connection structure is the optimal linker structure and still maintains a good function of degrading CAIX at a low concentration of the CAIX-targeted degrader.

[0020] A preparation method of a CAIX-targeted degrader includes the following steps:

[0021] S1. Boc-6-aminohexanoic acid, DCC, and N-hydroxysuccinimide are sequentially added to a reaction flask, stirred and dissolved with anhydrous N,N-dimethylformamide, reacted at room temperature, 3-azidopropylamine and DIEA are added to the solution, stirred at room temperature, separated and purified, and after drying, a white powder Boc-L1-Azide is obtained;

[0022] S2. The white powder Boc-L1-Azide is dissolved in TFA / DCM under ice bath conditions, stirred and reacted at room temperature, and the solvent is evaporated under reduced pressure to obtain a crude amine product with the Boc protecting group removed;

[0023] S3. The crude amine product, 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid, and DIEA are dissolved in anhydrous DMF, HATU is added, the reaction solution is stirred at room temperature, separated and purified, and after drying, Sul-L1-Azide is obtained;

[0024] S4. Dissolve Sul-L1-Azide, Alkyne-cRGD, CuSO4·5H2O and NaVc in DMF / H2O, react, separate and purify to obtain the CAIX targeting degrader (Sul-L1-RGD).

[0025] The preparation method of this degrader has a simple process and a high yield, which is beneficial to the production of enterprises. Using the above preparation method, a degrader Sul-Linker-RGD with the function of degrading carbonic anhydrase IX and the function of integrin αvβ3 receptor was synthesized. As a new type of carbonic anhydrase IX degrader, it provides a new strategy for the treatment of solid tumors based on hypoxic and acidic microenvironments and has great application potential in cancer therapeutic drugs.

[0026] Furthermore, in step S1, the mass ratio of Boc-6-aminohexanoic acid, DCC and N-hydroxysuccinimide is 2:1:1 - 1:2:2.

[0027] Furthermore, in step S2, the reaction time at room temperature is 1 - 3 hours.

[0028] Furthermore, in step S2, the concentration range of TFA / DCM is 10 - 30%.

[0029] Furthermore, in step S3, the mass ratio of 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid and DIEA is 1:5 - 1:20.

[0030] Furthermore, in step S3, the mass ratio of DIEA and anhydrous DMF is 1:10 - 1:5.

[0031] Application of a CAIX targeting degrader in the preparation of tumor vaccines or anti-tumor drugs. This CAIX targeting degrader can specifically target tumor regions, and simultaneously degrade carbonic anhydrase IX and integrin in the body of tumor patients, thereby changing the hypoxic and slightly acidic environment of the internal environment of tumor patients and playing a role in inhibiting the survival of hypoxic tumor cells.

[0032] Furthermore, the tumor includes breast cancer or colon cancer.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] (1) The CAIX targeting degrader of the present invention is a bifunctional molecular degrader, which has a ligand that specifically recognizes CAIX and a ligand that can specifically bind to the integrin receptor. The two are connected by a linker and can effectively degrade CAIX and integrin α v β3 receptor highly expressed on the surface of tumor cells. Integrin α vThe β3 receptor is highly expressed in tumor cells, and CAIX is highly expressed in highly invasive tumor cells rather than normal tissues. This degrader has high selectivity for tumor cells and strong specificity.

[0035] (2) The CAIX-targeted degrader of the present invention can effectively induce the degradation of carbonic anhydrase IX and integrin αvβ3 receptor under hypoxic conditions, inhibit the survival of tumor cells, degrade the entire pathogenic protein through lysosomes, thereby overcoming tumor drug resistance; thereby improving the hypoxic and acidic microenvironment unique to solid tumors, and thus playing a certain role in the treatment of cancer.

[0036] (3) Carbonic anhydrase IX, which is specifically bound by the degrader of the present invention, is highly expressed in highly invasive tumor cells rather than normal tissues, and has high selectivity for tumor cells, thereby reducing the dosage of the CAIX-targeted degrader, avoiding off-target effects caused by high doses, making it have low toxicity and high safety, and providing a new solution for the treatment or prevention of various cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is a schematic diagram of the degradation of CAIX by the CAIX-targeted degrader of the present invention;

[0039] Figure 2 It is a chemical molecular structure diagram of the CAIX-targeted degrader Sul-Linker-RGD of the present invention;

[0040] Figure 3 shows the degradation of CAIX in MDA-MB-231 cells by Sul-6-RGD "linker" at different concentrations or different treatment times;

[0041] Figure 4 is the result diagram of the degradation of CAIX by Sul-L1-RGD through the lysosomal pathway;

[0042] Figure 5 shows the effects of Sul-L1-RGD on the protein levels of Integrinβ3, β5, and β1;

[0043] Figure 6 It is the effect of Sul-L1-RGD on the survival of tumor cells under hypoxic and normoxic conditions. DETAILED DESCRIPTION OF THE INVENTION

[0044] PROTAC (Proteolysis Targeting Chimera) is an emerging targeted protein degradation strategy and one of the drug development technologies that have received significant attention in the field of cancer treatment. It utilizes the ubiquitin-proteasome system (UPS) to specifically recognize and effectively degrade target proteins. However, due to the fact that the UPS mainly targets some proteins within cells, its application has been restricted. As a complement to the UPS, endocytosis and the lysosomal pathway can degrade extracellular and membrane-associated proteins.

[0045] The present invention provides a new integrin-mediated lysosomal degrader of extracellular and membrane proteins, a CAIX-targeted degrader. Integrin, as a heterodimeric transmembrane protein receptor that can connect the intracellular and extracellular matrix (ECM), binds to its ligand and can form a receptor-ligand-mediated transmission system, thereby guiding secreted proteins or membrane proteins to the lysosome for degradation. The schematic diagram of the specific degradation process is shown in Figure 1 Figure. This CAIX-targeted degrader consists of three parts, including a target protein binding domain, an integrin binding domain, and a "Linker" structure that covalently links two active ligands together through special design. The CAIX-targeted degrader uses CAIX as the degradation target and induces the degradation of CAIX in an integrin- and lysosome-dependent manner. At the same time, it also has a significant degradation effect on integrin αvβ3 and significantly inhibits the survival of tumor cells under hypoxic conditions. The CAIX degrader of the present invention can be applied as a therapeutic drug for solid tumors in hypoxic and acidic microenvironments and has great application potential in cancer treatment.

[0046] The technical solutions of this patent will be further described in detail below in combination with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations for this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0047] Example 1

[0048] This example provides a CAIX-targeted degrader, which includes a CAIX target protein ligand, an integrin ligand, and a linker; the CAIX target protein ligand and the integrin ligand are connected through the linker.

[0049] In the present invention, the CAIX target protein ligand has a sulfonamide group. In this example, the CAIX target protein ligand is 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid, which is named Sul-6. 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid has a significant effect on inhibiting carbonic anhydrase IX. Therefore, only a small dose of the drug is required to achieve the function of inhibiting carbonic anhydrase IX. Thus, the toxic effect on normal cells is reduced, and the side effects of the CAIX-targeted degrader are reduced. The ligand specifically recognizing carbonic anhydrase IX in the CAIX-targeted degrader can also be other ligands, such as BMS, etc.

[0050] In the present invention, the integrin ligand can be selected from one or more of polypeptides with RGD sequences, cyclic RGD sequences, cyclopentapeptide sequences containing RGD, cyclopeptide cilengitide sequences, antibodies containing RGD sequences (such as the Etaracizumab sequence), and proteins containing RGD sequences (such as the BJ001 sequence). Integrin αvβ3 is overexpressed in a variety of tumor cells, making it an important target for tumor diagnosis and anti-tumor drug research. A segment of arginine–glycine–aspartic acid (RGD) sequence is contained in the natural ligands of αvβ3, which is involved in activities such as the proliferation and metastasis of tumor cells. Cyclopentapeptide (cyclo[(RGDf(NMe)V)]), also known as cilengitide, is an anti-tumor integrin inhibitor with high affinity and selectivity for integrin αvβ3, and also inhibits αvβ5 and α5β1; Etaracizumab is an αvβ3 integrin IgG1 monoclonal antibody, mainly targeting gastric cancer, renal cancer, melanoma, small intestine cancer, lymphoma, and psoriasis; BJ-001 is a tumor-targeted IL-15 fusion protein, and its domain contains a targeting molecule targeting tumor cells with high expression of integrin αvβ3, αvβ5, and αvβ6. The IL-15 structure can bind to the IL-2 / 15Rβγc receptor complex expressed on immune cells (especially NK and T cells) to activate the immune system and kill tumors. As an integrin recognition ligand, the present invention selects cyclic RGDyK (cRGD) containing the Arg-Gly-Asp sequence, which has high affinity for integrin αvβ3 and maintains selectivity for integrins such as αvβ5, αvβ6, and α5β1. The above sequences specifically binding to integrin receptors enable the CAIX-targeted degrader to degrade the entire pathogenic protein through lysosomes, which can overcome the drug resistance of tumors, thereby reducing the dosage of the CAIX-targeted degrader and avoiding the off-target effect caused by high doses, making it have low toxicity and high safety.

[0051] In this embodiment, the integrin ligand is a cyclic RGD sequence. The RGD sequence (Arg-Gly-Asp) Peptides is a cell adhesion sequence that can mimic cell adhesion proteins and bind to integrins. The RGD sequence is a cell attachment site for a large number of adhesive extracellular matrices, blood, and cell surface proteins, and nearly half of the more than 20 known integrins can recognize this sequence in their adhesive protein ligand regions. The integrin αvβ3 receptor specifically recognized by RGD is highly expressed in tumor cells, and CAIX specifically bound by sul-6 is highly expressed in highly invasive tumor cells rather than normal tissues, showing high selectivity for tumor cells. The cyclic RGD sequence enables the CAIX-targeted degrader to degrade the entire pathogenic protein through lysosomes, overcoming the drug resistance of tumors, thereby reducing the dosage of the CAIX-targeted degrader and avoiding off-target effects caused by high doses, making it have low toxicity and high safety.

[0052] In the present invention, the structure of the linker includes an alkyl chain containing one or more of an aromatic ring, a heterocyclic ring, a heteroatom, and a functional group.

[0053] In this embodiment, the structural formula of the linker is as follows:

[0054]

[0055] More preferably, the structural formula of the linker is as follows:

[0056] The performance of the CAIX-targeted degrader prepared by this connection structure in degrading CAIX and integrin reaches the highest level. As shown in the subsequent experimental examples, according to the experiments, the CAIX-targeted degrader still maintains a good function of degrading CAIX at low concentrations.

[0057] Please refer to the appendix Figure 2 , which is one of the chemical molecular structure diagrams of the CAIX-targeted degrader Sul-Linker-RGD; it contains a ligand Sul that can specifically recognize CAIX and a ligand cyclic RGD peptide that can specifically bind to integrins, and is connected by a linker L1; on the Figure 2 left side of which is shown the ligand Sul-6 that specifically recognizes CAIX, in the middle is the cyclic RGD peptide that can specifically bind to integrins, and on the right side are shown the chemical molecular formulas of three different structures of the linker, which connects the ligand Sul-6 and the cyclic RGD peptide. The CAIX-targeted degrader Sul-Linker-RGD takes CAIX as the degradation target and induces the degradation of CAIX in an integrin- and lysosome-dependent manner, and also has a significant degradation effect on integrin αvβ3, and significantly inhibits the survival of tumor cells under hypoxic conditions. It has high tumor targeting, less toxic and side effects on normal cells, and can use a smaller dose to obtain better effects.

[0058] Example 2

[0059] In this example, a preparation method of a CAIX-targeted degrader was provided. A series of CAIX degraders with different "linker" lengths, namely Sul-linker-RGD, were constructed through a novel integrin-facilitated target protein lysosomal degradation (IFLD) strategy.

[0060] Among the small molecule inhibitors with high affinity for targeting CAIX, a compound with strong inhibitory effect was selected as the small molecule ligand of CAIX, that is, 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid. Thus, a series of CAIX degraders were constructed. Taking Sul-L1-RGD as an example, the specific steps are as follows:

[0061] S1. Boc-6-aminohexanoic acid, DCC and N-hydroxysuccinimide were successively added to a reaction flask, stirred and dissolved with anhydrous N,N-dimethylformamide, reacted at room temperature, 3-azidopropylamine and DIEA were added to the solution, stirred at room temperature, separated and purified, and a white powder Boc-L1-Azide was obtained after drying.

[0062] S2. The white powder Boc-L1-Azide was dissolved in TFA / DCM under ice bath conditions, stirred and reacted at room temperature, and the solvent was evaporated under reduced pressure to obtain a crude amine product with the Boc protecting group removed.

[0063] S3. The crude amine product, 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid and DIEA were dissolved in anhydrous DMF, HATU was added, the reaction solution was stirred at room temperature, separated and purified, and Sul-L1-Azide was obtained after drying.

[0064] S4. Sul-L1-Azide, Alkyne-cRGD, CuSO4·5H2O and NaVc were dissolved in DMF / H2O, reacted, separated and purified to obtain the CAIX-targeted degrader Sul-L1-RGD.

[0065] In a further example, the mass ratio of Boc-6-aminohexanoic acid, DCC and N-hydroxysuccinimide in step S1 was 2:1:1 - 1:2:2, and the efficiency of synthesizing the crude amine product was relatively high within this material ratio range.

[0066] In a further example, the reaction time at room temperature in step S2 was 1 - 3 hours, and the coupling reaction could be completed within this time range.

[0067] In a further embodiment, the concentration range of TFA / DCM in step S2 is 10-30%, and the efficiency of synthesizing the crude amine product with TFA / DCM within this concentration range is relatively high.

[0068] In a further embodiment, the mass ratio of 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid to DIEA in step S3 is 1:5 - 1:20, and the efficiency of synthesizing the CAIX targeted degrader within this material ratio range is relatively high.

[0069] In a further embodiment, the mass ratio of DIEA to anhydrous DMF in step S3 is 1:10 - 1:5, and the efficiency of synthesizing the CAIX targeted degrader within this material ratio range is relatively high.

[0070] In a further embodiment, Sul-L2-Azide and Sul-L3-Azide were synthesized using the above steps respectively, and the final products Sul-L2-RGD and Sul-L3-RGD were ultimately synthesized.

[0071] The specific process is as follows:

[0072] First, Sul-L1-Azide needs to be synthesized. Boc-6-aminohexanoic acid (20 mg, 86.5 μM), DCC (17.8 mg, 86.4 μM), and N-hydroxysuccinimide (10.0 mg, 86.9 μM) were successively added to a reaction flask, dissolved by stirring with 1.5 mL of anhydrous N,N-dimethylformamide (DMF), and reacted at room temperature for 2 hours. Then, 3-azidopropylamine (8.7 mg, 86.9 μM) and DIEA (143.0 μL, 865.3 μM) were added to the solution, and stirring was continued at room temperature for 2 hours. It was separated and purified by HPLC, and after freeze-drying, a white powder Boc-L1-Azide was obtained. The white powder was dissolved in 20% TFA / DCM in an ice bath and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to obtain a crude amine product with the Boc protecting group removed. The crude deprotected amine, Sul-6 (24.2 mg, 86.3 μM), and DIEA (143.0 μL, 865.3 μM) were dissolved in 1 mL of anhydrous DMF, and then HATU (39.5 mg, 103.9 μM) was added. The reaction solution was stirred at room temperature for 2 hours, separated and purified by HPLC, and after freeze-drying, a white powder Sul-L1-Azide (22.5 mg, 54%) was obtained. Its mass spectrometry data was: HRMS(ESI+) m / z: calcd. for C15H26N9O5S2 [M+H]+ 476.1420, found 476.1493. Secondly, Alkyne-cRGD (9.0 mg, 12.6 μM), Sul-L1-Azide (6.0 mg, 12.6 μM), CuSO4·5H2O (1.89 mg, 7.6 μM), and NaVc (10.0 mg, 50.5 μM) were dissolved in a DMF / H2O (1.5 mL, 2:1) mixture. The solution was stirred at room temperature for 2 hours, separated and purified by HPLC, and after freeze-drying, a white powder Sul-L1-RGD (12 mg, 80%) was obtained. Its mass spectrometry data was HRMS(ESI+) m / z: calcd. for C48H73N18O14S2 [M+H]+ 1189.4917, found 1189.4990.

[0073] Through the same steps, by selecting different linkers, Sul-L2-Azide and Sul-L3-Azide, as well as the final products Sul-L2-RGD and Sul-L3-RGD, were synthesized respectively.

[0074] The mass spectrometry data is as follows:

[0075] Sul-L2-Azide, tR = 16.12, Yield = 52%, HRMS(ESI+) m / z: calculated for C18H32N9O5S2 [M+H]+ 518.1890, found 518.1962; Sul-L2-RGD, tR = 9.08, Yield = 86%, HRMS(ESI+) m / z: calculated for C51H79N18O14S2 [M+H]+ 1231.5386, found 1231.5459; Sul-L3-Azide, tR = 18.99, Yield = 51%, HRMS(ESI+) m / z: calculated for C21H38N9O5S2 [M+H]+ 560.2359, found 558.2286; Sul-L3-RGD, tR = 10.82, Yield = 84%, HRMS(ESI+) m / z: calculated for C54H85N18O14S2 [M+H]+ 1273.5856, found 1273.5929.

[0076] The preparation method of this CAIX-targeted degrader has a simple process and a high yield, which is beneficial to the production of enterprises. Using the above method, a bifunctional molecular degrader that promotes the lysosomal targeting and degradation of CAIX by integrin was prepared. A ligand that can specifically recognize CAIX and cRGD that can specifically bind to the integrin receptor were coupled through a "linker" structure to form a bifunctional compound, and this was used to prepare the CAIX-targeted degrader, which has extremely high application value in anti-tumor drugs.

[0077] In the following examples, the CAIX-targeted degrader Sul-L1-RGD with the best degradation effect and specific targeting of CAIX will be screened through Western blot experiments, and the optimal degradation concentration, time, and degradation pathway of Sul-L1-RGD will be further studied.

[0078] Experimental Example 1 Effect of different linkers on the degradation effect of CAIX-targeted degrader on CAIX protein

[0079] The breast cancer cells MDA-MB-231 were plated in 12-well plates. When the cell density reached 60% to 80%, they were treated with three small molecule compounds Sul-L1-RGD, Sul-L2-RGD, and Sul-L3-RGD with different "linker" lengths at different concentrations (concentration gradients of 5 nM, 25 nM, 50 nM, and 100 nM) for 8 h. Then, SDS lysis buffer was used to collect protein samples for Western blot experiments to detect the degradation of CAIX protein.

[0080] Please refer to Figure 3, which shows the Western blot results of Sul-6-RGD "linker" at different concentrations; A-C respectively show the Western blot analysis of the degradation of CAIX (CA9) induced by different concentrations of Sul-L1-RGD, different concentrations of Sul-L2-RGD (A), different concentrations of Sul-L3-RGD, and different induction times of Sul-L3-RGD (B). Here, MDA-MB-231 cells were treated with 5, 25, 50, 100 nM Sul-L1-RGD, Sul-L2-RGD, and Sul-L3-RGD for 8 h, Figure 3A , Figure 3B The first row of Figure 3A shows the concentration of the CAIX-targeted degrader used, the second row shows the time of using the CAIX-targeted degrader, the third row shows the Western blot result image of the remaining content of CAIX after inducing its degradation, the fourth row shows the content of the remaining CAIX compared with the control, and the fifth row shows the Western blot result image of the internal reference GAPDH in breast cancer cells MDA-MB-231. It can be seen from Figure 3A that when treated with 5 nM Sul-L1-RGD for 8 hours, the effect of degrading CAIX is the best, and the remaining rate of CAIX is only 53%. And within the concentration range of 5-100 nM, the remaining rate of CAIX after treatment with Sul-L1-RGD is lower than 60%; it can be seen from Figure 3A that when treated with 100 nM Sul-L2-RGD for 8 hours, the effect of degrading CAIX is the best, and the remaining rate of CAIX is 74%; it can be seen from Figure 3B that when treated with 100 nM Sul-L3-RGD for 8 hours, the effect of degrading CAIX is the best, and the remaining rate of CAIX is only 47%; it can be seen from Figure 3A -B that Sul-L1-RGD still maintains a good function of degrading CAIX at low concentrations. Figure 3C : Western blot analysis of the effect of different linker lengths on inducing the degradation of CAIX. Figure 3C The first row of Figure 3C shows the effect of Sul-L1-RGD on degrading CAIX at 5 nM, and the remaining rate of CAIX is 50%; the second row shows the effect of Sul-L2-RGD on degrading CAIX at 5 nM, and the remaining rate of CAIX is 70%; the third row shows the effect of Sul-L3-RGD on degrading CAIX at 5 nM, and the remaining rate of CAIX is 78%; further Figure 3C illustrates that Sul-L1-RGD still maintains a good function of degrading CAIX at low concentrations. Therefore, Sul-L1-RGD is selected as the best CAIX degrader through the above steps.

[0081] Experimental Example 2: Optimal Degradation Concentration and Time of CAIX by the Lysosomal Pathway of the CAIX Targeted Degrader

[0082] The breast cancer cells MDA-MB-231 were plated in 12-well plates. When the cell density reached 60% to 80%, they were first pretreated with Bafilomycin A1 (100 nM) or MG132 (5 μM) for 2 h, and then treated with Bafilomycin A1 or MG132 and Sul-L1-RGD (5 nM) for 8 h. Protein samples were collected using SDS lysis buffer for western blot experiments to detect the expression of CAIX protein.

[0083] Please refer to Figure 4, which is the result diagram of the degradation of CAIX by Sul-L1-RGD through the lysosomal pathway; Figure 4A The results show the degradation of CAIX mediated by Sul-L1-RGD at the specified concentration or specified time analyzed by Western blot in MDA-MB-231 cells. Among them, the first row shows the result of the degradation of CAIX by Sul-acid at 100 nM, and the remaining rate of CAIX is 92%; the second row shows the result of the degradation of CAIX by Sul-L1-RGD at 5 nM, and the remaining rate of CAIX is 51%, the result of the degradation of CAIX by Sul-L1-RGD at 25 nM, and the remaining rate of CAIX is 54%, the result of the degradation of CAIX by Sul-L1-RGD at 50 nM, and the remaining rate of CAIX is 53%, and the result of the degradation of CAIX by Sul-L1-RGD at 100 nM, and the remaining rate of CAIX is 50%;

[0084] Figure 4B The results show the degradation of CAIX mediated by Sul-L1-RGD at the specified concentration or specified time analyzed by Western blot in MDA-MB-231 cells. Among them, the first row shows the concentration of Sul-L1-RGD is 5 nM, the second row shows the time of using Sul-L1-RGD, the third row shows the Western blot result image of the breast cancer cells MDA-MB-231, and the fourth row shows the result of the degradation of CAIX. Among them, the remaining rate of CAIX is 56% when treated with Sul-L1-RGD for 4 h, 41% when treated for 8 h, 42% when treated for 12 h, and 47% when treated for 24 h. Therefore, the optimal treatment time for Sul-L1-RGD to degrade CAIX is 8 - 12 h.

[0085] Figure 4CShown are the results of analyzing the degradation of CAIX mediated by Sul-L1-RGD at specified concentrations or specified times in HT-29 cells by Western blot. The first row shows the concentration gradient of Sul-L1-RGD, the second row shows the time of using Sul-L1-RGD, the third row shows the Western blot result image of colon cancer HT-29 cells, and the fourth row shows the results of CAIX degradation. The results of the first five columns show that after 8 hours of treatment, when the concentration of Sul-L1-RGD is 5 nM, the remaining rate of CAIX is 48%, and when the concentration of Sul-L1-RGD is 25 nM, the remaining rate of CAIX is 49%, both lower than 50%. Therefore, the optimal concentration of Sul-L1-RGD when applied to HT-29 cells is 5 - 25 nM; the last five columns show the ability of Sul-L1-RGD to degrade CAIX at different treatment times when the concentration is 5 nM. In the range of 8 - 24 hours, the remaining rate of CAIX is in the range of 38 - 41%. It can be seen from the 9th column that the remaining rate of CAIX is the lowest at 38% when treated for 12 hours. Therefore, the optimal treatment time for Sul-L1-RGD to degrade CAIX when applied to colon cancer HT-29 cells is 8 - 24 hours. Figure 4D Western blot analysis of MDA-MB-231 cells treated with 5 nM Sul-L1-RGD and 100 μM lysosomal inhibitor bafilomycin A1 or 5 μM proteasome inhibitor MG132 for 8 hours. The results show that the lysosomal inhibitor can effectively inhibit the degradation of CAIX by Sul-L1-RGD, while the proteasome inhibitor cannot. Therefore, it is shown that Sul-L1-RGD degrades CAIX through the lysosomal pathway. Figure 4E Western blot analysis of CAIX levels in MDA-MB-231 cells treated with Sul-acid, cRGD, and Sul-L1-RGD at 5 nM for 8 hours. The results show that Sul-acid, cRGD, and linker L1 have a significant effect on degrading CAIX only after forming a CAIX-targeted degrader. Figure 4F Western blot analysis of the induction of CAIX degradation by different concentrations of Sul-L1-RGD in the presence of 100 μM cobalt chloride solution (simulating anoxic environment). The results show that under hypoxic conditions, different concentrations of Sul-L1-RGD can still induce CAIX degradation. According to the above results, it is shown that Sul-L1-RGD degrades CAIX through the lysosomal pathway and still has a strong ability to induce CAIX degradation under hypoxic conditions.

[0086] Experimental Example 3 Degradation effect of CAIX-targeted degrader on integrin protein

[0087] The breast cancer cells MDA-MB-231 were plated in 12-well plates. When the cell density reached 60% to 80%, after being treated with different concentrations of Sul-L1-RGD (5 nM; 25 nM; 50 nM; 100 nM) for different times (4 h; 8 h; 12 h; 24 h), protein samples were collected using SDS lysis buffer for western blot experiments to detect the degradation of different integrin proteins integrinβ1, integrinβ3, and integrinβ5.

[0088] Please refer to Figure 5 for the Western blot analysis of the effects of Sul-L1-RGD on the protein levels of Integrinβ3, β5, and β1; Western blot analysis of the degradation of integrinβ3 (A), integrinβ5 (B), and integrinβ1 (C) mediated by Sul-L1-RGD at the specified concentration or specified time.

[0089] Figure 5A The results of Western blot analysis of the degradation of Integrinβ3 mediated by Sul-L1-RGD at the specified concentration or specified time in MDA-MB-231 cells are shown. The first row shows the concentration gradient of Sul-L1-RGD, the second row shows the time of using Sul-L1-RGD, the third row shows the Western blot result image of integrinβ3 in breast cancer cells MDA-MB-231, and the fourth row shows the results of the degradation of integrinβ3. Among them, the results of the first five columns show that after treatment for 8 hours, when the concentration of Sul-L1-RGD is 5 nM, the remaining rate of integrinβ3 is 48%, when the concentration of Sul-L1-RGD is 25 nM, the remaining rate of integrinβ3 is 47%, when the concentration of Sul-L1-RGD is 50 nM, the remaining rate of integrinβ3 is 46%, and when the concentration of Sul-L1-RGD is 100 nM, the remaining rate of integrinβ3 is 45%, all of which are lower than 50%. Therefore, the optimal concentration of Sul-L1-RGD for degrading integrinβ3 in MDA-MB-231 cells is 5 - 100 nM; the last five columns show the ability of Sul-L1-RGD to degrade integrinβ3 at different times when the concentration is 5 nM. The remaining rate of integrinβ3 is in the range of 51 - 58% in the interval of 8 - 24 hours. It can be seen from the eighth column that the remaining rate of integrinβ3 is the lowest at 51% when treated for 8 hours. Therefore, the optimal treatment time for Sul-L1-RGD to degrade integrinβ3 in MDA-MB-231 cells is 8 - 24 hours.

[0090] Figure 5BThe figure shows the results of analyzing the degradation of Integrinβ5 mediated by Sul-L1-RGD at specified concentrations or specified times in MDA-MB-231 cells by Western blot. The first row shows the concentration gradient of Sul-L1-RGD, the second row shows the time of using Sul-L1-RGD, the third row shows the Western blot result image of integrin β5 in breast cancer cells MDA-MB-231, and the fourth row shows the results of the degradation of integrin β5. The results of the first five columns show that after 8 hours of treatment, when the concentration of Sul-L1-RGD is 5 nM, the remaining rate of integrin β5 is 64%, when the concentration of Sul-L1-RGD is 25 nM, the remaining rate of integrin β5 is 65%, when the concentration of Sul-L1-RGD is 50 nM, the remaining rate of integrin β5 is 73%, and when the concentration of Sul-L1-RGD is 100 nM, the remaining rate of integrin β5 is 47%. Therefore, the optimal concentration of Sul-L1-RGD for degrading integrin β5 when applied to MDA-MB-231 cells is above 100 nM; the last five columns show the ability of Sul-L1-RGD to degrade integrin β5 at different times when the concentration is 5 nM. The remaining rate of integrin β5 is in the range of 51-57% in the interval of 12-24 hours. It can be seen from the 9th column that the remaining rate of integrin β5 is the lowest at 51% when treated for 12 hours. Therefore, the optimal treatment time for Sul-L1-RGD to degrade integrin β5 when applied to MDA-MB-231 cells is 12-24 hours.

[0091] Figure 5CShown are the results of analyzing the degradation of Integrinβ1 mediated by Sul-L1-RGD at specified concentrations or specified times in MDA-MB-231 cells by Western blot. The first row shows the concentration gradient of Sul-L1-RGD, the second row shows the time of using Sul-L1-RGD, the third row shows the Western blot result image of integrinβ1 in breast cancer cells MDA-MB-231, and the fourth row shows the results of the degradation of integrinβ1. Among them, the results of the first five columns show that after 8 hours of treatment, when the concentration of Sul-L1-RGD is 5 nM, the remaining rate of integrinβ1 is 104%, when the concentration of Sul-L1-RGD is 25 nM, the remaining rate of integrinβ1 is 97%, when the concentration of Sul-L1-RGD is 50 nM, the remaining rate of integrinβ1 is 104%, and when the concentration of Sul-L1-RGD is 100 nM, the remaining rate of integrinβ1 is 69%. Therefore, the preferred concentration of Sul-L1-RGD for degrading integrinβ1 when applied to MDA-MB-231 cells is above 100 nM; the last five columns show the ability of Sul-L1-RGD to degrade integrinβ1 at different times when the concentration is 5 nM. The remaining rate of integrinβ1 is in the range of 94-105% in the interval of 4-8 hours. It can be seen from the seventh column that the remaining rate of integrinβ1 is the lowest at 94% when treated for 4 hours. Therefore, the optimal treatment time for Sul-L1-RGD to degrade integrinβ1 when applied to MDA-MB-231 cells is 4-8 hours. As Figure 5A shown in -C, it shows that Sul-L1-RGD has a significant effect on the protein level of Integrinβ3 and also has a certain effect on integrinβ5, while having almost no effect on integrinβ1.

[0092] Experimental Example 4 Survival of cancer cells after treatment with CAIX targeting degrading agent

[0093] Plate breast cancer cells MDA-MB-231 and colon cancer cells HT-29 in a 96-well plate. When the cell density reaches 50% to 60%, treat with 5 nM Sul-L1-RGD, Sul-acid, cRGD, SLC-0111 and 100 μM CoCl2 solution (simulating hypoxic environment) alone or in combination for different times (24 h, 48 h and 72 h), and perform a CCK8 experiment after 72 h to detect the cell survival situation.

[0094] Please refer to the appendix Figure 6, which is the result graph of the tumor cell survival experiment. CCK8 was used to analyze the effect of Sul-L1-RGD treatment on the survival of MDA-MB-231 (A) and HT-29 (B) cells under hypoxic conditions, and the effect of Sul-L1-RGD treatment on the survival of MDA-MB-231 cells under normoxic conditions. The abscissa shows the time of experimental treatment, and the ordinate shows the content of surviving cells; From Figure 6 As shown in A, under hypoxic conditions, in MDA-MB-231 cells, from the first day to the third day of the experiment, there was no significant difference in the cell survival of the Sul-acid group, cRGD group, and Sul-acid + cRGD group compared with the control group. After Sul-L1-RGD treatment, the survival of MDA-MB-231 cells decreased significantly, comparable to the treatment group with the carbonic anhydrase inhibitor SLC-0111, indicating that Sul-L1-RGD has the function of reducing tumor cell proliferation under hypoxic conditions; From Figure 6 As shown in B, in HT-29 cells, from the first day to the third day of the experiment, the cell survival in the CoCl2 + Sul-L1-RGD experimental group decreased significantly compared with the CoCl2 control group and the CTL control group, indicating that Sul-L1-RGD has the function of reducing tumor cell proliferation under hypoxic conditions. From Figure 6 As shown in C, in MDA-MB-231 cells, the cell survival of the Sul-acid group, cRGD group, and Sul-acid + cRGD group decreased slightly compared with the control group. Under normoxic conditions, Sul-L1-RGD treatment reduced the survival of MDA-MB-231 cells, with a higher reduction amplitude, comparable to the treatment group with the carbonic anhydrase inhibitor SLC-0111, indicating that Sul-L1-RGD has the function of reducing tumor cell proliferation under normoxic conditions.

[0095] The above results explored that the degradation of CAIX by Sul-L1-RGD is mediated by integrin αVβ3, and the specific recognition and selective targeted degradation of the αVβ3 receptor were verified. Finally, through the CCK8 experiment, it was verified that Sul-L1-RGD has an inhibitory effect on the survival of hypoxic tumor cells. In summary, Sul-L1-RGD can effectively degrade the highly expressed CAIX and integrin αvβ3 receptor on the surface of tumor cells, thereby improving the hypoxic and acidic microenvironment unique to solid tumors, and thus playing a certain role in cancer treatment: Therefore, using Sul-L1-RGD can effectively induce the degradation of CAIX under hypoxic conditions and inhibit the survival of tumor cells.

[0096] Compared with traditional tumor treatment drugs, the CAIX-targeted degrader in this protocol has many unique advantages. First of all, the targeted degrader can degrade the entire pathogenic protein in lysosomes, thus overcoming the drug resistance of tumors. Secondly, the integrin αvβ3 receptor specifically recognized by RGD is highly expressed only in tumor cells, and CAIX specifically bound by sul-acid is highly expressed only in highly invasive tumor cells rather than normal tissues. Therefore, the CAIX-targeted degrader has high selectivity for tumor cells and less impact on normal cells. In addition, the dosage of the CAIX-targeted degrader is small, so it avoids the off-target effect caused by high doses and has low toxicity and high safety. The CAIX-targeted degrader can effectively induce the degradation of CAIX under hypoxic conditions, inhibit the survival of tumor cells, has the function of targeting tumor regions and degrading carbonic anhydrase IX using integrin and lysosomes, and then changes the hypoxic and microacidic environment of the internal environment of tumor patients, playing a role in inhibiting the survival of hypoxic tumor cells. The CAIX-targeted degrader of the present invention can be used as a therapeutic drug for solid tumors with hypoxic and acidic microenvironments and has great application potential in cancer treatment.

[0097] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A CAIX targeted degrader, characterized in that, It includes a CAIX target protein ligand, an integrin ligand and a linker; the CAIX target protein ligand and the integrin ligand are connected through the linker.

2. The CAIX-targeting degrader according to claim 1, wherein The CAIX target protein ligand has a sulfonamide group.

3. The CAIX-targeted degrader according to claim 2, wherein, The CAIX target protein ligand is 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid.

4. The CAIX targeting degrader according to claim 1, wherein The integrin ligand includes one or more of a polypeptide with an RGD sequence, a cyclic RGD sequence, a cyclopentapeptide sequence containing RGD, a cyclic peptide cilengitide sequence, an antibody containing an RGD sequence, and a protein containing an RGD sequence.

5. The CAIX-targeted degrader according to claim 4, wherein The integrin ligand is a cyclic RGD sequence.

6. The CAIX targeting degrader according to claim 1, characterized in that, The structure of the linker includes an alkyl chain containing one or more of an aromatic ring, a heterocyclic ring, a heteroatom and a functional group.

7. The CAIX-targeted degrader according to claim 6, wherein The structural formula of the linker is as follows: L1 L and L3 8. The CAIX-targeted degrader according to claim 7, wherein, The structural formula of the linker is as follows: L1 9. A preparation method of a CAIX targeting degrader, characterized in that, It includes the following steps: S1. Boc-6-aminohexanoic acid, DCC and N-hydroxysuccinimide are successively added into a reaction flask, stirred and dissolved with anhydrous N,N-dimethylformamide, reacted at room temperature, 3-azidopropylamine and DIEA are added into the solution, stirred at room temperature, separated and purified, and a white powder Boc-L1-Azide is obtained after drying. S2. The white powder Boc-L1-Azide is dissolved in TFA / DCM under ice bath conditions, stirred and reacted at room temperature, and the solvent is evaporated under reduced pressure to obtain a crude amine product with the Boc protecting group removed. S3. The crude amine product, 4-[[5-(aminosulfonyl)-1,3,4-thiadiazol]amino]-4-oxobutyric acid and DIEA are dissolved in anhydrous DMF, HATU is added, the reaction solution is stirred at room temperature, separated and purified, and Sul-L1-Azide is obtained after drying. S4. Sul-L1-Azide, Alkyne-cRGD, CuSO4·5H2O and NaVc are dissolved in DMF / H2O, reacted, separated and purified to obtain a CAIX-targeted degrader.

10. Use of the CAIX-targeted degrader according to any one of claims 1-8 in the preparation of a tumor vaccine or an anti-tumor drug.

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