7-methoxy-5, 6-dimethyl acridone compound and medical application of 7-methoxy-5, 6-dimethyl acridone compound as STING synergistic agonist

Abstract

The invention belongs to the field of pharmaceutical chemicals, and relates to an acridone compound as shown in a formula I, a racemate or an optical isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof, and an application of the acridone compound as a novel STING synergistic agonist in preparation of STING-related antiviral or malignant tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and relates to a novel STING co-agonist drug and its use in preparing STING-related antiviral or malignant tumor drugs. Background Art

[0002] The human immune system is crucial in immunotherapy, serving as the body's first line of defense against invasion by foreign pathogens. Immune activation can combat pathogen infection and cancer. The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is a key mediator of the immune system, establishing a close link between innate and adaptive immunity. Pattern recognition receptors (PRRs) are fundamental components of the innate immune system, detecting tissue damage, cellular stress, and antigens. By detecting pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), they initiate downstream responses to type I interferons (IFNs) and proinflammatory cytokines. Microbial DNA and self-DNA from the nucleus or mitochondria are major PAMPs / DAMPs. As a PRRs, cGAS can recognize cytoplasmic double-stranded DNA and synthesize cyclic dinucleotides (CDNs) - 2', 3'-cGAMP, thereby activating the downstream STING protein and triggering a series of immune responses. At the same time, STING can also directly recognize CDNs synthesized by bacterial pathogens and be activated. Since the discovery of STING in 2008, many studies have shown that STING plays an important role in the elimination of various pathogens and diseases mediated by immunity. Activation of the cGAS-STING signaling pathway by infectious pathogens triggers the host immune response and promotes the transduction of inflammatory factors. Abnormal activation of STING can cause autoimmune and inflammatory diseases.

[0003] As a crucial component of the innate immune system, STING activation has the potential to stimulate and enhance both innate and adaptive immunity on a broader scale. This makes it suitable for the treatment of various types of cancer, and even non-cancerous diseases. Numerous clinical trials have evaluated the efficacy of STING agonists in immunotherapy, promoting optimization of dosage and route of administration, as well as innovative drug development with high target efficiency and minimal side effects.

[0004] Currently, most clinical research on STING agonists focuses on CDNs. However, the physical and chemical properties of CDNs are not ideal, and they typically require intratumoral administration. Various non-CDN small molecule STING agonists are currently under development. 1. Aminobenzimidazole compounds: In 2018, GlaxoSmithKline discovered this class of compounds, ABZI, through screening for small molecules that compete with 3H-cGAMP for STING binding. ABZI maintains a unique "open" STING conformation upon activation. Furthermore, its intravenous administration represents a breakthrough for CDN analogs, resulting in 80% tumor-free mice at the end of the experiment. Consequently, this class of compounds has become a hot topic for research and modification in recent years, aiming to improve their potency, selectivity, and pharmacokinetic and pharmacodynamic properties. 2. Benzothiophene compounds: Merck identified MSA-2, a compound with high membrane permeability and selective STING targeting, through cell-based phenotypic screening. MSA-2 can induce complete tumor regression in 80% to 100% of mice bearing colon cancer and induce long-term anti-tumor immunity, making it the first orally administered STING agonist. 3. 3-Imidazole-pyridazine compounds: In 2020, Chin et al. developed SR-717 through cell phenotypic screening and optimization. Subcutaneous injection of SR-717 in mice demonstrated anti-tumor activity (superior to anti-PD-1 or anti-PD-L1 therapy), promoted activation of CD8+ T cells, natural killer cells, and dendritic cells in relevant tissues, and promoted cross-antigen immunity. 4. Flavonoids: DMXAA and CMA were the earliest STING agonists, but clinical trials failed because they only activated murine STING. Compound 12b, modified by our research group, exhibits human activity, but its physicochemical properties are poor. 5. Benzothiazole compounds: These compounds bind to the N-terminal domain of the STING protein. G10, reported in 2020, can only activate human STING-R232 and STING-H232 proteins. C53, reported in 2022, showed stronger STING activation efficacy when co-administered with the endogenous ligand 2′,3′-cGAMP.

[0005] STING is widely distributed in cells throughout the body, and its overactivation can lead to severe autoinflammatory reactions and cytokine storms. Indiscriminately targeting and activating STING in different cells may lead to different therapeutic effects and even side effects. Although some new drug delivery technologies can enhance targeting to a certain extent, further clinical trial evaluation is still needed. Therefore, we need to solve the problem of mitigating the potential risks associated with these agonists while maintaining the therapeutic effect. The purpose of this invention is to discover a novel small molecule co-agonist of the STING pathway that can activate the STING pathway in conjunction with cGAMP at the site of lesions where trace amounts of cGAMP are produced, without causing any immune response in normal cells, thereby achieving the effect of targeted therapy, and can be used to prepare a new multifunctional drug for broad-spectrum antiviral or cancer immunotherapy. Summary of the Invention

[0006] Among the current mainstream STING agonists, 2′,3′-cGAMP, ABZI, MAS-02, SR-717, and flavonoids bind to the C-terminal domain of STING. ABZI forms a unique "open" conformation, while the other compounds induce STING to adopt a "closed" conformation. G10 and C53 bind to the N-terminal domain of STING. We investigated compounds that induce this "closed" conformation and found that the binding pockets of 2′,3′-cGAMP, MAS-02, and SR-717 overlap, while the binding pocket of flavonoids is located further down the C-terminus. Therefore, we divided the C-terminus into two binding pockets and investigated the combined effects of compounds in these pockets. We have found that compound 12b exhibits enhanced STING activation when combined with 2′,3′-cGAMP. Therefore, we plan to further optimize compound 12b to design a compound that does not activate or inhibit STING on its own, but exhibits a stronger agonist effect when combined with 2′,3′-cGAMP. Although STING protein is widely distributed throughout the body, it can only produce endogenous ligand 2′,3′-cGAMP at the lesion site. Our compound can enhance the effect of 2′,3′-cGAMP at the lesion site, thereby activating STING only at the lesion site, achieving targeted therapy and reducing treatment side effects.

[0007] The first aspect of the present invention relates to a compound represented by formula I, a racemate or optical isomer thereof, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0008]

[0009] in,

[0010] X is a C atom, an O atom, a S atom, a carbonyl group, a sulfinyl group or a sulfyl group;

[0011] Y is hydrogen, alkyl, haloalkyl, acetoxy or ester;

[0012] R1 is 1, 2 or 3 identical or different substituents optionally present on the phenyl ring, and R1 is selected from the following groups: hydrogen, halogen, hydroxy, alkyl, haloalkyl, alkoxy, amino, amino monosubstituted or disubstituted by alkyl, carboxyl, acetoxy, amide, phenyl.

[0013] In a preferred embodiment of the present invention, the compound of formula I, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate according to the present invention may be a compound represented by formula Ia.

[0014]

[0015] in,

[0016] n = 0 or 1;

[0017] R1 is one or two identical or different substituents optionally present on the benzene ring, R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-8 Alkyl, halogenated C 1-8 Alkyl, C 1-8 Alkoxy, amino, C 1-8 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-8 alkyl.

[0018] In a preferred embodiment of the present invention, the compound of formula I, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate according to the present invention may be of formula Ib,

[0019]

[0020] in,

[0021] R1 is one or two identical or different substituents optionally present on the benzene ring, R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-8 Alkyl, halogenated C 1-8 Alkyl, C 1-8 Alkoxy, amino, C 1-8 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-8 alkyl;

[0022] R2 is ethyl or hydrogen.

[0023] In a preferred embodiment of the present invention, the compounds represented by Formula I, Formula Ia, Formula Ib, or their racemates or optical isomers, or pharmaceutically acceptable salts, solvates, or hydrates, wherein R1 is one or two identical or different substituents optionally present on the benzene ring, and R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, amino, C 1-6 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-6 alkyl;

[0024] Preferably, R1 is one or two identical or different substituents optionally present on the benzene ring, and R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, C 1-4 Alkoxy, amino, C 1-4 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-4 alkyl;

[0025] Further preferably, R1 is one or two identical or different substituents optionally present on the phenyl ring, and R1 is selected from the following groups: hydrogen, fluorine, chlorine, bromine, hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, propoxy, amino, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, carboxyl, CH3(C=O)NH-, C2H5(C=O)NH-, phenyl;

[0026] More preferably, R1 is 1 or 2 identical or different substituents optionally present on the phenyl ring, and R1 is selected from the following groups: hydrogen, fluorine, chlorine, bromine, hydroxyl, methoxy, amino, diethylamino, propylamino, carboxyl, CH3(C=O)NH-, phenyl.

[0027] In a preferred embodiment of the present invention, the compounds represented by Formula I, Formula Ia, Formula Ib, or their racemates or optical isomers, or pharmaceutically acceptable salts, solvates, or hydrates, wherein R1 is optionally monosubstituted at the C1 position on its phenyl ring.

[0028] In a preferred embodiment of the present invention, the compounds represented by Formula I, Formula Ia, Formula Ib, or their racemates or optical isomers, or pharmaceutically acceptable salts, solvates, or hydrates, wherein R1 is optionally monosubstituted at the C2 position on its phenyl ring.

[0029] In a preferred embodiment of the present invention, the compound of formula I, or its racemate or optical isomer, or pharmaceutically acceptable salt, solvate, or hydrate, is selected from the following compounds:

[0030] 2-(2,7-dimethoxy-5,6-dimethyl-9-acridone)-4-acetic acid (Compound 1),

[0031] 7-methoxy-5,6-dimethyl-9-acridone-4-carboxylic acid (Compound 2),

[0032] 2,7-dimethoxy-5,6-dimethyl-9-acridone-4-carboxylic acid (Compound 3),

[0033] 7-methoxy-2,5,6-trimethyl-9-acridone-4-carboxylic acid (Compound 4),

[0034] 2-(7-methoxy-5,6-dimethyl-9-acridone)-10-acetic acid ethyl ester (Compound 5)

[0035] 2-(7-methoxy-5,6-dimethyl-9-acridone)-10-acetic acid (Compound 6),

[0036] 2-(2,7-dimethoxy-5,6-dimethyl-9-acridone)-10-acetic acid ethyl ester (Compound 7),

[0037] 2-(2,7-dimethoxy-5,6-dimethyl-9-acridone)-10-acetic acid (Compound 8),

[0038] 2-(7-methoxy-2,5,6-trimethyl-9-acridone)-10-acetic acid ethyl ester (Compound 9),

[0039] 2-(7-Methoxy-2,5,6-trimethyl-9-acridone)-10-acetic acid (Compound 10).

[0040] The second aspect of the present invention relates to the use of the compound represented by Formula I, Formula Ia, or Formula Ib, a racemate or optical isomer, or a pharmaceutically acceptable salt, solvate, or hydrate thereof according to any one of the first aspects of the present invention in the preparation of a medicament for treating and / or preventing diseases or conditions associated with viral infection. The viral infection includes, but is not limited to, infections caused by viruses such as rhinovirus, enterovirus, cardiovirus, hepatitis virus, influenza virus, SARS virus, Ebola virus, hemorrhagic fever virus, and human immunodeficiency virus. The disease or condition associated with viral infection is selected from respiratory diseases (including, but not limited to, common cold (e.g., summer cold), pharyngitis, tonsillitis, and croup), digestive system diseases, hemorrhagic fever diseases, meningitis / encephalitis, immunodeficiency diseases, hepatitis, and the like.

[0041] The third aspect of the present invention relates to the use of the compound represented by Formula I, Formula Ia, or Formula Ib, a racemate or optical isomer, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, as described in any one of the first aspects of the present invention, in the preparation of a medicament for treating and / or preventing malignant tumors associated with the STING pathway. The malignant tumors include, but are not limited to, melanoma, non-small cell lung cancer, renal cancer, gastric cancer, bladder cancer, head and neck cancer, pancreatic cancer, mesothelioma, and triple-negative breast cancer.

[0042] The features of any aspect of the present invention or any sub-aspect of such aspect are also applicable to any other aspect or any sub-aspect of such other aspect. In the present invention, for example, when referring to "the first aspect of the present invention", such "any sub-aspect" refers to any sub-aspect of the first aspect of the present invention, and similar references to other aspects have the same meaning.

[0043] Various aspects and features of the present invention are further described below.

[0044] All documents cited herein are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with those of the present invention, the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still intends to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail.

[0045] As used herein, the term "pharmaceutically acceptable" when describing, for example, a "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to a subject but also refers to a synthetic substance that has pharmaceutical use value.

[0046] The term "alkyl" as used herein refers to a saturated linear or branched monovalent hydrocarbon group, preferably having 1 to 12 carbon atoms, more preferably 1 to 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 3 carbon atoms. 1-8 "Alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a straight chain or branched chain alkyl group, and which may include its subgroups, such as C 1-6 Alkyl, C 1-4 Alkyl, C 1-3 Alkyl, C 1-2 Alkyl, C 2-5 Alkyl, C 2-4 Alkyl, etc. Typical examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, heptyl, octyl, etc.

[0047] As used herein, the terms "halogen", "halogen atom", "halo" and the like represent fluorine, chlorine, bromine or iodine, and particularly represent fluorine, chlorine or bromine.

[0048] The term "amino" as used herein means -NH2.

[0049] As used herein, the term "hydroxy" refers to -OH.

[0050] The term "carboxy" as used herein refers to -C(O)OH.

[0051] The term "haloalkyl" as used herein means an alkyl group which is mono- or poly-substituted by halogen, such as fluorine, chlorine, bromine or iodine. Preferred haloalkyl groups are chloromethyl, chloroethyl, dichloroethyl, trifluoromethyl, difluoromethyl, monofluoromethyl and the like.

[0052] The term "alkoxy" as used herein refers to a group -OR", wherein R" is an alkyl group as defined herein. Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexyloxy, 1,2-dimethylbutoxy, and the like.

[0053] The groups defined by the above terms herein may also be optionally replaced by -CN, -OH, -NH2, C 1-4 Alkyl, C 1-4 Alkoxy or halogen is mono- or poly-substituted.

[0054] The term "amido" as used herein refers to the group R'(C=O)NH-, wherein R' is an alkyl group as defined herein, preferably C 1-8 Typical examples of "acylamino" include, but are not limited to, formylamino and acetylamino.

[0055] When the compound name used herein is inconsistent with the chemical formula, the chemical formula shall prevail. As used herein, the term "effective amount" refers to a dose that can achieve treatment and / or prevention of the disease or condition described in the present invention in a subject.

[0056] As described herein, the term "pharmaceutical composition", which may also refer to a "composition", can be used to achieve treatment and / or prevention of the diseases or conditions described herein in a subject, particularly a mammal.

[0057] As described herein, the term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, horse, cow, or the like, that receives the compound of formula I or its pharmaceutical composition for treating and / or preventing the disease or condition described herein.

[0058] In the present invention, the viral infection includes but is not limited to infections caused by viruses such as rhinovirus, enterovirus, cardiovirus, hepatitis virus, influenza virus, SARS virus, Ebola virus, hemorrhagic fever virus, and human immunodeficiency virus (HIV).

[0059] In the present invention, the diseases or conditions related to viral infection are selected from respiratory diseases including but not limited to: common cold (e.g., summer cold), pharyngitis, tonsillitis and croup, digestive system diseases, hemorrhagic fever diseases, meningitis / encephalitis, immunodeficiency diseases, hepatitis, etc. In one embodiment of the present invention, it relates to a drug for preventing and / or treating diseases related to viral infection including rhinovirus, enterovirus, HIV, hepatitis virus, influenza virus, SARS virus, Ebola virus, hemorrhagic fever virus, etc., which comprises administering a preventive and / or therapeutically effective amount of at least one compound of Formula I or Formula II or a pharmaceutically acceptable salt or hydrate thereof to a patient in need of prevention and / or treatment of diseases related to viral infection including rhinovirus, enterovirus, HIV, hepatitis virus, influenza virus, SARS virus, Ebola virus, hemorrhagic fever virus, etc.

[0060] In the present invention, the malignant tumors include but are not limited to melanoma, non-small cell lung cancer, kidney cancer, gastric cancer, bladder cancer, head and neck cancer, pancreatic cancer, mesothelioma, triple-negative breast cancer, etc. In one embodiment of the present invention, it relates to a drug for preventing and / or treating melanoma, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancer, pancreatic cancer, mesothelioma, triple-negative breast cancer, etc., which comprises administering a preventive and / or therapeutically effective amount of at least one compound of Formula I or Formula II, or a pharmaceutically acceptable salt or hydrate thereof, to a patient in need of prevention and / or treatment of a disease related to melanoma, non-small cell lung cancer, kidney cancer, gastric cancer, bladder cancer, head and neck cancer, pancreatic cancer, mesothelioma, triple-negative breast cancer, etc.

[0061] According to the present invention, the pharmaceutical compositions of the compounds of the present invention can be administered by any of the following routes: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration via an explanted reservoir. Oral, intraperitoneal, or intravenous administration is preferred. Furthermore, to effectively treat central nervous system disorders, intraventricular administration is preferred to overcome the compound's potentially low blood-brain barrier penetration.

[0062] For oral administration, the compounds of the present invention may be formulated into any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions, or aqueous suspensions. Common carriers for tablets include lactose and corn starch, and lubricants such as magnesium stearate may also be added. Common diluents for capsule formulations include lactose and dried corn starch. Aqueous suspension formulations typically combine the active ingredient with a suitable emulsifier and suspending agent. If desired, sweeteners, flavorings, or coloring agents may be added to these oral dosage forms.

[0063] When administered rectally, the compounds of this invention are generally formulated in the form of suppositories, which are prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but melts at rectal temperature to release the drug. Such excipients include cocoa butter, beeswax, and polyethylene glycol.

[0064] When used topically, especially for treating affected areas or organs that are easily accessible by topical application, such as eyes, skin, or lower intestinal neurological diseases, the compounds of the present invention can be prepared into different topical preparations according to the different affected areas or organs, as described below:

[0065] When administered topically to the eye, the compounds of the present invention may be formulated as a micronized suspension or solution in an isotonic, sterile saline solution of a certain pH, with or without the addition of a preservative such as benzyl alkoxide chloride. Alternatively, for ophthalmic use, the compounds may be formulated in an ointment such as vaseline.

[0066] When applied topically to the skin, the compounds of the present invention can be formulated into suitable ointments, lotions, or creams, wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that can be used for ointments include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax, and water; carriers that can be used for lotions or creams include, but are not limited to, mineral oil, sorbitan monostearate, Tween 60, cetyl esters wax, hexadecene alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0067] For topical administration to the lower intestinal tract, the compounds of this invention may be formulated as rectal suppositories or in a suitable enema formulation as described above. Alternatively, a transdermal patch may be used.

[0068] The compounds of the present invention can also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oily suspensions, or sterile injectable solutions. Among these, acceptable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils, such as monoglycerides or diglycerides, can be used as solvents or suspending media.

[0069] It should also be noted that the specific dosage and method of use of the compounds of this invention for different patients will depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, potency of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 100 mg / kg body weight / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Combined administration of cGAMP and compounds activated the activity of the mSTING pathway.

[0071] Figure 2 Combined administration of cGAMP and compounds activated the activity of the hSTING pathway.

[0072] Figure 3 Cytotoxicity of compound 9 and cGAMP alone or in combination in 293T cells.

[0073] Figure 4 Toxicity of compound 9 and cGAMP alone or in combination to THP-1 cells.

[0074] Figure 5 Compound 9 binds to hSTING-R232.

[0075] Figure 6 Compound 9 synergized the secretion of IFNβ by cGAMP transfected into cells.

[0076] Figure 7 Compound 9 synergized the secretion of IFNβ by DNA transfected into cells.

[0077] Figure 8 Different STING agonists cooperate with cGAMP to activate the mSTING pathway.

[0078] Figure 9 Different STING agonists synergize with cGAMP to activate the hSTING pathway.

[0079] Figure 10 Summary of the binding sites of STING agonists and STING protein. DETAILED DESCRIPTION

[0080] The present invention can be further described by the following examples and test examples. However, the scope of the present invention is not limited to the following examples or test examples. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various changes and modifications may be made to the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods employed for achieving the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible herein.

[0081] In the following examples and test examples, the acridone compound or its pharmaceutically acceptable salt, ester, solvate, metabolite, metabolic precursor or prodrug is selected from any one of the compounds shown in the following table:

[0082]

[0083]

[0084]

[0085] In the following examples and test examples, the names and structural formulas of the endogenous STING agonist cGAMP and other STING agonists are shown in the following table:

[0086]

[0087] Example 1 The compound of the present invention activates the activity of the STING pathway at the cellular level when administered alone

[0088] 1. Test method:

[0089] The activity of secreted alkaline phosphatase can be detected by 293T reporter cells, thereby evaluating the activation of the STING-IRF pathway and indirectly evaluating the induction of type I IFN by the compound. 180 μL of 293T reporter cell (293TmSTING or 293T hSTING-R232) suspension (3×10 5 / well), then add 20 μL of 12.5 μg / mL cGAMP (positive reference) or 20 μL of 100 μM test compound, and incubate at 37°C, 5% CO2 for 48 hours. 20 μL of culture medium from each well is transferred to a new plate, and 180 μL of QUANTI-Blue detection solution is added to measure secreted alkaline phosphatase levels, i.e., optical density (OD). The OD values ​​of the test compound and the positive reference cGAMP are compared.

[0090] 2. Test results:

[0091] The biorating of the compound was obtained by comparing the OD value of the compound at a concentration of 100 μM with the OD value of 2′,3′-cGAMP at a concentration of 12.5 μg / mL in the same experiment. The experimental results are shown in Table 1.

[0092] Rating explanation: "-" represents poor activity, that is, the OD value of the test compound at 100 μM is lower than the OD value of 12.5 μg / mL 2',3'-cGAMP.

[0093] Table 1 Test results of compounds activating STING alone in cells

[0094]

[0095] Example 2 Activation of the STING pathway by the compounds of the present invention at the cellular level when administered in combination with the endogenous STING agonist cGAMP

[0096] 1. Test method:

[0097] The activation of the STING-IRF pathway by combined drug administration was evaluated using 293T reporter cells according to the method of Example 1. 180 μL of 293T reporter cell (293T mSTING or 293T hSTING-R232) suspension (3×10 5 / well), then add 10 μL of cGAMP (0.098, 0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25 μg / mL) and 10 μL of test compound (33, 100 μM), and incubate at 37°C, 5% CO2 for 48 hours. 20 μL of culture medium from each well was transferred to a new plate, and 180 μL of QUANTI-Blue detection solution was added to detect the level of secretory alkaline phosphatase, i.e., the optical density (OD) value, and calculate the half effective concentration (EC) of cGAMP combined with compound administration. 50 )value.

[0098] 2. Test results:

[0099] The experimental results of the compounds synergizing with cGAMP to activate mSTING are as follows Figure 1 The results showed that the endogenous STING ligand cGAMP activated the EC of mSTING in 293T cells. 50 The value was 5.38 μg / mL. Compounds 7, 8 and 9 could cooperate with cGAMP to activate the mSTING pathway. Among them, compound 7 (100 μM) had the best effect in cooperating with cGAMP. 50The value increased to 0.20μg / mL. The experimental results of the compound synergistically activating hSTING with cGAMP are as follows Figure 1 The results showed that the endogenous STING ligand cGAMP activated hSTING EC in 293T cells. 50 The value was 9.77 μg / mL. Compounds 7, 8 and 9 could cooperate with cGAMP to activate the hSTING pathway. Among them, compound 8 (100 μM) had the best effect in cooperating with cGAMP. 50 The value increased to 0.33 μg / mL.

[0100] Example 3 Toxicity of the compounds of the present invention and cGAMP alone or in combination at the cellular level

[0101] 1. Experimental methods:

[0102] Cell viability was measured using the CellTiter-Glo Fluorescent Cell Viability Assay Kit according to the manufacturer's instructions. 293T and THP-1 cells were incubated with DMSO (blank), compound 9 (100 μM), cGAMP (25 μg / mL), or compound 9 (100 μM) combined with cGAMP (25 μg / mL) for 48 hours. After removing the cell culture supernatant, 100 μL of CellTiter-Glo detection reagent was added to each well, and luminescence was recorded after 10 minutes.

[0103] 2. Experimental results:

[0104] The toxicity test results of compound 9 and cGAMP alone or in combination on 293T cells are shown in Figure 2. Figure 3 The results showed that compound 9 (100 μM) and cGAMP (25 μg / mL) alone or in combination had no toxicity to 293T cells. The toxicity test results of compound 9 and cGAMP alone or in combination on THP-1 cells are shown in Figure 2. Figure 4 The results showed that compound 9 (100 μM) and cGAMP (25 μg / mL) had no toxicity to THP-1 cells when administered alone or in combination.

[0105] Example 4 Affinity of the Compounds of the Invention for STING Protein

[0106] 1. Experimental methods:

[0107] The affinity between the STING protein and small molecules was determined using surface plasmon resonance (SPR). STING protein was diluted to a concentration of 50 μg / mL in 10 mM sodium acetate, pH 4.0, and then immobilized on a CM5 chip via amino coupling. Subsequently, arachidonic acid solutions of varying concentrations (2.34, 4.69, 9.375, 18.75, 37.5, 75, 150, and 300 μM) were flowed over the chip at 50 μL / min in PBS-P buffer (pH 7.4 containing 5% DMSO) at 25°C. The sample solution was injected onto the chip for 130 seconds, and after each binding reaction, the signal was allowed to dissociate for 150 seconds to return to baseline. Equilibrium and kinetic constants were calculated using a 1:2 binding model using BIA evaluation software.

[0108] 2. Experimental Results

[0109] The results of the SPR experiment are as follows Figure 5 As shown, the results showed that compound 9 could bind to the CTD region of hSTING-R232 protein with an affinity constant of approximately KD = 6.29 μM.

[0110] Example 5 Verification of intracellular mechanism

[0111] 1. Experimental methods:

[0112] On the one hand, cGAMP was transfected into THP-1 cells using Lipofectamine 2000, and the cells were then stimulated with test compounds to observe whether the compounds synergize with cGAMP to activate the cGAS-STING pathway, thereby secreting IFNβ, thereby eliminating the possibility that the compounds have a synergistic effect by promoting cGAMP transmembrane transport. On the other hand, DNA fragments were transfected into THP-1 cells using Lipofectamine 2000. cGAS recognizes foreign DNA and produces endogenous cGAMP. The cells were then stimulated with test compounds to observe whether the compounds synergize with endogenously produced cGAMP to activate the cGAS-STING pathway, thereby secreting IFNβ.

[0113] 180 μL of THP-1 cell suspension (5×10 5Lipofectamine reagent was diluted in Opti-MEM medium, and DNA or cGAMP was diluted in Opti-MEM medium. The diluted DNA or cGAMP was added to the diluted Lipofectamine transfection reagent and incubated at room temperature for 5 minutes to form a DNA / cGAMP-liposome complex. 10 μL of the DNA / cGAMP-liposome complex was then added to the cells. After incubation for 24 hours at 37°C, 5% CO2, 10 μL of compound 9 was added and the cells were cultured for 48 hours. Interferon-β secretion in the cell supernatant was measured using the Cisbio Human IFNβ Kit.

[0114] 2. Experimental results:

[0115] Figure 6 It shows that compound 9 can synergistically activate the STING pathway to secrete IFNβ by cGAMP transfected into cells. This experimental result shows that compound 9 does not have a synergistic effect by promoting the transmembrane transport of cGAMP. Figure 7 The results showed that, under stimulation by foreign DNA, THP-1 cells were able to recognize foreign DNA, triggering an immune response and secreting IFNβ, and that compound 9 was able to amplify this immune response. Since cGAS can recognize foreign DNA and synthesize cGAMP, these experimental results further demonstrate that compound 9 not only has a synergistic effect by promoting cGAMP transmembrane transport, but also that compound 9 can synergize with endogenously produced cGAMP to activate the cGAS-STING pathway.

[0116] Example 5 Synergistic Effect Test of Other STING Agonists

[0117] 1. Experimental methods:

[0118] Add 180 μL of 293TmSTING reporter cell suspension (3×10 5 10 μL of cGAMP and 10 μL of a STING agonist (MSA-02, SR-717, di-ABZI, DMXAA), compound 9, or DMSO were added and incubated at 37°C, 5% CO2 for 48 hours. 20 μL of culture medium from each well was transferred to a new plate, and 180 μL of QUANTI-Blue detection solution was added to measure secreted alkaline phosphatase levels, i.e., optical density (OD) values.

[0119] Add 180 μL of 293T hSTING-R232 reporter cell suspension (3×10 510 μL of cGAMP and 10 μL of a STING agonist (MSA-02, SR-717, di-ABZI, C53), compound 9, or DMSO were added and incubated at 37°C, 5% CO₂ for 48 hours. 20 μL of culture medium from each well was transferred to a new plate, and 180 μL of QUANTI-Blue detection solution was added to measure secreted alkaline phosphatase levels, i.e., optical density (OD) values.

[0120] 2. Experimental results:

[0121] Figure 8 It shows that the existing various STING agonists can only activate mSTING alone, and do not show synergistic effects when used in combination with cGAMP. However, acridinone compound 9 can synergize with cGAMP to cause a stronger mSTING activation effect. Figure 9 It shows that, except for C53, all existing STING agonists can only activate hSTING alone, and do not show synergistic effects when used in combination with cGAMP. However, acridinone compound 9 can synergize with cGAMP to induce a stronger hSTING activation effect. C53 binds to the N-terminal transmembrane region of the STING protein, but not at the same binding site as cGAMP ( Figure 10 ), thus synergizing STING. However, C53 itself has STING agonist activity, which can lead to widespread activation of the STING pathway in the body, causing a systemic inflammatory response. Acridinone compound 9, when administered alone, does not induce an immune response in normal cells. However, when cells in the lesion recognize damaged DNA and produce trace amounts of cGAMP, compound 9 synergizes with cGAMP to activate the STING pathway, triggering an immune response and achieving a targeted therapeutic effect.

Claims

1. The compound represented by formula I, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate, in, X is a C atom, an O atom, a S atom, a carbonyl group, a sulfinyl group or a sulfyl group; Y is hydrogen, alkyl, haloalkyl, acetoxy or ester; R1 is 1, 2 or 3 identical or different substituents optionally present on the phenyl ring, and R1 is selected from the following groups: hydrogen, halogen, hydroxy, alkyl, haloalkyl, alkoxy, amino, amino monosubstituted or disubstituted by alkyl, carboxyl, acetoxy, amide, phenyl.

2. The compound of claim 1, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate, which is a compound represented by formula Ia, in, n = 0 or 1; R1 is one or two identical or different substituents optionally present on the benzene ring, R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-8 Alkyl, halogenated C 1-8 Alkyl, C 1-8 Alkoxy, amino, C 1-8 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-8 alkyl.

3. The compound of claim 1, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate, which is a compound represented by formula Ib, in, R1 is one or two identical or different substituents optionally present on the benzene ring, R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-8 Alkyl, halogenated C 1-8 Alkyl, C 1-8 Alkoxy, amino, C 1-8 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-8 alkyl; R2 is ethyl or hydrogen.

4. The compound according to any one of claims 1 to 3, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate, wherein: R1 is one or two identical or different substituents optionally present on the benzene ring, R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-6 Alkyl, halogenated C 1-6 Alkyl, C 1-6 Alkoxy, amino, C 1-6 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-6 alkyl; Preferably, R1 is one or two identical or different substituents optionally present on the benzene ring, and R1 is selected from the following groups: hydrogen, halogen, hydroxyl, C 1-4 Alkyl, halogenated C 1-4 Alkyl, C 1-4 Alkoxy, amino, C 1-4 Alkyl mono- or di-substituted amino, carboxyl, R'(C=O)NH-, phenyl, wherein R' is C 1-4 alkyl; Further preferably, R1 is one or two identical or different substituents optionally present on the phenyl ring, and R1 is selected from the following groups: hydrogen, fluorine, chlorine, bromine, hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, propoxy, amino, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, carboxyl, CH3(C=O)NH-, C2H5(C=O)NH-, phenyl; More preferably, R1 is 1 or 2 identical or different substituents optionally present on the phenyl ring, and R1 is selected from the following groups: hydrogen, fluorine, chlorine, bromine, hydroxyl, methoxy, amino, diethylamino, propylamino, carboxyl, CH3(C=O)NH-, phenyl.

5. The compound according to any one of claims 1 to 4, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate, or hydrate, which is selected from the following compounds: 2-(2,7-dimethoxy-5,6-dimethyl-9-acridone)-4-acetic acid (Compound 1), 7-methoxy-5,6-dimethyl-9-acridone-4-carboxylic acid (Compound 2), 2,7-dimethoxy-5,6-dimethyl-9-acridone-4-carboxylic acid (Compound 3), 7-methoxy-2,5,6-trimethyl-9-acridone-4-carboxylic acid (Compound 4), 2-(7-methoxy-5,6-dimethyl-9-acridone)-10-acetic acid ethyl ester (Compound 5) 2-(7-methoxy-5,6-dimethyl-9-acridone)-10-acetic acid (Compound 6), 2-(2,7-dimethoxy-5,6-dimethyl-9-acridone)-10-acetic acid ethyl ester (Compound 7), 2-(2,7-dimethoxy-5,6-dimethyl-9-acridone)-10-acetic acid (Compound 8), 2-(7-methoxy-2,5,6-trimethyl-9-acridone)-10-acetic acid ethyl ester (Compound 9), 2-(7-Methoxy-2,5,6-trimethyl-9-acridone)-10-acetic acid (Compound 10).

6. Use of the compound according to any one of claims 1 to 5, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate or hydrate in the preparation of a medicament for treating and / or preventing diseases or conditions associated with viral infection.

7. Use of the compound according to any one of claims 1 to 5, its racemate or optical isomer, or its pharmaceutically acceptable salt, solvate or hydrate in the preparation of a medicament for treating and / or preventing malignant tumors associated with the STING pathway.

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