Targeted STING protein degradation agent and application thereof

Abstract

The invention provides a compound, the general formula of which is A-L-B. In the general formula, A is a chemical group which specifically binds or activates STING (stimulating interferon gene) protein; b is a chemical group specifically combined with LC3 (microtubule-associated protein-1 light chain 3) protein; l is a chemical linker. The compound can effectively mediate the degradation of STING protein and is used for treating inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of molecular drug technology, and in particular to a degrader targeting the STING protein and its application. Background Technology

[0002] The cGAS-STING pathway, as a cytoplasmic DNA sensing system, activates a signaling cascade of type I interferon (IFN-I) and various pro-inflammatory factors by recognizing exogenous pathogen DNA or abnormal host DNA (such as mitochondrial DNA leakage due to mitochondrial damage or DNA generated by genomic instability). The core mechanism of this pathway involves cyclic guanosine monophosphate-AMP synthase (cGAS) binding to cytoplasmic DNA and catalyzing the synthesis of the second messenger cGAMP, which in turn activates the STING protein localized in the endoplasmic reticulum. Through a TBK1 kinase-mediated phosphorylation cascade, this ultimately induces the transcriptional activation of interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB). Studies have shown that precise spatiotemporal control of STING signaling is crucial for maintaining immune homeostasis, and its overactivation has been confirmed to be closely related to the pathogenesis of various autoimmune diseases and chronic inflammatory diseases. This has led to small-molecule intervention strategies targeting STING becoming a research hotspot.

[0003] Currently, STING inhibitors can be divided into two categories based on their mechanism of action: one is competitive antagonists, which block the binding of the endogenous agonist cGAMP by occupying the cyclic dinucleotide (CDN) binding site; the other targets the conserved cysteine ​​residues (Cys88 / Cys91) in the transmembrane region of STING, inhibiting their palmitoylation modification and thus blocking STING activation. However, these traditional "site-driven" inhibitors require maintaining high concentrations to continuously occupy the target site, which may lead to adaptive resistance and signal reactivation, and they cannot target the inactive conformation of STING protein.

[0004] In recent years, autophagy-targeted chimeric (ATTEC) technology, as a novel "event-driven" strategy, bridges target proteins (such as STING) with the key autophagy protein LC3 using bifunctional molecules, forming a ternary complex that induces the target to be encapsulated by autophagosomes and ultimately degraded via the lysosomal pathway. Compared with traditional inhibitors, ATTEC blocks signal transduction at its source by clearing target proteins rather than inhibiting their activity. It has advantages such as low-dose, long-lasting effects, overcoming drug resistance, and targeting undrug-forming proteins, providing a breakthrough direction for the treatment of inflammatory diseases. Summary of the Invention

[0005] This invention relates to STING-ATTEC, a molecular drug targeting the autophagy pathway to degrade STING protein, and its application in the treatment of inflammation-related diseases. The STING-ATTEC molecule employs a dual-ligand structure: one end is a small molecule STING protein inhibitor, C170, which specifically binds to overactivated STING protein; the other end is a small molecule ligand, GW5074, for LC3 protein, which binds to LC3 protein on the autophagosome membrane; the two are linked by a triazole-linked alkyl chain, forming a linker for spatial synergistic effects. This molecular design mediates the recruitment and close co-localization of STING protein and LC3, thereby inducing selective encapsulation and degradation of STING protein by autophagosomes, thus reducing intracellular STING protein levels and inhibiting downstream inflammatory signaling pathways. This molecule exhibits specific degradation of highly expressed STING protein under inflammatory conditions, demonstrating good targeting and potential therapeutic value.

[0006] In one aspect, the present invention provides a compound having the general formula ALB, wherein:

[0007] A is a chemical group that specifically binds to or activates the STING (stimulating interferon gene) protein;

[0008] B is a chemical group that specifically binds to LC3 (microtubule-associated protein 1 light chain 3) protein;

[0009] L is a chemical linker selected from the following groups or combinations thereof: straight-chain or branched alkyl groups, preferably containing 2-20 carbon atoms; polyethylene glycol, preferably containing 1-10 ethylene glycol units; 1,2,3-triazole ring or its derivatives, preferably formed by an azide-yne ​​cycloaddition reaction; amide group, ether group, ester group; aromatic ring or heterocyclic ring, preferably benzene ring, pyridine ring or piperazine ring; biodegradable group, preferably disulfide bond or hydrazine bond.

[0010] In one embodiment of the present invention, the linker comprises a 1,2,3-triazole ring, preferably a 1,4-substituted triazole or a 1,5-substituted triazole. Further, the 1,2,3-triazole ring may be bonded to an alkyl chain, polyethylene glycol, or an amide group.

[0011] In one embodiment of the present invention, the connector includes:

[0012] R1 is selected from C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, or polyethylene glycol chains containing 1-5 ethylene glycol units, and preferably further includes substituted functional groups containing amine, hydroxyl, or carboxyl groups; preferably, it has a length of 2-15 atoms and is used to connect the STING protein binding group.

[0013] R2 is selected from C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, or hybrid linkers, wherein the hybrid linker comprises a combination of alkyl, amide, or ether groups, preferably with a length of 2-15 atoms, and is used to link the LC3 protein binding group.

[0014] In one embodiment of the present invention, R1 is selected from one of the following structures:

[0015]

[0016] In one embodiment of the present invention, R2 is selected from one of the following structures:

[0017]

[0018] In one embodiment of the present invention, the connector is selected from one of the following structures:

[0019]

[0020] In one embodiment of the present invention, the A group is selected from the following structures:

[0021]

[0022] Among them, R3, R4, and R5 are each selected from hydrogen atoms (-H), C1-C6 straight-chain or branched alkyl groups (such as methyl-CH3, ethyl-C2H5) or halogen atoms (such as chlorine-Cl);

[0023] R6 is selected from hydrogen atom (-H), C1-C6 straight-chain or branched alkyl (such as methyl-CH3, ethyl-C2H5), halogen atom (such as chlorine-Cl) or hydroxyl (-OH);

[0024] R7 is selected from an oxygen atom or a hydroxyl group (-OH).

[0025] In one embodiment of the present invention, the A group is selected from the following structures:

[0026]

[0027] In one embodiment of the present invention, the B group is selected from the following structures:

[0028]

[0029] Wherein, R8 is a C1-C6 straight-chain or branched alkyl group (such as methyl-CH3, ethyl-C2H5), and preferably the B group is selected from the following structures:

[0030]

[0031] In one embodiment of the present invention, the compound is selected from the following structures:

[0032]

[0033] In a second aspect, the present invention provides a method for preparing the above-mentioned compound, comprising: generating the compound by reacting compound 17 and compound 5 via a click chemistry reaction; the structures of compound 17 and compound 5 are as follows:

[0034]

[0035] Preferably, the reaction conditions for compounds 17 and 5 are as follows: using sodium ascorbate as a reducing agent, a CuAAC reaction occurs under the action of a copper catalyst. The solvent is DMF, the temperature is room temperature, and the reaction time is 10-12 h. Preferably, the catalyst is CuSO4.

[0036] In one embodiment of the present invention, the method for synthesizing compound 17 is as follows:

[0037]

[0038] In one embodiment of the present invention, compounds 13 and 14 undergo a dehydration reaction under the action of the catalyst piperidine to generate compound 15. Preferably, methanol is used as the solvent, and the reaction conditions are reflux at 95°C for 11-12 hours.

[0039] In one embodiment of the present invention, compounds 15 and 18 undergo a nucleophilic substitution reaction under the action of a strong base, NaH, to generate compound 16. Preferably, the solvent used is DMF, and the reaction conditions are: NaH is added at 0°C, stirring at room temperature for 30 min, and then stirring for 3-4 h after adding compound 18.

[0040] In one embodiment of the present invention, compound 16 undergoes a substitution reaction with azidetrimethylsilane under the action of catalyst KF to generate compound 17.

[0041] In one embodiment of the present invention, the preparation method of compound 5 is as follows:

[0042]

[0043] In one embodiment of the present invention, compound 1 undergoes an amino protection reaction under the action of a protecting agent to generate compound 2, wherein the protecting agent is (Boc)₂O (tert-Butoxycarbonyl anhydride). The reaction conditions are: temperature 0°C, reaction time 10-12 h.

[0044] In one embodiment of the present invention, compound 3 reacts with compound 2 under the action of an activator and a coupling agent to generate compound 4. Optionally, the activator is N-hydroxysuccinimide (NHS), and the coupling agent is N,N'-dicyclohexylcarbodiimide (DCC). The reaction conditions are: room temperature, and the reaction time is 10-12 h.

[0045] In one embodiment of the present invention, compound 4, after being deprotected by a deprotecting agent, is activated by a coupling agent and a base to form an active ester, and a terminal alkyne group is introduced to generate compound 5. The reaction conditions are: room temperature, time 12-14 h.

[0046] In one embodiment of the present invention, the deprotecting agent is trifluoroacetic acid (TFA), the coupling agent is EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), the additive is HOBt (1-hydroxybenzotriazole), and the base is DIPEA (N,N-diisopropylethylamine).

[0047] In a third aspect, the present invention provides the use of the above-mentioned compound in the preparation of a medicament for treating inflammatory diseases.

[0048] In one embodiment of the present invention, the compound can effectively mediate the degradation of STING protein.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The STING-ATTEC molecular drug provided by this invention constructs a dual-ligand small molecule structure with targeted autophagy degradation function by linking the STING small molecule inhibitor C170 with the autophagosome protein LC3 ligand GW5074. Compared with the prior art that only uses STING inhibitors or LC3 ligands, this invention has the following significant technical advantages and practical effects:

[0051] Traditional small-molecule inhibitors of STING (such as C170) can temporarily block STING activity, but they are unlikely to significantly reduce its protein expression level, especially under persistent inflammatory conditions, where STING protein may still be highly expressed, maintaining the inflammatory response. Furthermore, the LC3 ligand GW5074 itself does not possess the ability to directly regulate STING expression. Therefore, neither of these inhibitors, when used alone, has a significant effect on reducing STING protein levels.

[0052] The molecular design of this invention employs a triazole-linked alkyl chain as the linker structure, ensuring sufficient spatial flexibility between the two ligands while also exhibiting good stability and biocompatibility. This contributes to improved conformational adaptability and functional synergy efficiency within cells. The triazole structure can be rapidly constructed via click chemistry, with simple synthetic steps, facilitating modification and industrial-scale development.

[0053] In summary, this invention, by constructing a pure small molecule-mediated dual-ligand structure STING-ATTEC, not only innovatively achieves targeted degradation of the STING protein, but also verifies its specific role in inflammatory states through cell experiments. This overcomes the limitation of existing single-target drugs being unable to achieve protein degradation, and provides a novel small molecule drug strategy for the treatment of STING-related chronic inflammation and autoimmune diseases. Attached Figure Description

[0054] Figure 1 These are the results of a Western blot cell experiment. Detailed Implementation

[0055] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0056] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0057] Example 1: Synthesis of the compound

[0058] 1. Synthesis of Compound 5

[0059]

[0060] (1) Synthesis of compound 2

[0061] Compound 1 (1.0 g, 9.3 mmol, 4.0 equiv.) was dissolved in 30 mL of DCM in a 150 mL reaction flask. Then, (Boc)₂O (0.5 g, 2.3 mmol, 1.0 equiv.) dissolved in 30 mL of DCM was slowly added dropwise using a constant pressure funnel at 0 °C. The reaction was carried out at 0 °C for 12 h, during which the reaction solution gradually changed from clear and transparent to milky white. After the reaction was complete, the pH of the solution was adjusted to approximately 2 with 2N HCl. The solution was extracted with water and DCM, and the aqueous phase was retained. The pH was then adjusted to approximately 11 with saturated NaOH, and the solution was extracted again with water and DCM. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to directly obtain a brown solid, compound 2 (1.3 g, 70%).

[0062] (2) Synthesis of compound 4

[0063] Compound 3 (1.0 g, 6.4 mmol, 1.0 equiv.) was dissolved in 20 mL of DCM and 3 mL of LDM in a 100 mL reaction flask. NHS (0.9 g, 7.7 mmol, 1.2 equiv.) and DCC (1.6 g, 7.7 mmol, 1.2 equiv.) were added at room temperature, and the mixture was stirred and activated for 1 h. The filtrate was filtered and retained. Compound 2 (1.6 g, 7.7 mmol, 1.2 equiv.) and TEA (0.8 g, 7.7 mmol, 1.2 equiv.) were added to the filtrate, and the reaction was carried out at room temperature for 12 h. The reaction solution was a turbid, deep yellow color. After the reaction was complete, the DCM was first evaporated to dryness, and then extracted with water and EA. 10 mL of 2N HCl was added to remove excess compound 2. The organic phase was dried over anhydrous sodium sulfate and filtered. After evaporating the solvent, the mixture was repeatedly concentrated with DCM and filtered. The residue was dried to obtain a yellow solid compound 4 (1.7 g, 75%).

[0064] (3) Synthesis of compound 5

[0065] Compound 4 (100.0 mg, 0.3 mmol, 1.2 equiv.) was dissolved in 8 mL of DCM in a 50 mL reaction flask, and 2 mL of TFA was added. The reaction was carried out at room temperature for 20 min, during which the reaction solution changed from dark yellow to light yellow. After the reaction was completed, the solvent was directly evaporated. 4-Pentynic acid (23.5 mg, 0.3 mmol, 1.0 equiv.) was dissolved in 10 mL of DMF in a 50 mL reaction flask, and EDCI (55.0 mg, 0.3 mmol, 1.2 equiv.), HOBt (38.9 mg, 0.3 mmol, 1.2 equiv.), and DIPEA (68.2 mg, 0.6 mmol, 2.2 equiv.) were added. The reaction was activated at room temperature for 1 h. Then, the compound 4 after the Boc protecting group was removed was dissolved in DMF and added to the reaction flask. The reaction was carried out at room temperature for 12 h, during which the reaction solution was dark yellow. After the reaction was completed, the mixture was extracted with water and EA. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness to give a yellowish-brown solid compound 5 (71.5 g, 91%).

[0066] 2. Synthesis of Compound II-2

[0067]

[0068] The specific steps are as follows:

[0069] Synthesis of Compound 15: Compound 13 (1.0 g, 3.5 mmol, 1.0 equiv) was dissolved in 20 mL of EtOH in a 150 mL reaction flask. Compound 14 (1.5 g, 7.0 mmol, 2.0 equiv) was added, along with piperidine (0.45 g, 5.3 mmol, 1.5 equiv). The mixture was refluxed at 95 °C for 12 h. After the reaction was complete, a large amount of ice water was added, resulting in the precipitation of a large amount of yellow solid. The crude product was obtained by filtration. The crude product was dissolved in a small amount of THF, heated to 75 °C, and THF was gradually added until completely dissolved. After cooling to room temperature, PE was slowly added, precipitating the solid. The precipitate was filtered, dried, and compound 15 was obtained, which was then directly proceeded to the next step.

[0070] Synthesis of Compound 16: Compound 15 (500 mg, 1.1 mmol, 1 equiv) was dissolved in DMF in a 150 mL reaction flask and stirred at 0 °C. NaH (132 mg, 5.5 mmol, 5 equiv) was slowly added simultaneously. After addition, the mixture was moved to room temperature and stirred for 30 min. Then, Compound 18 (536.6 mg, 2.2 mmol, 2 equiv) was added, and the mixture was stirred for 4 h. After the reaction was complete, the mixture was extracted with water and EA. The organic phase was dried over anhydrous sodium sulfate, filtered, and subjected to column chromatography (PE:EA = 5:1) to obtain a brown liquid, compound 16 (567 mg, 89%).

[0071] Synthesis of Compound 17: KF (34.8 mg, 0.6 mmol, 2.0 equiv.) and azidotrimethylsilane (69 mg, 0.6 mmol, 2.0 equiv.) were dissolved in 10 mL DMF in a 50 mL reaction flask and reacted at room temperature for 1 h. Compound 16 (180 mg, 0.3 mmol, 1.0 equiv.) was added to the reaction flask and reacted at 70 °C for 12 h. The reaction solution was colorless and clear. After the reaction was completed, the mixture was extracted with water and EA. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness before being directly added to the next step.

[0072] Synthesis of Compound II-2: Compound 5 (73.0 mg, 0.2 mmol, 1.0 equiv.) was dissolved in 10 mL of DMF in a 50 mL reaction flask. An anaerobic environment was maintained under nitrogen protection. CuSO4 (8.0 mg, 0.1 mmol, 0.2 equiv.) and sodium ascorbate (261.0 mg, 1.32 mmol, 6.0 equiv.) were dissolved in 1.0 mL of water and added to the reaction flask sequentially and rapidly. The reaction was carried out at room temperature for 1 h. Compound 17 (133.1 mg, 0.2 mmol, 1.0 equiv.) was dissolved in 5 mL of DMF and added to the reaction flask. The reaction was carried out at room temperature for 12 h. After the reaction was completed, the mixture was extracted with water and EA. The organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness before column chromatography (DCM:MeOH = 50:1) to give a yellow solid, compound II-2 (129.5 mg, 63%).

[0073] Test case

[0074] An in vitro inflammation model was induced by stimulating primary macrophages (BMDM) with 100 ng / ml LPS for 6 hours. Western blot cell experiments were then conducted. Figure 1 As can be seen, the expression level of STING protein in cells treated with LPS was significantly upregulated, and inflammation was significantly relieved, indicating a significant inflammatory response. After treatment with STING-ATTEC, the STING protein level was significantly downregulated, indicating that this molecule can effectively mediate the degradation of STING protein. In the control group, treatment with C170 or GW5074 alone did not show a significant decrease in STING protein expression. This result clearly demonstrates that STING inhibition or LC3 recruitment alone is insufficient to induce protein degradation; only by linking the two through a suitable linker to form the STING-ATTEC complex can the target protein effectively approach the autophagy system, thereby triggering its selective degradation.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A compound having the general formula ALB, wherein: A is a chemical group that specifically binds to or activates the STING (stimulating interferon gene) protein; B is a chemical group that specifically binds to LC3 (microtubule-associated protein 1 light chain 3) protein; L is a chemical linker selected from the following groups or combinations thereof: straight-chain or branched alkyl groups, preferably containing 2-20 carbon atoms; polyethylene glycol, preferably containing 1-10 ethylene glycol units; 1,2,3-triazole ring or its derivatives, preferably formed by an azide-yne ​​cycloaddition reaction; amide group, ether group, ester group; aromatic ring or heterocyclic ring, preferably benzene ring, pyridine ring or piperazine ring; biodegradable group, preferably disulfide bond or hydrazine bond.

2. The compound according to claim 1, characterized in that: The linker comprises a 1,2,3-triazole ring, preferably a 1,4-substituted triazole or a 1,5-substituted triazole; further, the 1,2,3-triazole ring may be combined with an alkyl chain, polyethylene glycol or an amide group.

3. A compound according to claim 2, characterized in that: The connector includes: R1 is selected from C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, or polyethylene glycol chains containing 1-5 ethylene glycol units, and preferably further includes substituted functional groups containing amine, hydroxyl, or carboxyl groups; preferably, it has a length of 2-15 atoms and is used to connect the STING protein binding group. R2 is selected from C1-C10 alkyl, C6-C10 aryl, C5-C10 heteroaryl, or hybrid linkers, wherein the hybrid linker comprises a combination of alkyl, amide, or ether groups, preferably with a length of 2-15 atoms, and is used to link the LC3 protein binding group.

4. A compound according to claim 3, characterized in that: R1 is selected from one of the following structures: Preferably, R2 is selected from one of the following structures: Preferably, the connector is selected from one of the following structures:

5. A compound according to claim 1, characterized in that: The A group is selected from the following structures: Among them, R3, R4, and R5 are each selected from hydrogen atoms (-H), C1-C6 straight-chain or branched alkyl groups (such as methyl-CH3, ethyl-C2H5) or halogen atoms (such as chlorine-Cl); R6 is selected from hydrogen atom (-H), C1-C6 straight-chain or branched alkyl (such as methyl-CH3, ethyl-C2H5), halogen atom (such as chlorine-Cl) or hydroxyl (-OH); R7 is selected from an oxygen atom or a hydroxyl group (-OH); Preferably, the A group is selected from the following structures:

6. A compound according to claim 1, characterized in that: The B group is selected from the following structures: Wherein, R8 is a C1-C6 straight-chain or branched alkyl group (such as methyl-CH3, ethyl-C2H5), and preferably the B group is selected from the following structures: Preferably, the compound is selected from the following structures:

7. A method for preparing the compound according to any one of claims 1-6, characterized in that: The process includes the following steps: reacting compound A and compound B via a click chemistry reaction to generate the compound; the structures of compound 17 and compound 5 are as follows: Preferably, the reaction conditions for compounds 17 and 5 are as follows: using sodium ascorbate as a reducing agent, a CuAAC reaction occurs under the action of a copper catalyst; the solvent is DMF, the temperature is room temperature, and the reaction time is 10-12 h; preferably, the catalyst is CuSO4.

8. The preparation method according to claim 7, characterized in that: The method for synthesizing compound 17 is as follows: Compounds 13 and 14 undergo a dehydration reaction under the action of the catalyst piperidine to generate compound 15; preferably, the solvent used is methanol, and the reaction conditions are reflux at 95°C for 11-12 hours. Compounds 15 and 18 undergo a nucleophilic substitution reaction under the action of a strong base NaH to generate compound 16. Preferably, the solvent used is DMF, and the reaction conditions are: NaH is added at 0°C, stirring at room temperature for 30 min, and stirring for 3-4 h after adding compound 18. Compound 16 undergoes a substitution reaction with azidotrimethylsilane in the presence of catalyst KF to generate compound 17.

9. The preparation method according to claim 7, characterized in that: The method for synthesizing compound 5 is as follows: Compound 1 undergoes an amino protection reaction under the action of a protecting agent to generate compound 2. The protecting agent is (Boc)2O (tert-Butoxycarbonyl anhydride). The reaction conditions are: temperature 0℃, reaction time 10h-12h. Optionally, compound 3 reacts with compound 2 under the action of an activator and a coupling agent to generate compound 4; optionally, the activator is N-hydroxysuccinimide (NHS) and the coupling agent is N,N'-dicyclohexylcarbodiimide (DCC); the reaction conditions are: room temperature, and the reaction time is 10h-12h. Compound 4, after being deprotected by a deprotecting agent, is activated by a coupling agent and a base to form an active ester and introduces a terminal alkyne group to generate compound 5. The reaction conditions are: room temperature, time 12-14 h. The deprotecting agent may be trifluoroacetic acid (TFA), the coupling agent may be EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), the additive may be HOBt (1-hydroxybenzotriazole), and the base may be DIPEA (N,N-diisopropylethylamine).

10. The use of the compound according to any one of claims 1-6 in the preparation of a medicament for treating inflammatory diseases; Preferably, the compound can effectively mediate the degradation of STING protein.

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