Application of pentafluorobenzene compound in preparation of ZBP1 inhibitor

Abstract

The invention provides application of pentafluorobenzene compounds in preparation of ZBP1 inhibitors, and belongs to the field of medicinal chemistry and biological medicine. The pentafluorobenzene compound disclosed by the invention belongs to a ZBP1 small-molecule inhibitor, can inhibit the release of inflammatory factors by inhibiting the activity of ZBP1, and has an application prospect in treating myocardial infarction diseases. Formula I

Description

Technical Field

[0001] This invention belongs to the fields of medicinal chemistry and biomedicine, specifically relating to pentafluorobenzene compounds with ZBP1 inhibitory activity and their applications. Background Technology

[0002] Myocardial infarction, one of the leading causes of cardiovascular death worldwide, hinges on the rapid restoration of myocardial blood flow. While reperfusion therapy significantly reduces acute-phase mortality, it inevitably triggers myocardial ischemia / reperfusion injury (MI / R)—a paradoxical phenomenon that has become a key bottleneck restricting the long-term prognosis of myocardial infarction patients. The oxidative stress burst, uncontrolled inflammatory response, and mitochondrial dysfunction triggered instantaneously by reperfusion collectively lead to secondary cardiomyocyte death, plunging patients into a clinical dilemma of "open blood vessels, but unstoppable cardiac damage." Currently, clinical interventions for MI / R are extremely limited. Although some anti-inflammatory and antioxidant drugs (such as allopurinol and recombinant human superoxide dismutase) have shown some early cardioprotective effects in trials, large-scale clinical studies have confirmed that they do not significantly benefit the reduction of major adverse cardiac events (such as heart failure, re-infarction, and death) or the prolongation of survival in patients.

[0003] In recent years, ZBP1 (Z-DNA binding protein 1), an important molecule in the field of innate immunity and inflammation regulation, has come into the research spotlight. ZBP1 was initially found to be highly expressed by interferon-γ and serves as a key bridge connecting pathogen recognition and inflammatory response. Its core mechanism lies in the fact that when cells are stressed or damaged, abnormally accumulated Z-type nucleic acids (Z-DNA / Z-RNA) can specifically activate ZBP1, thereby strongly initiating the NF-κB signaling pathway and driving the transcription and release of a large number of inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α, forming a "cytokine storm." In the context of myocardial infarction / reperfusion (MI / R), ischemia, hypoxia, and reperfusion themselves can lead to abnormalities in the structure of nuclear and mitochondrial nucleic acids, producing endogenous Z-type nucleic acids, thus abnormally activating the ZBP1 pathway. This is likely a significant initiating factor for uncontrolled inflammatory responses after reperfusion. Therefore, specifically inhibiting ZBP1 holds promise for curbing the inflammatory cascade at its source and providing deep protection for the myocardium. Compared to broad-spectrum anti-inflammatory strategies, targeting ZBP1 may be more precise and safer.

[0004] However, to date, no ZBP1 small molecule inhibitors have been reported to have entered clinical trials globally, and this target remains a "blue ocean" of potential. Developing the first ZBP1 inhibitor not only has significant scientific value but also has the potential to revolutionize clinical treatment paradigms: it aims not only to reduce immediate myocardial damage after reperfusion but also to improve long-term cardiac remodeling and survival outcomes in patients with myocardial infarction, addressing a key challenge in current clinical practice. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the application of pentafluorobenzene compounds in the preparation of ZBP1 inhibitors.

[0006] The specific technical solution of the present invention is as follows: This invention provides the use of pentafluorobenzene compounds of Formula I, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or solvates thereof, or their crystal forms, in the preparation of ZBP1 inhibitors: Formula I in, L is selected from either none or S; R1 is selected from phenyl or hydrogen; R2 is independently selected from hydrogen, C 1~3 Alkoxy, -NHC(O)R3; R3 is a phenyl 6-8 membered nitrogen heterocycle; n is selected from 1, 2, or 3; Alternatively, R1 and R2 together with the phenyl group they are attached to form a fused tricyclic ring, wherein the fused tricyclic ring is 9H-xanthonyl ( ).

[0007] Furthermore, the pentafluorobenzene compounds are selected from compounds having the following structures: , , , , .

[0008] Furthermore, the ZBP1 inhibitor is a drug for the prevention and / or treatment of diseases associated with abnormal ZBP1 activity.

[0009] Furthermore, the disease associated with abnormal ZBP1 activity is myocardial infarction.

[0010] Furthermore, the disease associated with abnormal ZBP1 activity is myocardial ischemia-reperfusion injury.

[0011] Furthermore, the intended use is to protect cardiomyocytes from hypoxia / reoxygenation damage.

[0012] Furthermore, the drug is a pharmaceutical preparation made by using the above-mentioned pentafluorobenzene compounds, or their pharmaceutically acceptable salts, or their stereoisomers, or their solvates, or their crystal forms as active ingredients, plus a pharmaceutically acceptable carrier.

[0013] Furthermore, the pharmaceutical preparation may be in the form of tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

[0014] Preferably, the pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following: Hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, citric acid, or ferulic acid.

[0015] "Pharmaceutically acceptable salts" refer to salts of compounds prepared by reacting a compound with a relatively non-toxic acid or base, containing specific substituents. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, etc.); and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid. Acids such as citric acid, tartaric acid, and methanesulfonic acid; organic acid salts also include salts of organic acids such as amino acids (e.g., arginine) and glucuronic acid. Certain compounds contain both basic and acidic functional groups, thus allowing them to be converted into either a base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubilities in polar solvents.

[0016] Pharmaceutically acceptable salts can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of both. Non-aqueous media such as ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile are generally preferred.

[0017] "Pharmaceutically acceptable carriers" are excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0018] The pharmaceutical products described in this invention can be prepared using any method known to those skilled in the art, based on the disclosed information. Examples include conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0019] The medicaments described in this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The medicaments of this invention can also be controlled-release or sustained-release formulations (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the aforementioned drug include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0020] Compared with the prior art, the present invention has the following significant advantages: This invention, based on the ZBP1 target, efficiently identified and validated a class of pentafluorobenzene compounds as small-molecule inhibitors of ZBP1 through a strategy of "AlphaFold structure prediction—multi-level virtual screening—cell model validation—BLI binding affinity assay". In vitro experiments confirmed that the selected compounds exhibited significant protective activity against a cardiomyocyte hypoxia / reoxygenation injury model. Among them, compound I-3 not only showed the best cellular activity but also, as verified by BLI technology, can directly bind to the ZBP1 protein (K). D =22.231 μM). The pentafluorobenzene compounds of the present invention have significant ZBP1 inhibitory activity and can effectively inhibit inflammatory factors, showing promise for the treatment of myocardial ischemia-reperfusion injury.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 The results of the CCK8 assay were used to detect cell viability.

[0024] Figure 2 Results of lactate dehydrogenase (LDH) assay to examine cytotoxicity.

[0025] Figure 3 The results of the biological membrane layer reflected light interferometry (BLI) experiment were used to examine the binding of compound I-3 to the ZBP1 protein. Detailed Implementation

[0026] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0027] Example 1: Virtual screening of ZBP1 inhibitors This invention utilizes computer-aided drug design methods to virtually screen and evaluate the binding affinity of candidate compounds. The results show that compounds (I-1 to I-5) have high affinity potential with the ZBP1 protein. Basic information and molecular docking scores for each compound are shown in Table 1.

[0028] Table 1. Basic information and molecular docking scores of the compounds Example 2: In vitro cell activity evaluation of the compounds of the present invention 1. Experimental Methods A hypoxia / reoxygenation (H / R) injury model was established using mouse HL-1 cardiomyocytes to evaluate the potential protective effects of compounds I-1–I-5 against ZBP1-mediated H / R cardiomyocyte injury. The specific procedures were as follows: HL-1 cells were cultured in glucose-free medium under 1% O2 conditions (hypoxia) for 4 hours, with compounds numbered I-1, I-2, I-3, I-4, and I-5 (final concentration 10 μM) added to the medium. Subsequently, the medium was replaced with high-glucose medium and cultured for another 6 hours under 21% O2 conditions (reoxygenation), maintaining the same compound concentrations throughout. After H / R treatment, cell viability was assessed using the CCK-8 assay, and cytotoxicity was evaluated using a lactate dehydrogenase (LDH) release assay.

[0029] 2. Experimental Results The results of the CCK-8 experiment showed that compounds I-3 and I-4 could significantly inhibit the decrease in cardiomyocyte survival rate caused by H / R ( Figure 1 The LDH release assay further confirmed that compounds I-3 and I-4 had a significant protective effect against H / R-induced cytotoxicity. Figure 2 Based on the combined results of the two tests, compound I-3 exhibited the best in vitro cell protective activity.

[0030] Example 3: Evaluation of the in vitro cell activity of the compounds of the present invention 1. Experimental Methods The binding affinity of compound I-3 to ZBP1 protein was determined using bio-layer interferometry (BLI) via a ForteBioOctet RED96 system. Purified GST-tagged protein and GST-ZBP1 fusion protein were diluted to 10 μg / mL with PBS buffer (pH 7.4). Compound I-3 was serially diluted with PBST containing 5% DMSO (PBS + 0.02% Tween-20) at five concentration gradients (1.25 μM to 40 μM). GST and GST-ZBP1 proteins were immobilized using a GST probe, and then binding and dissociation experiments were performed sequentially with different concentrations of the compound solution and the immobilized biosensor. Experimental data were analyzed using ForteBio Data Analysis 10.0 software, with background signals from the GST control and buffer subtraction subtracted, and the binding curves were aligned and baselined (55–59.8 s). A 1:1 combined model was used for curve fitting, and the combined dynamic parameters were calculated.

[0031] 2. Experimental Results BLI experimental results confirmed that compound I-3 can specifically bind to ZBP1 protein. Figure 3 Through fitting analysis, the equilibrium dissociation constant (K) of the two was measured. D The value was 22.231 μM.

[0032] In summary, this invention successfully identified several potential small molecule inhibitors of ZBP1 from a commercial compound library using a virtual screening strategy based on AlphaFold protein structure prediction. Through multi-level molecular docking and induced fit docking optimization, five candidate compounds with diverse structures and excellent docking scores were finally selected. Further in vitro cell experiments showed that compounds I-3 and I-4 exhibited significant protective activity in a cardiomyocyte hypoxia / reoxygenation injury model, with compound I-3 showing particularly outstanding activity. Biomembrane interferometry directly verified the specific binding of compound I-3 to the ZBP1 protein, with a binding affinity K0. D The value was 22.231 μM. These results confirm the reliability of the virtual screening process used in this invention and provide a promising lead compound, I-3, for the development of novel cardioprotective drugs targeting ZBP1.

Claims

1. Use of the pentafluorobenzene compound of Formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a crystal form thereof, in the preparation of ZBP1 inhibitors: Formula I in, L is selected from either none or S; R1 is selected from phenyl or hydrogen; R2 is independently selected from hydrogen, C 1~3 Alkoxy, -NHC(O)R3; R3 is a phenyl 6-8 membered nitrogen heterocycle; n is selected from 1, 2, or 3; Alternatively, R1 and R2 together with the phenyl group to which they are attached form a fused tricyclic ring, wherein the fused tricyclic ring is 9H-xanthonyl.

2. The use according to claim 1, characterized in that, The pentafluorobenzene compounds are selected from compounds having the following structures: 、 、 、 、 。 3. The use according to claim 1, characterized in that, The ZBP1 inhibitor is a drug for the prevention and / or treatment of diseases associated with abnormal ZBP1 activity.

4. The use according to claim 3, characterized in that, The disease associated with abnormal ZBP1 activity is myocardial infarction.

5. The use according to claim 4, characterized in that, The disease associated with abnormal ZBP1 activity is myocardial ischemia-reperfusion injury.

6. The use according to claim 5, characterized in that, The purpose is to protect myocardial cells from hypoxia / reoxygenation damage.

7. The use according to claim 3, characterized in that, The drug is a pharmaceutical preparation made by using a pentafluorobenzene compound as described in any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a crystal form thereof as the active ingredient, plus a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that, The pharmaceutical preparations are in the form of tablets, capsules, powders, pills, granules, injections, oral liquids, syrups, inhalers, ointments, patches, or suppositories.

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