WO2021068951A1

WO2021068951A1 - Medical use of benzenesulfonamide compound and pharmaceutical composition thereof

Info

Publication number: WO2021068951A1
Application number: PCT/CN2020/120280
Authority: WO
Inventors: 王琛; 洪泽; 余文颖; 孙宏斌; 梅家豪; 李晨辉; 刘星; 孙立; 柳军
Original assignee: 中国药科大学
Priority date: 2019-10-12
Filing date: 2020-10-12
Publication date: 2021-04-15
Also published as: CN112057443A; CN112057443B

Abstract

A medical use of a benzenesulfonamide compound and a pharmaceutical composition thereof. In particular, the present invention relates to a compound represented by one of formulas I to V or a pharmaceutically acceptable salt or solvate thereof. The present invention can be used to prepare a STING inhibitor or a drug for inhibiting the activation of STING signaling pathway and to prepare a drug for preventing or treating a disease mediated by STING.

Description

Medical use of benzenesulfonamide compound and its pharmaceutical composition

Technical field

The present invention belongs to the field of biomedicine, and specifically relates to the medical use of benzenesulfonamide compounds or their pharmaceutically acceptable salts or solvates and pharmaceutical compositions thereof, which can act on transmembrane protein 173 (TMEM173), also known as STING ( Stimulator of interferon gene) and inhibit its signal pathway. Therefore, such compounds can be used to prepare drugs for the prevention or treatment of STING-mediated diseases.

Background technique

The activation of the body's innate immune system is mediated by pattern recognition receptors (PRRs) expressed on the cell membrane and in the cytoplasm that recognize non-self pathogen-related molecular patterns (PAMPs). After identifying PAMPs, PRRs activate downstream immune signal transmission, promote inflammation and the release of immune cytokines, and further activate the body's adaptive immune system to co-kill and eliminate invading pathogenic microorganisms (Cold Spring Harbor Symposia on Quantitative Biology, 1989, 54 :1-13.). Similarly, damage-associated molecular patterns (DAMPs) secreted by cells under abnormal conditions (stress, damage, aging, or death) will also be recognized by pattern recognition receptors to activate the immune system and promote Cell regeneration and repair after injury (Science, 2002, 296(5566): 301-305).

As a classic PAMP or DAMP molecule, DNA in the cytoplasm plays an important role in the activation of the innate immune system (Curr Opin Immunol, 2018, 55: 31-37.). The DNA receptor protein cGAS (Cyclic AMP-GMP synthase) in the cytoplasm can recognize abnormally exposed cytoplasmic DNA molecules, and use ATP and GTP as substrates to catalyze the synthesis of 2',3'-cGAMP molecules (2'-3'/ 3'-5'cyclic GMP-AMP). The signal molecules c-di-AMP and c-di-GMP formed in the metabolism of 2’, 3’-cGAMP and bacteria are called cyclic dinucleotides (CDNs). These CDNs molecules can specifically bind to the "V"-shaped pocket formed by the dimer of the endoplasmic reticulum regulatory protein STING (also known as TMEM173, MITA, ERIS or MPYS) (Nature, 2008, 455(7213): 674 -678; Nature, 2011, 478(7370): 515-518), thereby inducing the multimerization activation of STING protein (Cell, 2013, 154(4): 748-62). The multimerized STING protein transfers from the endoplasmic reticulum to the Golgi compartment, and in this process recruits the downstream kinase protein TBK1 and the transcription factor IRF3. TBK1 catalyzes the phosphorylation of STING and IRF3 after autophosphorylation is activated (Nature, 2019 , 567(7748):394-398; Nature, 2019,569(7758):718-722). Phosphorylated IRF3 further dimerizes into the nucleus to promote the expression of type I interferons and related immune factors (interferon stimulated genes, ISGs). In addition, STING protein can also activate the NF-κB signaling pathway by recruiting TBK1 and TRAF6 molecules, and promote the expression of inflammatory factors such as TNF-α and IL-6 (J Virol, 2014, 88(10): 5328-41).

Although the activation of the STING-mediated innate immune signaling pathway plays an important role in the body's resistance to the invasion of pathogenic microorganisms, continuous activation of the STING pathway can lead to the occurrence and development of a variety of autoimmune and inflammatory diseases (Nature Immunology, 2017) ,18(7):716-724). AGS syndrome (Aicardi-Goutières syndrome) is a rare systemic autoimmune disease caused by TREX1, RNASE H2, SAMHD1, ADAR or IFIH1 gene mutations (Am J Med Genet A, 2015, 167A(2): 296 -312). Studies in animal models have found that the gene mutations of nucleic acid metabolizing enzymes TREX1, RNASE H2 and SAMHD1 lead to cytoplasmic DNA aggregation and activate the cGAS-STING signaling pathway, and further knocking out the expression of STING protein in diseased mice can significantly improve the progression of the disease ( J Exp Med, 2016, 213(3): 329-36; Proc Natl Acad Sci U S A, 2015, 112(42): E5699-705; Nature, 2018, 557(7703): 57-61). TREX1 gene mutations have also been found in patients with systemic lupus erythematosus (SLE), and 50%-60% of SLE patients have high expression of type I interferon in the serum (Nat Rev Rheumatol, 2018, 14(4) :214-228). It was found in patients with Bloom syndrome that mutations in the BLM protein lead to the formation of micronuclei, which in turn activates the STING signaling pathway to induce high expression of IFNs and ISGs in the patient's serum (J Exp Med, 2019, 216(5): 1199-1213). SAVI (STING-associated vasculopathy with onset in infection) disease is a systemic autoimmune disease induced by abnormal activation of individual amino acids in the STING protein (N Engl J Med, 2014, 371(6): 507-518) . In addition, in inflammation-related disease models, such as skin cancer induction (Nat Commun, 2014, 5: 5166), tumor metastasis (Nature, 2018, 553(7689): 467-472), and progeria (Nature, 2017, 550(7676):402-406), sepsis (Shock, 2017, 47(5): 621-631), acute pancreatitis (Gastroenterology, 2018, 154(6): 1822-1835), non-alcoholic fat Liver and liver fibrosis (Gastroenterology, 2018, 155(6): 1971-1984; Proceedings of the National Academy of Sciences, 2017, 114(46): 12196-12201), pneumonia (Nature Communications, 2018, 9(1) ), chronic nephritis and renal fibrosis (Cell Metabolism, 2019, DOI: 10.1016/j.cmet.2019.08.003), ischemic reperfusion injury (Nature Medicine, 2017, 23(12): 1481-1487; EMBO Molecular Medicine,2020,7;12(4):e11002), Parkinson's disease (Nature,2018,561(7722):258-262), Brain trauma (Journal of Neuroscience,2020,40(2):424-446) , Trauma or other diseases induced spinal cord injury (Cell, 2020, 183:1-14; Biochemical and Biophysical Research Communications, 2019, 517(4): 741-748) and Huntington's disease (Journal of Clinical Investigation, 2020, 130( 6): 3124-3136; Proceedings of the National Academy of Sciences, 2020, 117(27): 15989-15999), familial amyotrophic lateral sclerosis (Nature, 2020, 585(7823): 96-101), etc. , And inhibiting the activation of the STING signaling pathway can significantly improve the occurrence and development of the above-mentioned diseases. In short, the development of inhibitors targeting STING protein has broad clinical application prospects.

At present, there are only a handful of STING small molecule inhibitors reported in the literature, with weaker activity and greater side effects (Cell Reports 2018, 25, 3405-3421; ACS Med. Chem. Lett, 2019, 10(1), 92-97) . Therefore, there is an urgent clinical need to develop new STING small molecule inhibitors.

Summary of the invention

Purpose of the invention: In view of the problems existing in the prior art, the present invention provides any one or more of the following formulas I to V, or a pharmaceutically acceptable salt or solvate thereof, for preparing and inhibiting the STING signal pathway Use in activated drugs and in preparing drugs for preventing or treating STING-mediated diseases. Through virtual screening, biological activity evaluation and mechanism research, the present invention found that the benzenesulfonamide compounds represented by the following formulas I to V or their pharmaceutically acceptable salts or solvates can specifically inhibit the activation of the STING signal pathway.

The invention also provides a pharmaceutical composition for preventing and treating STING-mediated diseases.

Technical solution: In order to achieve the above object, the benzenesulfonamide compounds or pharmaceutically acceptable salts or solvates of any one or more of formulas I to V described in the present invention are used in the preparation of inhibitors of STING signaling pathway. Use in medicine:

The use of any one or more of the benzenesulfonamide compounds of formula I to V or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicine for preventing or treating STING-mediated diseases.

Further, the STING-mediated diseases include infectious diseases, inflammatory diseases, autoimmune diseases, organ fibrotic diseases, ischemic cardiovascular and cerebrovascular diseases, neurodegenerative diseases, brain trauma, spinal cord injury, cancer or One or more of the cancerous syndromes.

In certain embodiments, the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat infectious diseases, including: Mycobacterium tuberculosis infection, chlamydia infection, herpes virus (herpes simplex virus) infection , Adenovirus infection, hepatitis B virus infection, orthomyxovirus infection and coronavirus infection.

In certain embodiments, the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat inflammatory diseases, including: metabolic inflammation-related diseases (such as insulin resistance, metabolic syndrome, type 1 or Type 2 diabetes, hyperlipidemia, obesity, atherosclerosis, myocardial ischemia, myocardial infarction, arrhythmia, coronary heart disease, hypertension, heart failure, myocardial hypertrophy, myocarditis, ischemic encephalopathy, stroke, hemorrhage Encephalopathy, cerebral hemorrhage, cerebral edema, diabetic cardiomyopathy, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy and diabetic ulcer, non-alcoholic fatty liver, non-alcoholic steatohepatitis, alcoholic fatty liver, cirrhosis, gout, stroke Or cerebral infarction, etc.), musculoskeletal inflammation (hands, wrists, elbows, shoulders, neck, knees, ankles and feet joint inflammation, such as osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, acute and chronic infectious Arthritis, etc.), ocular inflammation (keratitis, scleritis, conjunctivitis, etc.), digestive system inflammation (colitis, hepatitis, cholangitis, cholecystitis, pancreatitis, gastritis, enteritis, inflammatory bowel disease, proctitis) , Nervous system inflammation (meningitis, neuromuscular rigidity, multiple sclerosis, CNS vasculitis), degenerative neurological diseases (Parkinson's disease, Huntington's disease, familial amyotrophic lateral sclerosis), vascular system or lymph System inflammation (vasculitis, lymphangitis, phlebitis), reproductive system inflammation (cervicitis, endometritis, epididymitis, orchitis, urethritis), respiratory system inflammation (pneumonia, asthma, chronic obstructive pulmonary disease, chronic Bronchitis, emphysema, bronchiolitis obliterans, idiopathic pulmonary fibrosis, cystic fibrosis lung disease), organ fibrotic diseases (liver fibrosis, kidney fibrosis, pulmonary fibrosis, myocardial fibrosis), others Inflammatory diseases include appendicitis, myocarditis, mumps, gingivitis, prostatitis, peritonitis, pleurisy, vasculitis, phlebitis, edema, nephritis, spinal cord injury including trauma or other diseases caused by spinal cord injury, brain injury.

In certain embodiments, the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat autoimmune diseases. Including: Aicardi-Goutières syndrome (AGS), infantile-onset STING-related vasculitis (SAVI), retinal vascular disease with cerebral protein dystrophy (RCVL), systemic lupus erythematosus (SLE), familial frostbite lupus ( CHBL), Behcet’s disease, Chagas’ disease, psoriasis, multiple sclerosis, scleroderma and Behcet’s disease, etc.

In certain embodiments, the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat T cell-mediated hypersensitivity reactions with inflammatory components, including urticaria, skin allergies, and allergies. Rhinitis, contact dermatitis and respiratory allergies.

In certain embodiments, the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat cancer and tumor metastasis in various tissues and organs of the body, including but not limited to lung, bone, pancreas, liver, Cancer of the kidney, head, uterus, ovary, stomach, colon, esophagus, small intestine, endocrine system, prostate, bladder, cervix, and vagina. Such as liver cancer, kidney cancer, cervical cancer, lung cancer, skin cancer, uterine cancer, adenocarcinoma, prostate cancer, sarcoma, osteosarcoma, thyroid cancer, non-small cell lung cancer, esophageal cancer, chronic myeloid leukemia, chronic lymphocytic leukemia , Acute myeloid leukemia, acute lymphocytic leukemia, multiple myeloma, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma.

In certain preferred embodiments, the STING-mediated disease is psoriasis, stroke, myocardial infarction, brain injury, trauma or other disease-induced spinal cord injury, Parkinson's disease, Huntington's disease, familial muscular atrophy Lateral sclerosis, non-alcoholic steatohepatitis, systemic lupus erythematosus, Aicardi-Goutières syndrome, rheumatoid arthritis, inflammatory bowel disease, STING-related vascular disease (SAVI) or diabetes occurring in infancy complication.

The pharmaceutically acceptable salt of the compound of formula I to V is a salt formed by a metal ion or a pharmaceutically acceptable amine, ammonium ion or choline.

The compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used alone or in combination with other therapeutic agents. As immunomodulators, the compounds of the present invention can be used in monotherapy or in combination with other therapeutic agents to treat diseases related to abnormal STING protein function, including infectious diseases, inflammatory diseases, autoimmune diseases, and organ fibrotic diseases , Ischemic cardiovascular and cerebrovascular diseases, brain trauma, spinal cord injury, neurodegenerative diseases, cancer or precancerous syndrome.

The compounds of the present invention can be used as pharmaceutical salts. The salt may be the acid salt of at least one of the following acids: galactonic acid, D-glucuronic acid, glycerophosphoric acid, hippuric acid, isethionic acid, lactobionic acid, maleic acid, 1,5-naphthalene Disulfonic acid, naphthalene-2-sulfonic acid, pivalic acid, terephthalic acid, thiocyanic acid, cholic acid, n-dodecyl sulfuric acid, benzenesulfonic acid, citric acid, D-glucose, glycolic acid, lactic acid, Malic acid, malonic acid, mandelic acid, phosphoric acid, propionic acid, hydrochloric acid, sulfuric acid, tartaric acid, succinic acid, formic acid, hydroiodic acid, hydrobromic acid, methanesulfonic acid, niacin, nitric acid, orotic acid, oxalic acid, bitter Acid, L-pyroglutamic acid, saccharinic acid, salicylic acid, gentisic acid, p-toluenesulfonic acid, valeric acid, palmitic acid, sebacic acid, stearic acid, lauric acid, acetic acid, adipic acid, carbonic acid, Benzenesulfonic acid, ethanedisulfonic acid, ethyl succinic acid, fumaric acid, 3-hydroxynaphthalene-2-carboxylic acid, 1-hydroxynaphthalene-2-carboxylic acid, oleic acid, undecylenic acid, ascorbic acid, camphor acid , Camphor sulfonic acid, dichloroacetic acid, ethane sulfonic acid. On the other hand, the salt can also be a compound of the present invention and metal (including sodium, potassium, calcium, etc.) ions or pharmaceutically acceptable amines (including ethylenediamine, tromethamine, etc.), ammonium ions or choline. The salt formed.

The present invention includes various deuterated forms of the compounds of the present invention. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom.

The pharmaceutical composition for preventing or treating STING-mediated diseases of the present invention comprises any one of formula I to V benzenesulfonamide compound or a pharmaceutically acceptable salt or solvent compound thereof as an active ingredient and a pharmaceutically acceptable compound. Accepted excipients. The adjuvants that can be arbitrarily mixed can be changed according to the dosage form, administration form, and the like. Examples of excipients include excipients, binders, disintegrating agents, lubricants, flavoring agents, flavoring agents, coloring agents or sweetening agents, and the like. The pharmaceutical composition can be in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches, and other pharmacologically conventional preparations.

The compound of the present invention can be purchased from EnamineStore, and can also be prepared by referring to the method described in the examples or an improved method thereof. The HPLC purity of all compounds is above 95%.

The present invention screens compounds that may bind to the STING protein through a computer-assisted virtual screening method, and further verifies the biological activity of the compounds at the cellular level. As a result, it was found that the compound of formula I (SN-011, CAS: 2249435-90-1), the compound of formula II (SN-001, CAS: 727699-84-5), the compound of formula III (SN-005), the compound of formula IV ( SN-006) and the compound of formula V (SN-010) can inhibit the STING signaling pathway with high efficiency, specificity and low toxicity. For example, the half inhibition rate of the compound of formula I on IFN-β activation in mouse primary bone marrow-differentiated macrophages and human primary foreskin fibroblasts is about 100 nM and 500 nM, respectively. Further mechanism studies have found that the compound of formula I can significantly activate the multimerization of the STING protein induced by DNA in the cytoplasm, inhibit its transfer from the endoplasmic reticulum to the Golgi apparatus, and inhibit the recruitment of downstream adaptor proteins TBK1 and IRF3, leading to transcription factors Phosphorylation of IRF3 and NF-κB and their nuclear transcription levels are significantly reduced, thereby inhibiting the downstream expression of type I interferon, IL-6 and TNF-α. In addition, the present invention obtained and analyzed the co-crystal structure of human STING protein and the compound of formula I, and the results showed that multiple conservative amino acid sites in the STING protein have a binding effect with the compound of formula I, including Tyr167, Ser241, Ser243, Glu260 And Thr263. It is worth noting that these sites are involved in the binding of STING protein to its endogenous ligand molecule 2'3'-cGAMP. And further competitive Pulldown experiments confirmed that the compound of formula I binds to the STING protein in a non-covalent and reversible manner. The surface ion resonance molecular interaction instrument (SPR) detected that the affinity between SN-011 and the human STING protein was 4.03nM, while the affinity between 2'3'-cGAMP and the STING protein was 9.23nM. This indicates that compared with 2'3'-cGAMP, the compound of formula I has stronger binding ability to STING protein.

Accordingly, the present invention provides the use of the compounds represented by formulas I to V or their pharmaceutically acceptable salts or solvates in the preparation of drugs for inhibiting the activation of the STING signal pathway.

The present invention selects the autoimmune disease model of mouse Trex1 gene knockout, the in vitro cell model of SAVI, the rat and mouse stroke models induced by middle cerebral artery embolism (MCAO), and the non-alcoholic mice induced by high-fat diet The fatty liver model and the mouse psoriasis model induced by imiquimod are used for the pharmacodynamic evaluation of the compounds of the present invention. The results of the study taking the compound of formula I as an example show that:

1. The compound of formula I can significantly improve the spontaneous inflammatory damage of multiple tissues and organs in Trex1 knockout mice, inhibit the activation of the body's self-reactive adaptive immune system, and prolong the long-term survival rate of diseased mice.

2. In cells overexpressing SAVI-related STING point mutants, the compound of formula I can significantly inhibit the expression of cell type I interferons and related ISGs and inflammatory cytokines.

3. In the rat stroke model prepared by MCAO, the compound of formula I can significantly down-regulate the expression of type I interferon and its related ISGs and inflammatory cytokines in the brain tissue of the ischemic area, and significantly improve the neurobehavior of stroke animals Learn function.

4. In the mouse stroke model prepared by MCAO, immediate administration of the compound of formula I can significantly down-regulate the expression of inflammatory cytokines in the brain tissue of the ischemic area, and significantly improve the memory and motor functions of the mice; the compound of formula I is used in stroke After 24 hours of administration, it can still significantly improve the motor function of stroke mice.

5. In the non-alcoholic fatty liver model of mice induced by a long-term high-fat diet, the compound of formula I can significantly improve the lipid accumulation and inflammatory infiltration of the liver of mice, and improve liver damage.

6. In the establishment of a psoriasis-like inflammation model induced by imiquimod in BALB/c mice, the compound of formula I can significantly improve psoriasis-like inflammation.

Based on the above research results, the present invention provides the use of a compound of formula I to V or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicine for preventing or treating STING-mediated diseases.

Beneficial effects: Compared with the prior art, the present invention has the following advantages:

(1) The present invention finds for the first time that any one of the benzenesulfonamide compounds of formula I to V or a pharmaceutically acceptable salt or solvate thereof can be used as a new type of small molecule inhibitor targeting STING with high efficiency, specificity and low efficiency. Toxically inhibit the activation of the STING signaling pathway, so it can be used to prepare drugs that inhibit the activation of the STING signaling pathway.

(2) The compound of the present invention has a clear mechanism of action. It inhibits the activation of the STING signal pathway by directly binding to the STING protein and maintaining its dimer conformation in a resting state. It is particularly important that the compounds of the present invention have significant in vivo curative effects in disease models such as inflammatory diseases, autoimmune diseases, organ fibrosis diseases and ischemic cardiovascular and cerebrovascular diseases, and are therefore expected to be used in the preparation of prevention or treatment of the above-mentioned diseases. Drugs for STING-mediated diseases.

Description of the drawings

Figure 1 is a graph of the inhibitory effect of small molecule compounds containing benzenesulfonamide structure on the STING signaling pathway obtained through virtual screening and the structures of some compounds: (A) MEF cells were incubated with 10μM test compounds. After 6 hours of incubation, lipids The cells were transfected with 5μg ISD for 6 hours, and the cells were collected to detect the expression of Ifnβ gene; (B) MEF cells were incubated with 10μM test compound, and the cells were collected after 6 hours of incubation to detect the expression of Ifnβ gene; (C) Compound SN-001～SN -004 and the chemical structure of the negative control compound SN-100; all data are the average of three parallel experiments and the variance is the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

Figure 2 is a graph showing the in vitro biological activity of compound SN-001 (compound of formula II): (A～D) 5～20μM compound SN-001 and SN-100 were incubated with L929 cells for 6 hours, liposomes were transfected with HT- DNA 4μg stimulation for 6 hours, harvested cells to detect Ifnβ, Ifnα4, Cxcl10 and Il6 gene expression; (E～H)5～20μM compound SN-001 and SN-100 were incubated with THP-1 cells for 6 hours, HSV-1 was used 40μL of the virus was stimulated for 6 hours, and the cells were collected to detect the expression of IFNβ, IFNα4, CXCL10 and IL6 genes; (I) 10μM compounds SN-001 and SN-100 were incubated in L929 cells for 6 hours, respectively, liposome transfected with HT-DNA 4μg stimulation After 4 hours, the specific antibody immunoblotting method used to collect the protein from the cells was used to detect the expression level of the corresponding protein; (J～K) 10μM compounds SN-001 and SN-100 were incubated in L929 cells for 6 hours, and liposomes were transfected with HT- DNA 8μg stimulation for 4 hours, immunofluorescence marks the transcription factors IRF3 and P65 into the nucleus, the nuclear position is indicated by DAPI staining, and the length of the ruler is 25μm; (L～N) MEF, L929 and THP-1 cells were incubated for 5～ 20μM compound SN-001 12 or 24 hours later, use MTS reagent to detect the effect of SN-001 on cell viability; all data are three parallel experiments and the average value is taken and the variance is the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

Figure 3 is a graph showing the in vitro biological activity effects of compound SN-011 (compound of formula I) and related compounds: (A) 10μM SN-001 and related compounds SN-005～SN-011 were incubated with L929 cells for 6 hours, and liposome-transformed Stimulate with HT-DNA 4μg for 6 hours, collect cells to detect the expression of IFN-β gene; (B) Compound structure of compound SN-001 and related compounds SN-005～SN-011; (C～E) 1μM compound SN-011 After incubating the MEF cells for 6 hours, use the classical agonist ISD upstream of STING 5μg, HT-DNA 4μg, HSV-1 40μl, c-di-GMP 1μg and 2'3'-cGAMP 1μg to stimulate, and then detect the cell Ifnβ after the stimulation is completed. , The expression of Cxcl10 and Il6 genes; all data are three parallel experiments and the average value is taken and the variance is used as the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

_{Figure 4 is a graph showing the IC 50} test effect of compound SN-011 (compound of formula I) in MEF, BMDM and HFF cells: (A～C) MEF, BMDM and HFF cells were incubated with gradient concentrations of compound SN-011 (5nM) ~5μM) 6 hours later, use 2'3'-cGAMP 1μg to stimulate for 3 hours. After the stimulation is completed, detect the expression of IFN-β gene, and plot the inhibition rate of gene expression to fit the IC ₅₀ value; (D~ F) MEF, BMDM and HFF cells were incubated with 1.25～20μM compound SN-011 for 24 or 48 hours, and MTS reagent was used to detect the effect of SN-011 on cell viability; ns, no significant difference; *, P<0.05;**,P<0.01;

Figure 5 is a graph showing the effect of compound SN-011 (compound of formula I) specifically inhibiting the activation of the STING signaling pathway: (A～B) SN-011 was incubated in STING knockout MEF cells for 6 hours, and then used in the cells. Add LPS 10μg, poly(I:C)100μg, Sendai virus 10μl, IFN-β100U stimulation, or liposome transfection with CpG-DNA 5μg, poly(I:C)5μg, and detect Ifnβ, Il6, Tnfα after the stimulation is completed, Expression of Isg15 and Isg56 genes; (C) HFF cells were incubated with 1～10μM SN-011 for 12 or 24 hours. After incubation, the expression of cGAS, STING, TBK1, IRF3 proteins were detected respectively; (D～E) HFF cells were incubated separately 1～10μM SN-011 12 or 24 hours, after incubation, the expression of cGAS (MB21D1) and STING (TMEM173) genes were detected respectively; all data are three parallel experiments and the average value is taken as the error bar: ns, no significant Sexual difference; *,P<0.05; **,P<0.01;

Figure 6 is a diagram showing the effect of compound SN-011 (compound of formula I) on inhibiting STING and the activation of downstream signaling pathways mediated by it: (A) 1μM SN-011 or SN-100 after incubating HFF cells overnight, use 2'3'- cGAMP 1μg stimulated for 1 hour. After the stimulation was completed, the cells were collected, and the phosphorylation and multimerization activation of STING protein were detected by Western blotting; (B) 1μM SN-011 or SN-100 incubated with HFF cells overnight, HSV-1 virus 40μl Infect the cells for 4 hours, collect the cells after the stimulation is completed, and use the immunoprecipitation method to detect the interaction between STING and its downstream proteins TBK1 and IRF3; (C) 1μM SN-011 or SN-100 after incubating HFF cells overnight, use 2'3' -cGAMP 1μg stimulation for 2 hours, harvest the cells, use the protein specific antibody immunoblotting method to detect the expression level of the corresponding protein; (D) 1μM SN-011 or SN-100 after incubating HFF cells overnight, use 2'3'-cGAMP After 2 hours of stimulation with 1μg, the cells were fixed, and the IRF3 protein was detected by immunofluorescence method into the nucleus. The nucleus position was indicated by DAPI staining. The length of the ruler was 25μm; (E) 1μM SN-011 incubated Hela cells overnight, HSV-1 virus 40μl Infect the cells for 3 hours. After the stimulation is completed, the transfer of STING protein to the Golgi apparatus is detected by immunofluorescence. The Golgi apparatus position is indicated by GM130 staining, and the length of the ruler is 25μm;

Figure 7 is a diagram of the co-crystal structure of compound SN-011 (compound of formula I) and hSTING-CTD (149-379) protein: (A) a schematic diagram of the co-crystal structure of SN-011 and hSTING-CTD (149-379) protein, The two monomer molecules in the dimer structure of the STING protein are represented by green and blue respectively, and the SN-011 molecule in the pocket formed by the dimer is represented by a rod-shaped skeleton structure; (B) SN-011/hSTING-CTD (149-379) Co-crystal structure (purple) and apo-hSTING-CTD (149-379) crystal structure (4EMU) (yellow) comparison diagram; (C) SN-011/hSTING-CTD (149-379) Schematic diagram of comparison between the co-crystal structure (purple) and the crystal structure (4LOH) (yellow) of 2'3'-cGAMP/hSTING-CTD(149-379); (D)SN-011/hSTING-CTD(149-379) The way the co-crystallization results are stacked in the crystal lattice; (E) Schematic diagram of the interaction between the amino acids on the protein and small molecules after the hSTING-CTD(149-379) protein is combined with SN-011;

Figure 8 is the verification diagram of the binding affinity and binding site of compound SN-011 (compound of formula I) with STING protein: (A～E) SPR method detects small molecules SN-011, SN-100 and 2'3'-cGAMP Binding curve with hSTING-CTD (149-379) protein, and detect the changes in the affinity between the protein and SN-011 molecules after the mutation of the key binding sites Ser241A, Ser243A and Thr263A; (F～G) Flag-hSTING will be expressed And its point mutants Tyr167, Ser241A, Ser243A, Glu260A and Thr263A plasmids were transfected into HEK293T cells with 5μg each. SN-011 (10μM) was added 12 hours after transfection to continue incubating for 12 hours. After the incubation was completed, the cells were collected to detect the IFNβ gene. Calculate the inhibition rate according to the gene expression value; (H～I) Transfect 5μg each of the plasmids expressing Flag-hSTING and its point mutants Tyr167, Ser241A, Ser243A, Glu260A and Thr263A into HEK293T cells and transfect them After 12 hours, add SN-011 (10μM) and continue to incubate for 9 hours. After incubation, add 2'3'-cGAMP 1μg to stimulate for 3 hours, collect cells to detect the expression of IFNβ gene, and calculate the inhibition rate based on the gene expression value; all data All three parallel experiments are averaged and the variance is used as the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

Figure 9 is a graph showing the effect of compound SN-011 (compound of formula I) on inhibiting the ^{high expression of IFNs and ISGs in Trex1-/-} mouse primary BMDM cells: (A) Heat map analysis SN-011 (500nM) after 12 hours of incubation, RNA-Sequencing sequencing results of gene expression in wild-type (WT) and Trex1 ^-/- mouse BMDM cells; a total of 72 ISG genes are displayed on the heat map, and two independent repeated experiments are selected for each treatment group; (B～E) Trex1 ^-/- BMDM cells were incubated with DMSO, SN-011 (500nM) or SN-100 (500nM) respectively, and the cells were collected after 12 hours of overnight incubation to detect the expression of Ifnβ, Cxcl10, Isg15 and Il6 genes; the gene expression of WT BMDM was The control group calculates ^{the growth factor of related gene expression in Trex1 -/-} BMDM cells; all data are the average of three parallel experiments and the variance is used as the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

Fig. 10 is a diagram showing the effect of compound SN-011 (compound of formula I) on reducing the ^{inflammation in multiple tissues and organs of Trex1 -/-} mice: (A～D) WT and Trex1 ^-/- mice were injected intraperitoneally with PBS or SN-011 ( 5mg/kg), 3 times a week after continuous injection for 1 month, the mouse heart, stomach, tongue and muscle tissues were taken to detect the expression of Ifnβ, Cxcl10, Isg15 and Il6 genes in the tissues; (E) Take the above tissues , After fixation, perform paraffin section and H&E staining to observe the inflammatory cell infiltration of each tissue. At least 6 mice in each group are counted. The average value of the experiment is taken and the variance is used as the error bar: ns, no significant difference; * ,P<0.05;**,P<0.01;

Figure 11 is a graph showing the effect of compound SN-011 (compound of formula I) on improving ^{systemic autoimmune symptoms in Trex1 -/-} mice: (A) WT and WT after injection of SN-011 (5 mg/kg) 1 month The spleen of Trex1 ^-/- mice. After spleen cells were separated, flow cytometry was used to detect activated T cells (CD4 ⁺ CD69 ⁺ , CD8 ⁺ CD69 ⁺ ) and memory T cells (CD4 ⁺ CD44 ^high CD62L ^low , CD8 ⁺ CD44) in the spleen ^high CD62L ^low) content; (B) statistical analysis of the experimental group versus mouse spleen cells with different subtypes of the content of T; (C) injection of SN-011 (5mg / kg) WT 1 month and after Trex1 ^{- /} -Statistical analysis chart of the survival rate of mice; (D) Take ^{the sera of WT and Trex1 -/-} mice 1 month after injection of SN-011, and use the anti-nuclear antibody detection kit immunofluorescence method to detect the mouse serum For the content of anti-nuclear antibodies in each group, the data of at least 6 mice in each group shall be counted. The average value of the experiment and the variance shall be the error bar: ns, no significant difference; *, P<0.05; **, P<0.01 ；

Figure 12 is a graph of the pharmacokinetic evaluation effect of compound SN-011 (compound of formula I) in mice: (A) A single intraperitoneal injection of SN-011 (5 mg/kg) in C57BL6 mouse plasma within 24 hours Drug concentration; (B) Statistical table of pharmacokinetic parameters, the blood drug concentration of 3 mice at each time point is taken for statistics;

Figure 13 is a diagram of the toxicological evaluation effect of compound SN-011 (compound of formula I) in mice: (A) WT mice were injected intraperitoneally with PBS or SN-011 (1 mg/kg), injected 3 times a week for 10 consecutive injections Figure of statistical analysis of body weight after one week; (B) statistical analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of the above-mentioned mice; (C) blood urea nitrogen (BUN) in the serum of the above-mentioned mouse, Statistical analysis chart of serum creatinine (CREA); (D) The mouse heart, kidney, stomach and liver were taken from the above-mentioned mice. After fixation, they were paraffin sectioned and H&E stained to observe the pathological changes of each tissue. Each group was at least 6 The data of only mice are counted, and the average value of the experiment is taken and the variance is used as the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

Figure 14 is a graph showing the effect of compound SN-011 (compound of formula I) on inhibiting the expression of inflammatory genes induced by SAVI-related STING point mutants: (A～C) HEK293T cells were transfected and expressed Flag-hSTING and SAVI-related point mutants V155M, N154S, G166E, C206Y, R281Q and R284G plasmids each 5μg, after 12 hours of transfection, incubate compound SN-011 (10μM), after compound incubation for 12 hours, collect cells to detect the expression of IFN-β, CXCL10 and TNF-α genes (D) HEK293T cells were transfected with 5μg plasmids expressing Flag-hSTING and SAVI-related point mutants V155M, G166E, C206Y, R281Q and R284G, 6 hours after transfection, incubate compound SN-011 (10μM), and incubate the compound After 12 hours, the cells were collected, and the recruitment of STING to the downstream target proteins TBK1 and IRF3 was detected by immunoprecipitation; (E) HEK293T cells were transfected to express Flag-hSTING and SAVI-related point mutants V155M, G166E, C206Y, R281Q and 5μg each of R284G plasmid, incubate compound SN-011 (10μM) after 6 hours of transfection, collect cells after 12 hours of compound incubation, and detect the phosphorylation expression of TBK1 and IRF3 protein by immunoblotting; (F) Transfection in HEK293T cells Plasmids expressing Flag-hSTING and SAVI-related point mutants V155M, G166E, C206Y, R281Q, and R284G are each 5 μg. After 6 hours of transfection, the compound SN-011 (10 μM) is incubated. After the compound is incubated for 12 hours, the cells are collected with non-denatured coagulation. Gel electrophoresis method to detect the multimerization expression of STING; all data are the average of three parallel experiments and the variance is used as the error bar: ns, no significant difference; *, P<0.05; **, P<0.01;

Figure 15 is a graph showing the effect of compound SN-011 (compound of formula I) on improving brain damage induced by acute cerebral ischemia in rats: (A) Middle cerebral artery occlusion (MCAO) induced acute cerebral ischemia in rats for 24 hours, using TCC The normal area of the infarct area of the brain detected by the staining method is red, and the infarct area is white; (B～C) Rat middle cerebral artery occlusion for 6 hours and 24 hours respectively to score the behavior of rats; (D～I) Brain Twenty-four hours after the middle artery occlusion induced acute cerebral ischemia in rats, the cerebral cortex tissues were taken to detect the expression of Ifnβ, Ifnα4, Cxcl10, Mcp1, Tnfα and Il6 genes; rats were injected into the abdominal cavity at the time of modeling and 12 hours after modeling. The experimental results of dose SN-011 (1mg/kg) and high dose SN-011 (3mg/kg), each group of 4 rats were statistically analyzed, ns, no significant difference; *, P<0.05; ** ,P<0.01, compared with the MCAO group in the administration group;

Figure 16 Compound SN-011 (compound of formula I) is a diagram of the effect of improving the brain injury of mice induced by acute cerebral ischemia: (A) The mouse is injected intraperitoneally with low dose SN-011 (1mg/kg) immediately after the middle cerebral artery is blocked. And high-dose SN-011 (2mg/kg), 24 hours later, take the cerebral cortex tissues to detect the expression of Mcp-1, Il6 and Tnfα genes; (B) The mice were intraperitoneally injected with low-dose SN immediately after the middle cerebral artery was blocked. -011 (1mg/kg) and high-dose SN-011 (2mg/kg). After 72 hours, tissues from the cerebral cortex were taken to detect the expression of Mcp-1, Il6 and Cxcl10 genes; (C) Middle cerebral artery occlusion in mice Immediately or 24 hours later, intraperitoneal injection of SN-011 (2mg/kg) was continued until the seventh day after the stroke. Rotary rod experiment was used to measure the changes in the motor function of the mice; (D) After the middle cerebral artery of the mice was blocked SN-011 (2mg/kg) was injected intraperitoneally immediately or 24 hours later, and the administration was continued until the seventh day after stroke. Morris water maze test was used to measure the changes in memory function of mice. The experimental results of no less than 5 mice in each group were statistically analyzed, n.s., no significant difference; *, P<0.05; **, P<0.01, compared the administration group with the vehicle group after MCAO;

Figure 17 is a graph showing the effect of compound SN-011 (compound of formula I) on improving the progression of non-alcoholic fatty liver in mice induced by high-fat diet (HFD): (A～B) NAFLD model in mice induced by high-fat diet and treatment Changes in serum ALT and AST contents of mice in each group after treatment; (C～D) contents of TC and TG in serum of mice in each group; (E) changes in body weight of mice in each group; (F) mice in each group Changes in liver weight; (G) Changes in visceral fat weight (perirenal fat) of mice in each group; (H～I) Contents of TC and TG in liver of mice in each group; (J～O) Livers of mice in each group Changes in Ifnβ, Cxcl10, Mcp-1, Tnfα, Fas and Srebp-1c gene expression in the middle of the blood; (P) H&E staining results analysis of liver pathological sections of mice in each group; C57BL6 mice were induced by high-fat diet for 20 weeks, starting from the 10th Intervention was performed by intraperitoneal injection of low-dose SN-011 (1mg/kg) and high-dose SN-011 (2mg/kg) starting from the first week. The experimental results of at least 8 mice in each group were statistically analyzed, and there was no significant difference in ns; *, P<0.05; **, P<0.01, comparison between the administration group and the HFD group;

Figure 18 is a graph showing the effect of compound SN-011 (compound of formula I) on the right ear and back of mice with imiquimod-induced psoriasis-like inflammation: control group, model group, SN-011 250mg/kg group and SN-011 The appearance of mice in the 50mg/kg group;

Figure 19 is a graph showing the effect of compound SN-011 (compound of formula I) on the ear thickness of mice with imiquimod-induced psoriasis-like inflammation: control group, model group, SN-011 250mg/kg group and SN-011 50mg/ Right ear thickness of mice in kg group, *P<0.05, **P<0.01, ***P<0.001, n=8;

Figure 20 is a graph showing the effect of compound SN-011 (compound of formula I) on the right ear and back of mice with imiquimod-induced psoriasis-like inflammation: A. H&E staining image of mouse ear skin; B. Mouse back skin H&E staining image, scale bar 50μm (x200); C. Take mouse ear skin for H&E staining, and analyze the thickness of spine, scale bar 20μm (x400); D. Take mouse back skin for H&E staining, and analyze the thickness of spine , Scale bar 20μm (x400), *p<0.05, **p<0.01,***p<0.001.n=8;

Figure 21 is a schematic diagram of the mechanism of compound SN-011 (compound of formula I) inhibiting the activation of the STING signaling pathway.

Detailed ways

The present invention will be further described below in conjunction with the drawings and embodiments. The present invention is not limited by these embodiments. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.

1. Chemical synthesis reagents and materials

The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained from the market.

The structure of the compound is determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS). The NMR measurement was performed with a (Bruker) nuclear magnetometer, and the measurement solvent was deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ), and the internal standard was tetramethylsilane (TMS).

Column chromatography generally uses 200-300 mesh silica gel from Qingdao Ocean Chemical Plant Branch as the carrier.

The known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from Leyan, Beide Pharmaceutical, Aladdin, Anaiji and other companies.

2. Cells and cell culture

The cell lines used in the present invention include human kidney embryonic cells HEK293T, mouse embryonic fibroblasts MEF and L929, human cervical cancer cells Hela, human primary foreskin fibroblasts HFF and the like. Unless otherwise specified, all cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco), and L929 was cultured in 1640 medium. Bone marrow-derived macrophages BMDM are differentiated from mouse femoral hematopoietic stem cells. The differentiation time is generally 7 days. The conditioned medium containing L929 supernatant is used for culture, and the corresponding stimulation can be carried out after the differentiation is completed.

3. Evaluation of mouse strains and in vivo pharmacodynamics, pharmacokinetics and toxicology

C57BL/6J mice aged 6 to 8 weeks were purchased from the Institute of Model Animals of Nanjing University, and Trex1 heterozygous mice were donated by Dr. Nan Yan (University of Texas Southwestern Medical Center). Homozygous knockout mice were obtained by crossing heterozygous mice (Cell Reports 2018, 25, 3405-3421). BALB/c mice (for anti-psoriasis drug efficacy test), female, 56-62 days old, provided by Zhejiang Weitong Lihua Experimental Animal Co., Ltd. In the experiment, rats and mice were kept in the SPF animal room of the Drug Safety Evaluation Center of China Pharmaceutical University. Animal experiments are carried out in strict accordance with the operating rules formulated by the Animal Management Committee of China Pharmaceutical University.

In the ^{spontaneous autoimmune disease model of Trex1 -/-} mice, the mice were intraperitoneally injected with the test compound SN-011 from the 4th week at a dose of 5 mg/kg mouse body weight, with a final concentration of 2% Tween20 and 2% DMSO promotes the dissolution of the compound, and the dissolving solution is PBS, injected three times a week for 30 consecutive days.

To evaluate the pharmacokinetics experiment of SN-011 in C57BL6J mice, after intraperitoneal injection of 5 mg/kg SN-011, the hourly plasma was taken at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours. For the detection of blood drug concentration.

Toxicological evaluation experiment of compound SN-011 in mice. C57BL6J mice were injected intraperitoneally with 1mg/kg SN-011 three times a week for 10 consecutive weeks. After the injection, the serum biochemical indicators and the pathological changes of tissues and organs in the mice were detected. .

In a rat model of acute cerebral ischemia induced by middle artery occlusion, male SD rats weighing 280-300 g were given compound SN-011 before and 12 hours after surgery, the low dose was 1 mg/kg, the high dose It is 3mg/kg. The relevant indicators were tested 24 hours after the operation. The rats in the experimental group were evaluated for behavioral scores at 6 hours and 24 hours after the operation. The scoring rules are as follows: grab the tail and bend it to the left for 1 point. Catch the tail on the ground and only make a circle to the left for 2 points, and release the tail to only make a circle to the left for 3 points. Paralysis of the left half of the body is 4 points.

In a mouse model of acute cerebral ischemia induced by middle artery occlusion, male C57/6J mice weighing 25-30g were taken immediately after surgery or given compound SN-011 for 24 hours after surgery, once a day for continuous administration To the end of the experiment, the low dose was 1 mg/kg and the high dose was 2 mg/kg.

In a mouse model of non-alcoholic fatty liver (NAFLD) induced by a high-fat diet (HFD), male C57BL6J mice aged 4-6 weeks were fed with high-fat diet (research diets, 60% Kcal High-Fat Diets, D12492) ). After 10 weeks of feeding on high-fat feed, the compound SN-011 was injected intraperitoneally, with a low dose of 1 mg/kg and a high dose of 2 mg/kg. The drug was administered three times a week for 10 weeks. The model group was given a PBS solution containing 2% Tween20 and 2% DMSO. After the completion of the 20th week, the relevant indicators were tested.

In the pharmacodynamic experiment of the compound to improve the psoriasis-like inflammation induced by imiquimod, there were 32 BALB/c mice, female, 56-62 days old. Raised in a normal environment, free to eat and drink. Preparation of compound SN-011 ointment: 250 mg/kg group: 1) Dissolve 560 mg of compound SN-011 in 1 ml of DMSO; 2) Add lanolin (11.2 g) and petroleum jelly (11.2 g) to 1 mL of ether, Dissolve by heating at 34°C; 3) Add the solution of 1) to the mixture of 2), then add 3ml of ethanol and stir evenly, and heat for several minutes; 4) Dissolve 0.05g of methyl paraben in 0.4ml of ether and Add 0.02g of Propyl Perginate to the mixture of 3), scrub with 200l of ether for 1-2 times and then add to the mixture; 5) Heat the mixture of 4) at 88°C for 60 minutes, distill off the ethanol and ether, and prepare Get ointment. 50mg/kg group: 1) Dissolve 112mg of compound SN-011 in 500l of DMSO; 2) Add lanolin (11.2g) and petrolatum (11.2g) to 1mL of ether, and heat to dissolve at 34°C; 3) Add the solution of 1) to the mixture of 2), then add 3ml of ethanol, stir evenly, and heat for several minutes; 4) Add 0.05g of methyl paraben and 0.02g of propylparaben dissolved in 0.4ml of ether to 3. In the mixture of ), scrub with 200 l ether for 1-2 times and then add to the mixture; 5) The mixture of 4) is heated at 88°C for 60 minutes, and the ethanol and ether are evaporated to obtain an ointment. Thirty-two BALB/c mice were randomly divided into blank control group (Control group), model control group (Model group), SN-011 250mg/kg group and SN-011 50mg/kg group according to their body weight, with 8 mice in each group. Use a shaver to remove hair on the back, exposing a 2cm×3cm skin area. 5% Imiquimod (IMQ) cream (local dose 62.5 mg) was administered daily to the right ear and back. From the first day to the fifth day of the experiment, the model was administered in the morning in the afternoon, and from the sixth day to the seventh day of the experiment, both in the morning and afternoon, and the model was made at noon. The experimental period was 7 days. Starting from the application of imiquimod cream to make the model, the mice were photographed every day and the right ear and back of the mice were observed. The thickness of the right ear of each mouse was measured every day. The skin specimens of the mouse back and right ear were soaked in 4% paraformaldehyde and embedded in paraffin. Stain with hematoxylin and eosin (H&E). Psoriasis lesion area and disease severity (PASI) score, right ear thickness, etc. were processed by graphpad prism 5 software, and multiple groups were processed by one-way analysis of variance (One-Way ANOVA).

4. Evaluation of mouse tissue sections and related pathology

Take mouse tissue sections and H&E staining to evaluate tissue damage and inflammatory cell infiltration. The specific experimental steps are as follows: 1) After the mouse dosing cycle is completed, take the various organs and tissues of the mouse and fix them with 4% PFA overnight for later use; 2) Take the material: take the fixed tissue according to the needs of the observation site. Carry out the corresponding trimming, put it into the embedding frame, gradient dehydration; 3) After dehydration, embed the tissue with paraffin to save the wax block of the tissue sample; 4) Repair section: After trimming the complete tissue section, you can slice it. The thickness of the slice is 5μm; 5) After placing the slice on the water, pick it up with a glass slide, and bake the slice at 56°C to dissolve the paraffin on the sample; 6) H&E staining after gradient rehydration, gradient dehydration, mounting and storage 7) Observe the pathological changes of the tissue under an upright microscope, just take pictures and record.

5. Detection of serum biochemical indicators

The biochemical indicators of mouse serum include ALT, AST, TC, TG, GLU, BUN and CREA. The above-mentioned indicators are tested by the Dimention X-Pand plus biochemical analyzer at the Center for Drug Safety Evaluation of China Pharmaceutical University.

6. Detection of triglyceride (TG) and total cholesterol (TC) content in mouse liver

The content of TC and TG in the liver of mice was tested using the TG content detection kit (solarbio, BC0625) and the TC content detection kit (solarbio, BC1985) in accordance with the instructions.

7. Detection of T cell content in spleen

Flow cytometry was used to detect the effect of SN-011 on the content ^{of CD4 +} and CD8 ⁺ T cells in the spleen ^{of Trex1 -/- mice.} The experimental procedures are as follows: 1) After passing the 200 mesh filter, the spleen cells are washed into the centrifuge tube with 10ml of PBS, resuspend the cells, and centrifuge at 1200rpm for 5 minutes; 2) Lyse the cells with 4ml of red blood cell lysate; 3) Block , Add 20μl of Fc blocking to each tube, block at 4℃ for 10 minutes, centrifuge at 1200rpm, wash once with PBS, and resuspend the cells in PBSA (1% BSA in PBS); 4) Antibody staining, 1:100 dilution of antibody into PBSA and mix well After mixing with cells, Antibody mix for T memory cells (CD4-FITC, CD8-PE, CD44-APC, CD62L-Brilliant Violet 421), Antibody mix for T activator cells (CD4-FITC, CD8-PE, CD69-APC) 5) After the antibody is added, mix well, incubate at 4°C for 30 minutes in the dark, add PBS and wash again, resuspend with 500μl of PBS, and then test the content of corresponding T cell subtypes on the machine (Attune NxT cytometer, Thermo Fisher scientific).

8. Detection of autoimmune antibodies in mouse serum

Use the ANA (HEp-2) antigen substrate slide kit (MBL-BION) detection kit to detect serum antinuclear antibodies (ANA). The method is as follows: 1) Take out the matrix sheet covered with HEp-2 cells from the kit and equilibrate at room temperature 2) Dilute the serum with 2% BSA in PBS 1:50 and incubate HEp-2 cells with it. After reacting at room temperature for 30 minutes, wash off the serum with PBS; 3) Use absorbent paper to absorb the water around the pores and add FITC Label the anti-mouse IgG secondary antibody, and wash off the secondary antibody with PBS after reacting for 30 minutes at room temperature; 4) Mount the slide with Dako anti-fluorescence quenching mounting tablet and check it under a microscope.

9. Molecular docking method to screen small molecule compounds that bind to STING protein

The hSTING-CTD crystal structure PDB:4EF5 is used for molecular docking screening, the molecular docking software uses DOCK3.7, and the virtual screening uses small molecule virtual database ZINC15 (http://zinc15.docking.org). The flexible docking program used in this experiment calculates the energy score of the molecular energy between the small molecule ligand and the protein receptor, that is, evaluates the van der Waals energy and electrostatic energy between the small molecule and the receptor by scoring. The experiment process is as follows: 1) Protein crystal structure: select the hSTING-CTD structure file 4KSY from the PDB database, delete the ligand present on the protein, remove the metal ions and solvent molecules in the structure under the Dock Pre framework and add to the structure Add the hydrogen atom and charge to complete the preparation of the receptor protein molecule; 2) Preparation of the small molecule of the ligand: After downloading the data containing the structure of the small molecule from ZINC15, use the Marvin software to hydrogenate the small molecule structure and use the Corina software to convert the small molecule. The molecule is transformed into a 3D space structure, and the energy state of the small molecule monomer is calculated with AMSOL software; 3) Upload the prepared ligand and protein crystal files to the computer server; 4) When the 2',3'-cGAMP is bound to the protein Grid generated in the pocket is used to evaluate the energy state in the space area; 5) Calculate the energy state of the grid point: AMBER is used to calculate van der Waals energy, QNIFFT is used to calculate electrostatic energy; 6) Virtual screening: the structure of small molecules in the database Do molecular docking in the pocket of the receptor grid, check the docking results, and judge the possibility of protein and compound binding.

10. Evaluation of cell viability

To detect the toxicity of compounds in cells, use Promega's Cell Proliferation Assay kit. The experimental process is as follows: 1) According to the speed of cell growth, different numbers of cells are put into the 96-well plate one day in advance; 2) When the cells grow to an appropriate density above 75%, incubate the corresponding compounds with different concentrations. 3) After the compound incubation is completed, 20 μl of MTS working solution is added to each well to react for 1 hour, and the absorbance value is detected at a wavelength of 490 nm and the influence of the compound on the cell survival rate is calculated.

11. Transfection of cells

There are two expression methods for transfecting the target plasmid into cells, calcium chloride-HEPES calcium transfection system and liposome transfection system. Calcium transfection is mainly for 293T cells, and liposome transfection mainly uses ISD and HT-DNA. Wait for the stimulus to transfer into the cytoplasm. The calcium transfer method is as follows: When the cells grow to a density of about 70%, start calcium transfer, mix the target plasmid with CaCl ₂ and add the HEPES solution by pipetting to mix it, then add dropwise to the cells, and transfect for 24 hours to make the plasmid in Intracellular expression. The method of liposome transfection is as follows: add the stimulus and lipo 2000 to the Opti-MEM solution at a ratio of 1:1, and then add the stimulus to the lipo2000 solution after standing for 5 minutes, mix and add it to the cells. Complete the transfection. After 6 hours of stimulation, the cells can be collected for subsequent experimental operations.

12. CDNs activate the intracellular STING signaling pathway

The cells grow adherently in the culture plate, and incubate the test compound with the corresponding concentration overnight. After the incubation is completed, use a _{buffer containing 50mM HEPES (pH=7.0), 100mM KCl, 3mM MgCl 2} , 0.1mM DTT, 85mM sucrose to dissolve the CDNs Stimulants, and (0.2% (m/v) BSA, 1mM ATP, 0.1mM GTP, 10μg/ml Digitonin), mixed with working solution to incubate the cell membrane, make CDNs molecules enter the cytoplasm, and then activate STING on the endoplasmic reticulum Proteins and CDNs stimulated the cells for 3 hours and then collected the cells to detect the expression of related genes.

13. Test the half inhibition rate (IC ₅₀ ) of the compound in the cell

HFF cells grow adherently in the culture plate. When the cells grow to 80% abundance, start the incubation of the compound, and incubate the test compound separately (the compound is dissolved in DMSO, and the final concentration of the compound in the medium: 1 and 10μM _{。IC 50} test concentration: 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.039μM) overnight, the blank control group only added DMSO. After the incubation is completed, the cells are treated with a digoxigenin solution containing a 2'3'-cGAMP stimulant (final concentration of 2'3'-cGAMP: 1μg/mL), so that the 2'3'-cGAMP molecule enters the cytoplasm and activates the internal STING protein on the plasma net, 2'3'-cGAMP stimulated the cells for 3 hours and then harvested the cells to detect the expression of the Ifnb gene. The inhibitory rate of the compound on the STING signaling pathway after stimulation of 2'3'-cGAMP at concentrations of 1 and 10 μM was calculated based on the Ifnb gene expression multiple: 1-[Ifnb (compound group)/Ifnb (DMSO group)]. The IC ₅₀ value is obtained by curve fitting the ratio of inhibition of Ifnb gene expression between the compound treatment group and the DMSO blank control group.

14. Plasmid construction

STING, TBK1, IRF3, cGAS related sequence information is queried in Genbank of NCBI. The plasmid cDNA is obtained from the thymus cDNA library by PCR and cloned into the corresponding eukaryotic expression vector to obtain the corresponding plasmid. All plasmid point mutants use QuickChange XL site-directed mutagenesis methods (Stratagene). The plasmid cloning method is as follows: 1) Design the cloning primer corresponding to the plasmid fragment, protect the base-enzyme digestion sequence-target primer (20-25bp); 2) Use DNA polymerase KOD to carry out PCR amplification of the target gene sequence; 3) The amplified DNA product is recovered by agarose gel; 4) the recovered product and the used vector are subjected to double enzyme digestion and ligation; 5) the ligation product is plated, the positive clones are picked, and the plasmid is extracted and sequenced to verify the correctness of the target gene sequence. Complete the cloning of the plasmid.

15. Detect the expression of the target protein by western blotting

The experimental procedure of western blotting is as follows: 1) Cells are lysed with protein lysis buffer (0.5% TritonX-100, 1mM EDTA, 1% Cocktail dissolved in TBS buffer) and centrifuged at high speed, and the supernatant protein lysis buffer is taken for detection For protein concentration, use the BCA method to detect protein concentration after detecting the protein concentration by using the BCA method to detect the protein concentration. Level the volume and concentration of the sample according to the protein concentration; 2) Add 5×loading buffer to the sample to prepare the sample, and run the SDS-PAGE of the corresponding concentration according to the molecular weight of the target protein Gel; 3) Transfer membrane, wet transfer the protein separated on the gel to PVDF membrane; 4) Block, block common protein with 5% skim milk, block phosphorylated protein with 5% BSA; 5) Incubate with primary antibody, The antibody is diluted according to the corresponding concentration and incubated at 4°C for 2 hours or overnight, and then washed 6 times with TBST buffer for 6 minutes each time to remove non-specific adsorption; 6) Incubate with the secondary antibody, and the secondary antibody of the corresponding attribute shall be in accordance with the corresponding concentration After incubating for 1 hour at room temperature after dilution, wash 6 times with TBST buffer for 6 minutes each time to wash off non-specific adsorption; 7) Color development, incubate with ECL luminescent solution and use Bio-Rad's ChemiDoc to detect the chemiluminescence strip of the protein With the expression of reactive protein.

16. Non-denaturing polyacrylamide gel electrophoresis analysis of protein dimerization and multimerization expression levels

The experimental method is as follows: 1) Configure a non-denaturing polyacrylamide gel of the corresponding concentration, and the gel does not contain SDS; 2) Put the configured non-denaturing gel in a buffer (25mM Tris, 192mM Glycine, pH8.4, inner tank Add 0.2% sodium deoxycholate) to pre-electrophoresis at 40mA for 30 minutes, balance the non-denaturing gel with the electrophoresis solution; 3) Use protein lysis buffer (0.5% TritonX-100, 1mM EDTA, 1% Cocktail in TBS) Buffer) After lysis, add 5×native loading buffer for sample preparation; 4) Electrophoresis conditions are 25mA constant current for 1-1.5 hours, and then follow the standard Western blotting method for detection.

17. Co-immunoprecipitation method to detect protein-protein interactions

The protein interaction is detected by co-immunoprecipitation (Co-IP) as follows: 1) Add protein lysate (0.5% TritonX-100, 1mM EDTA, 1% Cocktail dissolved in TBS buffer) to the cells, lyse and sonic The cell lysate was clarified and centrifuged at 13000 rpm at 4°C for 15 minutes to collect the supernatant protein solution; 2) To specifically identify the target protein, add 1 μl of the target protein antibody to the protein lysate, shake well at 4°C and incubate for 4 hours; 3) catch the purpose For protein, add 20μl of pre-prepared Protein G Agarose to the protein lysing solution, return to 4℃ and incubate for 2 hours to allow Beads to bind to the antibody. Use buffer (0.5% TritonX-100, 1mM EDTA) to wash away non-specific adsorbed proteins on agarose 5) Sample preparation, add protein 5×loading buffer to the tube, denature the protein at 95℃ for 6 minutes, and then detect the expression of the protein of interest according to the standard Western blot method to determine whether there is any interaction between the target protein and the protein of interest .

18. Immunofluorescence

The immunofluorescence experiment operation of the cells is as follows: 1) Spread the cells on a cover glass and grow to a suitable density; 2) After the cells are incubated and stimulated with the corresponding compound, 4% paraformaldehyde is added to fix at room temperature for 1 hour, and then washed twice with PBS Permeate the membrane with rupture buffer (0.25% Triton, 1mM EDTA dissolved in PBS) for 20-30 minutes; 3) Transfer the slide to the dark spot, add blocking solution (5% BSA dissolved in PBS) to block for at least 1 hour and incubate Load the primary antibody (diluted 1:1000 in PBST) for the corresponding detection protein at 4°C overnight; 4) After the primary antibody is incubated, add PBS to wash off non-specific adsorption and then add the fluorescently labeled secondary antibody, and incubate for 1 hour at room temperature in the dark; 5) After incubation, stain the cell nucleus with DAPI for 1 minute; 6) After nuclear staining, mount the slide with anti-fluorescence quencher, store in the dark, and observe the intracellular protein under the 63x NA1.4 oil microscope of the laser confocal microscope Carl Zeiss LSM700的Fluorescence distribution.

19. Prokaryotic expression and purification of His-hSTING-CTD (149-379) protein

The experimental methods for expressing the target protein fragment in E. coli are as follows: 1) Construct an expression vector plasmid containing the target protein fragment His-STING-149-379; 2) 1μg plasmid is transformed, transferred, and used at 18℃ with a final concentration of 0.5mM IPTG induces a large amount of protein expression in E. coli; 3) After harvesting the bacteria in buffer (25mM Tris8.0, 150mM NaCl, pH=8.0), sonicate the bacteria for 15 minutes until the bacterial solution is slightly clear; 4) High-speed centrifugation (18000rpm, 30 minutes), collect the upper protein solution; 5) Hang a 1.5ml Ni column to adsorb the target protein on the Ni column, and then wash off non-specific binding; 6) Over-gradient imidazole, 80mM, 250mM, 500mM gradient imidazole The protein is eluted from Ni-NTA Agarose; 7) Run SDS-PAGE gel and stain with Coomassie brilliant blue to detect the expression of the target protein; 8) Collect the target protein solution, use a 50KD ultrafiltration tube to concentrate the protein solution and then concentrate the protein solution Pass the Akata protein purification system to remove the contaminated protein and collect the target protein; 9) The His-STING-149-379 protein used in the crystallization experiment is pre-cut with TEV with His-tag tag, and then purified by a size exclusion chromatography column without tag. The target protein is concentrated to a concentration of 15mg/ml; 10) The purified target protein is concentrated to a suitable concentration, aliquoted, quick-frozen in liquid nitrogen, and stored at -80°C.

20. Co-crystallization of SN-011 and hSTING-CTD (149-379) protein and data processing and structure analysis

15mg/ml of STING-CTD (149-379) protein was dissolved in 25mM Tris-HCl, pH 7.5, 150mM NaCl solution, and 1μL of SN-011(50mM) was added to 100μL of protein solution. After incubating for 1 hour, Centrifuge at high speed to take the supernatant protein solution for crystallization. Use the hanging drop method to mix 1 μL of protein solution and 1 μL of pool solution (0.1M HEPS-NaOH, pH 7.0, 0.1M sodium formate, 25% PEG3350), and grow crystals in a 16 degree environment. After the crystals grow, use the The 30% PEG 3350 cryopreservation solution stores the collected crystals in liquid nitrogen. X-ray diffraction is performed at the Shanghai National Light Source Center. The collection, integration and processing of diffraction data use HKL3000 software, and use apo-STING (PDB: 4F5W) as a template to analyze the structure using a molecular replacement method. The co-crystal structure is adjusted using COOT software and the structure is refined using CCP4 software.

21. Surface plasmon resonance technology (SPR) detects the affinity between SN-011 and His-STING-CTD (149-379) protein

The SPR experiment temperature was 25°C, and the experiment instrument was Biacore T200SPR instrument (GE Healthcare). Purified 2μg/ml hSITNG-CTD protein and its mutants were fixed on the Sensor Chip CM5 (carboxymethylated dextran surface) chip using Amine Coupling Kit under the condition of ph=5.5. SN-01 gradient dilution (nM): 15.6, 7.81, 3.91, 1.85, 0.93 and cGAMP gradient dilution (nM): 125, 62.5, 31.3, 15.6, 7.81, flowing through the chip surface at a speed of ^{30 μl min -1, and combining solution} The departure time is all 90s. The buffers used in the experiment are all PBS containing 0.05% Tween 20. All data were repeated twice under the same conditions. The final affinity data was obtained by fitting a 1:1 binding model in the software Biacore T200 Evaluation software version 3.0 (GE Healthcare).

22. Rotarod test

Use XR1514 model rat and mouse rotary rod fatigue tester (purchased from Shanghai Xinruan Information Technology Co., Ltd.) to test, set the speed to 24r/min, the longest recording time is 5min, and all over 5min is registered as 5min. Train the mice 3 times before the formal experiment. The formal experiment is to record the movement time of the mouse on the rotating rod, and the average value of the three tests is the final result.

23, Morris water maze test

1) Put the mouse into the water with its head facing the pool wall, place the platform in the fourth quadrant, and put the mouse into the pool facing the pool wall from any point of the four starting points on the pool wall. Record the time(s) for the animal to find the underwater platform. In the first few training sessions, if this time exceeds 60s, guide the mouse to the platform. Let it stay on the platform for 10 seconds.

2) Remove the mouse and wipe dry. Each mouse enters the water from a different quadrant for training every day, with an interval of 15-20 minutes between the two trainings, and trains for 5 consecutive days.

3) On the second day after the last acquired training, the platform was removed and the 60s exploration training started. Place the mouse into the water from the opposite side of the original platform quadrant. Record the time spent by the mouse in the target quadrant (the quadrant where the platform was originally placed) and the ratio of the movement distance to the total time and movement distance, as a detection index of spatial memory;

24. RNA extraction and real-time fluorescence quantitative PCR technology to detect the expression of the target gene

The extraction process of total RNA in cells or tissues is as follows: 1) After taking appropriate amount of cells and tissues to fully lyse with TRIzol (Invitrogen), add chloroform to extract the RNA in the lysate 1:5, and centrifuge at 12000g for 15 minutes at 4°C; 2) Add the same volume of isopropanol to the upper aqueous solution to precipitate the RNA in the solution; 3) Use 1ml of 75% ethanol to remove impurities for the RNA precipitation; 4) After the RNA is dry and transparent, dissolve it with a suitable amount of DEPC water at 55°C, OD260 Check the RNA concentration. After the total RNA extraction is completed, it can be used for subsequent fluorescence quantitative PCR to detect the expression of the target gene. The experimental steps are as follows: RNA is mixed with the primer Oligo dT and transcribed into cDNA under the action of the reverse transcriptase kit. After the primers, DNA polymerase and the fluorescent dye FastStart Universal SYBR GREEN MASTER MIX (Roche) are mixed, they are amplified and detected in the ABI QuantStudio 3 instrument. GAPDH is the internal reference for gene expression, and the 2- ^ΔΔCT method is used for relative quantitative calculation of the target gene. expression. The primer sequences used to detect the target gene are shown in Table 1 below:

Table 1. qPCR primer sequences

25, RNA-Sequencing

After separating bone marrow cells from the femurs of WT or Trex1 ^-/- mice at 6-8 weeks, the cells were induced to differentiate into BMDMs using conditioned medium containing L929 supernatant. After induction, incubate SN-011 (500nM) for 12 hours according to the experimental group design, extract the RNA from the cells and integrate the RNA into the cDNA library according to the standard Illumina RNA-seq protocol. The generated cDNA library was sequenced with Illumina HiSeq2000 in a 1×100bp run. After the sequencing results were processed, they were compared with the mouse genome, and heat maps were performed to reflect the differences in gene expression and further cluster analysis.

Example 1

N-(3-((4-Fluorophenyl)sulfonamido)-4-hydroxyphenyl)-[1,1'-biphenyl]-4-carboxamide (Compound I)

Compound 1 (2.00g, 13.0mmol) and pyridine (1.54g, 19.5mmol, 1.57mL) were dissolved in 40mL of dichloromethane, and 4-fluoro dissolved in 20mL of dichloromethane was added dropwise at 0°C. Benzenesulfonyl chloride 1A (3.04g, 15.68mmol), mixed and stirred at 25°C for 12 hours. Subsequently, the reaction was identified as complete by thin layer chromatography (TLC), and the mobile phase was petroleum ether: ethyl acetate = 2:1. The solvent was evaporated under reduced pressure, and the crude product was purified by silica gel chromatography (mobile phase is petroleum ether: ethyl acetate = 10:1～2:1, V/V) to obtain compound 2 as a pale yellow solid product (2.74g, 67.6% yield). ¹ H NMR:(DMSO-d ₆ ,400MHz)δ8.04(d,J=2.4Hz,1H),7.94～7.92(m,1H),7.83～7.80(m,2H),7.41～7.37(m, 2H), 6.89(d,J=8.8Hz,1H).LCMS(m/z): 312.02[M+H] ⁺ .

Compound 2 (1.50 g, 4.80 mmol) was dissolved in 15 mL of methanol, 10% Pd/C (0.1 g) was added, and the reaction was carried out under a hydrogen atmosphere at 25° C. for 16 hours. The reaction product was identified by LC-MS to determine that the reaction was complete. The reaction solution was filtered with a celite packed column, and the solvent was removed under reduced pressure to obtain 1.4 g of compound 3 as a yellow solid product. ¹ H NMR: (DMSO-d ₆ ,400MHz)δ8.44(br.s,1H), 7.81～7.77(m,2H), 7.37～7.33(m,2H), 6.51(d,J=2.4Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H), 6.19 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 4.60 (br.s, 1H), 3.17 (s, 1H).LCMS( m/z):282.05[M+H] ⁺ .

990 mg of compound 3 was dissolved in 10 mL of dichloromethane, then 229 mg of imidazole was added and 507 mg of TBSCl (tert-butyldimethylchlorosilane) dissolved in 1 mL of dichloromethane was added dropwise. The reaction was stirred at 15°C for 12 hours, and the reaction product was identified by LC-MS to judge that the reaction was complete. The reaction solution was washed with water (20 mL x 3), dried over anhydrous Na ₂ SO ₄ , and the solvent was evaporated under reduced pressure. The crude product was _{purified by a chromatographic column packed with Al 2} O ₃ (mobile phase was dichloromethane:methanol=10:1, V/V) to obtain compound 4 as a reddish brown solid. ¹ H NMR: (CDCl ₃ ,400MHz)δ7.78～7.75(m,2H),7.10～7.06(m,2H),6.97(d,J=2.8Hz,1H),6.78(s,1H),6.52 (d,J=8.4Hz,1H),6.28(dd,J1=2.8Hz,J2=8.8Hz,1H),3.48(s,2H),0.93(s,9H),0.08(s,6H).LCMS (m/z):396.13[M+H] ⁺ .

In 5mL of dichloromethane was added 185mg of compound 4A, 215mg of EDCl (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), 139mg HOBt (1-hydroxybenzo three Azole) and 181 mg of DIPEA (N,N-diisopropylethylamine), and finally 370 mg of compound 4 was added. The reaction was stirred at 15°C for 16 hours, and the reaction product was identified by LC-MS to judge that the reaction was complete. The reaction product was diluted with 10 mL of dichloromethane, washed with water (20 mL x 3), dried with anhydrous Na ₂ SO ₄ , and the solvent was evaporated under reduced pressure to obtain 400 mg of red-brown solid crude product compound 5, which was directly unpurified For subsequent reactions.

630 mg of compound 5 and 176 mg of Et ₃ N.3HF were added to 15 mL of dichloromethane, and the reaction was stirred at 15° C. for 3 hours. The reaction product was identified by LC-MS to determine that the reaction was complete. The reaction solution was diluted with 25 mL of dichloromethane, washed with water (20 mL x 2), dried over anhydrous Na ₂ SO ₄ , and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (mobile phase: petroleum ether: ethyl acetate = 5:1) to obtain off-white solid compound I (260 mg, 51.5% yield). ¹ H NMR: (DMSO-d ₆ ,400MHz)δ10.13(s,1H),9.38(s,2H),8.03(d,J=8.4Hz,2H),7.83～7.71(m,7H),7.51 ～7.35(m,6H),6.70(d,J=8.8Hz,1H).LCMS(m/z):462.10[M+H] ⁺ .

Example 2

N-(3-((4-Fluorophenyl)sulfonylamino)-4-methoxyphenyl)-[1,1'-biphenyl]-4-carboxamide (Compound II)

Compound 6 (0.54g, 3.2mmol), pyridine (0.5mL) were added to dichloromethane (10mL), and 4-fluorobenzenesulfonyl chloride (1A: 0.744g, 3.84mmol) in dichloromethane ( 10mL) solution, after dripping, react at room temperature for 8h. The solvent was evaporated under reduced pressure, ethyl acetate (50mL) and water (20mL) were added, and the solution was shaken. The organic phase was washed with 1N HCl (10mL), water (20mL) and saturated brine (20mL), anhydrous Na ₂ SO _{4 was} dried, filtered, and the solvent was evaporated under reduced pressure to obtain the residue. The crude product was purified by silica gel column chromatography (mobile phase: petroleum ether: ethyl acetate = 10:1～2:1, V/V) to give a pale yellow The solid product compound 7 (0.74 g, 71%).

Compound 7 (0.5 g, 1.53 mmol), 10% Pd/C (50 mg) was added to MeOH (20 mL), and _{reacted in H 2} atmosphere for 12 h. The Pd/C was filtered off, the filter cake was washed with MeOH, and the filtrate was evaporated to remove the solvent under reduced pressure to obtain compound 8 (0.43 g) as a yellow-brown solid product.

Compound 4A (119mg, 0.60mmol) and DIPEA (0.16mL, 0.96mmol) were added to THF (4mL), HATU(O-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethylurea hexafluorophosphate) (291 mg, 0.77 mmol), after reacting at room temperature for 1 h, compound 8 (237 mg, 0.80 mmol) was added, and reacting at room temperature for 5 h. Add ethyl acetate (20mL) and water (10mL), separate the layers, wash the organic phase with 1N HCl (5mL), water (10mL) and saturated brine (10mL), dry with anhydrous Na ₂ SO ₄ , filter, and reduce The solvent was removed by pressure evaporation to obtain the residue, which was purified by silica gel column chromatography (mobile phase: petroleum ether: ethyl acetate = 5:1) to obtain an off-white solid compound II (117 mg, 41%). ¹ H NMR: (DMSO-d ₆ ,400MHz)δ10.25(s,1H),9.59(s,1H),8.15(d,J=8.4Hz,2H),7.87～7.73(m,7H),7.65 ～7.35(m,6H),6.88(d,J=8.8Hz,1H), 3.48(s,3H).LCMS(m/z):477.5[M+H] ⁺ .

Example 3

4-([1,1'-biphenyl]-4-carboxamido)-2-((4-fluorophenyl)sulfonamido)phenyl acetate (Compound V)

The compound I (200 mg, 0.43 mmol) prepared in Example 1 was dissolved in THF (5 mL), pyridine (52 μL, 0.65 mmol) was added, acetyl chloride (37 μL, 0.52 mmol) was added dropwise at room temperature, and the reaction was carried out at room temperature for 10 hours. It was quenched with water (10 mL), extracted with EtOAc (30 mL), and the organic phase was washed with 1N HCl (10 mL), water (10 mL) and saturated brine (10 mL), dried over anhydrous Na ₂ SO ₄ , filtered, and evaporated under reduced pressure. Solvent to obtain a white solid, which was purified by silica gel column chromatography (DCM/MeOH=10/1) to obtain a white solid, Compound V (76 mg, 35%). ¹ H NMR(300MHz,DMSO-d ₆ )δppm 10.39(s,1H),10.01(s,1H),8.06(d,J=8.3Hz,2H),7.96(d,J=2.2Hz,1H), 7.86-7.8 (m, 4H), 7.77 (d, J = 7.4 Hz, 2H), 7.62 (dd, J = 8.8, 2.3 Hz, 1H), 7.52 (dd, J = 7.4, 7.4 Hz, 2H), 7.44 (d,J=5.3Hz,1H),7.40(dd,J=7.9,7.9Hz,2H),7.01(d,J=8.8Hz,1H),2.14(s,3H).ESI-MS: m/ z 461.1[M-CH ₃ CO] ^- .

Example 4

4'-((3-((4-Fluorophenyl)sulfonamido)-4-methoxyphenyl)carbamoylcarboxamido)-[1,1'-biphenyl]-4-ylacetic acid Ester (Compound III)

Refer to the method of Example 2 to prepare compound III: ESI-MS: m/z 491.3[M-CH ₃ CO] ^- .

Example 5

N-(4-Methoxy-3-(benzenesulfonamido)phenyl)-[1,1'-biphenyl]-4-carboxamide (Compound IV)

Refer to the method of Example 2 to prepare compound IV: ESI-MS: m/z 459.1 [M+H] ⁺ .

Example 6

Virtual screening and in vitro activity evaluation based on the crystal structure of STING protein

The present invention is based on the analysis of the crystal structure of the C-terminal domain of the human STING protein (hSTING-CTD-139-379, PDB: 4EF5), and carries out computer simulation to screen potential small molecules that bind to STING. Use DOCK3.7 software to screen small molecules in the virtual compound database of ZINC15 (http://zinc15.docking.org) with hSTING-CTD structures. After the screening, a total of 152 small molecules respond to the binding of hSTING-CTD. Through analysis and screening of small molecule compounds, it is found that a class of small molecule compounds containing 1-sulfa-3-amide-benzene ring skeleton structure has a good response to the hSTING-CTD structure. On this basis, a small molecule compound with this characteristic structure was purchased from EnamineStore for in vitro biological activity evaluation. The results showed that in MEF cells or L929 cells induced and stimulated by DNA analogues such as ISD or HT-DNA, the compound of formula I (SN-011), compound of formula II (SN-001), compound of formula III (SN-005), The compound of formula IV (SN-006) and compound of formula V (SN-010) can significantly inhibit the expression of Ifnb and other immune-inflammatory factor genes (Figure 1A, Figure 2A ~ 2H, Figure 3A, Figure 3C, Figure 3E), and do not affect The expression of Ifnb gene in resting state (Figure 1B). The compound SN-100 (CAS:1384744-19-7) does not affect the expression of IFN-β gene in MEF cells induced by ISD. Therefore, this compound was selected as a negative control compound for subsequent experiments. In addition, in L929 cells stimulated by HT-DNA, SN-001 significantly inhibited the phosphorylation expression of STING, TBK1, IRF3, P65, IκBα and the dimerization of IRF3 protein (Figure 2I), followed by the import of transcription factors IRF3 and P65. The nucleus was also significantly reduced after incubating the compound (Figures 2J～2K), and the negative control compound SN-100 did not affect the phosphorylation expression of the above-mentioned protein and the incorporation of transcription factors into the nucleus. In order to exclude the influence of SN-001's cytotoxicity on its biological activity, the MTS method was used to detect the effect of the compound on cell viability in MEF, L929 and THP-1 cells. The results showed that SN-001 (5μM～20μM) was incubated to It did not affect cell viability for 24 hours (Figure 2L～2N). Figure 1C and Figure 3B show the structures of some of the compounds purchased after passing the virtual screening. It should be noted that the compounds such as ZINC72311784 in Figures 1A and 1B are all commercially available compounds (the structures of some compounds are not shown). The CAS numbers of some of the compounds in Figures 1 and 3 are as follows: SN-001/ZINC08686914 (CAS:727699-84-5), SN-003/ZINC15418850 (CAS:1385921-05-0), SN-004/ZINC00991157 (CAS: 681834-84-4), SN-007 (CAS: 568569-75-5), SN-008 (CAS: 2249106-01-0), SN-100/ZINC78992473 (CAS: 1384744-19-7).

In MEF cells, the classical agonists of STING signaling pathway ISD, HT-DNA, HSV-1, c-di-GMP and 2'3'-cGAMP are used for stimulation, and the pre-incubation of compound SN-011 (compound of formula I) can Significantly down-regulate the expression of Ifnb, Cxcl10 and Il-6 genes induced by the above stimuli (Figure 3C ~ 3E).

The in vitro biological activity of the compound was _{further evaluated by evaluating the half inhibition rate (IC 50} ) of SN-011 (compound of formula I) in different cells. In MEF (mouse embryonic fibroblasts), BMDM (mouse bone marrow primary macrophages) and HFF (human primary foreskin fibroblasts), the IC ₅₀ values of SN-011 are 127.5±5.5nM and 121.7, respectively ±32.2nM and 502.8±94.5nM (Figure 4A-4C). SN-011 also has good safety in vitro. It is specifically shown that incubating the compound in MEF, BMDM and HFF cells up to a concentration of 20μM for 48 hours, and none of them showed any effect on cell viability (Figure 4D～4F) . In STING knockout MEF cells, pre-incubation with SN-011 does not affect the IFR3 and NF-κB downstream genes Ifnb, Tnf-α and Il-6 activated by LPS, CpG-DNA, poly(I:C) and Sendai Virus The expression of (Figure 5A ~ 5B). In addition, pre-incubation with SN-011 did not affect Ifnβ's activation of the expression of Isg15 and Isg56 genes in STING knockout MEF cells (Figure 5B).

The above results indicate that the compounds of formula I to V of the present invention can efficiently, safely and specifically inhibit the activation of the cGAS-STING signal pathway.

Example 7

SN-011 (compound of formula I) inhibits the activation of STING protein and its downstream signal transduction mediated by it

In the resting state, the STING protein is anchored in the endoplasmic reticulum in the form of a homodimer with its N-terminal transmembrane region (amino acid 1-137). When STING and its ligand CDNs molecules such as 2',3'-cGAMP After binding, the spatial conformation of the STING dimer changes, which is specifically expressed as the C-terminal structure (amino acid 138-379) rotates 180° with respect to the transmembrane region, and multiple dimer molecules are arranged side by side. Tetrameric and higher multimeric forms (Nature, 2019, 567(7748):389-393). The multimerized STING protein is transferred from the endoplasmic reticulum to the Golgi apparatus. In this process, the STING multimer recruits the kinase protein TBK1 with the PLPLRT/SD sequence of its C-terminal region and promotes the autophosphorylation activation of TBK1 (Ser172) (Nature , 2019, 567(7748): 394-398; Nature, 2019, 569(7758): 718-722). Activated TBK1 further phosphorylates the serine in the pLxIS366 sequence of the STING polymer, thereby promoting the recruitment of the transcription factor IRF3. IRF3 is recruited into the polymer complex and further activated by phosphorylation of TBK1 (Science, 2015, 347(6227): aaa2630 Proceedings of the National Academy of Sciences, 2016,113(24):E3403-E3412), this is the activation of protein-level STING and the process of mediating downstream signal transmission. Based on this, the present invention found that pre-incubation with SN-011 can significantly inhibit the multimerization and phosphorylation of STING in HFF cells induced by 2',3'-cGAMP (Figure 6A) and its transfer to the Golgi apparatus (Figure 6A). Figure 6E). In addition, SN-011 can significantly reduce the recruitment of STING protein to downstream TBK1 and IRF3 (Figure 6B), which is consistent with 2',3'-cGAMP-induced phosphorylation of TBK1, IRF3, P65 and IκBα proteins and IRF3 The level of dimerization (Figure 6C) and the nucleation of IRF3 were significantly reduced after HFF cells were pre-incubated with SN-011 (Figure 6D). In contrast, the negative control compound SN-100 did not affect the changes in the above-mentioned protein levels. As a control, we further tested the effect of SN-011 on the stability of cGAS, STING, TBK1 and IRF3 proteins in the resting state of cells, and the results showed that SN-011 did not affect the ground state expression of the above-mentioned proteins in HFF cells (Figure 5C), and The expression of cGAS gene (MB21D1) and STING gene (TMEM173) (Figure 5D ~ 5E). This indicates that the mechanism of action of SN-011 is to directly affect the function of the protein rather than the stability of the STING signaling pathway protein. The above studies show that SN-011 can inhibit the activation of STING protein in cells and the downstream signal transduction mediated by it.

Example 8

Co-crystal structure of SN-011 (compound of formula I) and STING-CTD (149-379) protein

In order to study the structural basis of the binding between compound SN-011 and STING protein, the present invention obtained and analyzed the co-crystal structure of SN-011 and STING-CTD (149-379) protein, with a resolution of

The invisible C-terminal region (amino acid 341-379) in the co-crystal structure may be due to the steric instability of the structure. In the co-crystal structure, each corresponding structural unit contains a homodimer formed by two STING protein monomers and two SN-011 molecules bound in the binding pocket of protein dimer CDNs (Figure 7A). From the structural point of view, the SN-011 molecule binds to the surface of the STING dimer in anti-parallel, the biphenyl ring on the small molecule structure is bound to the bottom of the dimer pocket, and the 4-fluoro-benzenesulfonamide group extends to the protein two. The top of the polymer pocket. Similar to the crystal structure of apo-STING-CTD protein, STING-CTD (149-379) and SN-011 still maintain a "V"-shaped dimer structure after binding, and the distance of His185 amino acids on both sides of the pocket is

(Figure 7B). In contrast, after STING-CTD protein binds 2'3'-cGAMP and c-di-AMP, the monomers on both sides of its pocket will approach each other under the traction of small molecules, making the protein dimer pocket closed. "U" shape and the distance of His185 amino acids on both sides of the pocket is shortened to

(Figure 7C) (Cell, 2019, 178:1-12). Subsequently, we further analyzed the way the STING-CTD protein was stacked in the crystal lattice after SN-011 was combined. The co-crystal structure was stacked in the crystal lattice in a similar "head-to-tail correspondence" manner (Figure 7D), and this type of stacking also appeared. In the unactivated STING structure (ACS Med Chem Lett, 2019, 10(1): 92-97), and this stacking method is significantly different from the STING-CTD protein after the combination of 2'3'-cGAMP and c-di-AMP The stacking method of "side by side" in the lattice (Nature, 2019, 567(7748): 389-393). In summary, after binding to the SN-011 molecule, the spatial conformation of the STING protein did not change significantly, and it still maintained its dimer conformation in the resting state.

Further structural analysis showed that SN-011 is stabilized in the pocket by forming hydrogen bonds and stacking with specific amino acids in the STING protein dimer pocket. The biphenyl ring in the structure of SN-011 forms a π-π stacking effect with the benzene ring of the amino acid side chain of Tyr167 of the protein. The benzene ring connected to the phenolic hydroxyl group in the SN-011 structure can also form a conjugate or hydrogen bond with the surrounding Glu260 and Ser241 amino acids for stability. The phenolic hydroxyl and sulfonamide bonds in SN-011 can further form hydrogen bonds with the hydroxyl on the side chain of Ser243 amino acid (Figure 7E). Based on this, the co-crystal structure of SN-011 and STING protein analyzed by the present invention not only clarifies the position of the compound in the protein, but also provides information on the way of interaction between the two and the binding site.

The above results indicate that the compound of the present invention can inhibit the activation of the STING signal pathway by directly binding to the STING protein and maintaining its dimer conformation in a resting state.

Example 9

Mutations in the amino acid sites that bind to SN-011 (compounds of formula I) affect the binding of small molecules and their biological activities

In order to further determine the effect of the amino acid sites analyzed by the co-crystal structure on the binding of SN-011, surface plasmon resonance (Surface Plasmon Resonance, SPR) was used to detect the effect of mutations of related amino acid sites on the affinity between small molecules and proteins. SPR test results show that the affinity between SN-011 and hSTING-CTD protein is about 4.03nM, which is better than the binding affinity of 2'3'-cGAMP (Kd～9.23nM). It is worth noting that the single mutant S243A and triple mutant proteins S241A, S243A and T263A of the hSTING-CTD binding site significantly reduced the interaction between hSTING-CTD and SN-011, with Kd of 176.2nM and 1.36μM, respectively (Fig. 8A～8E). hSTING-CTD-Y167A and hSTING-CTD-E260A cannot be purified in Escherichia coli due to solubility problems, so the effect of Y167 and E260 on the affinity between small molecules and proteins cannot be evaluated in this experimental system.

Subsequently, the hSTING protein and its related point mutant proteins were overexpressed in HEK293T cells to evaluate the effect of the binding site mutation on the biological activity of SN-011. The experimental results show that compared with wild-type hSTING, SN-011 has a significantly lower inhibitory activity on the IFN-β gene expression induced by its point mutants S241A, S243A, T263A and 3A, and under 2'3'-cGAMP stimulation conditions Mutation of the above amino acid sites can also weaken the inhibitory activity of SN-011 (Figures 8F-8I). The hSTING-Y167A and hSTING-E260A mutants cannot activate downstream IFN-β gene expression after overexpression in HEK293T cells, nor do they respond to exogenous 2'3'-cGAMP stimulation (Figure 8G, 8I), so this cannot be evaluated. The effect of two site mutations on the biological activity of SN-011. The above research results indicate that the compound of the present invention binds to the STING protein through several key amino acid residues (Tyr167, Glu260, Ser241 and Ser243), thereby inhibiting the activation of the STING signal pathway. Therefore, the benzenesulfonamide compounds represented by formulas I to V of the present invention or their pharmaceutically acceptable salts or solvates can be used to prepare drugs that inhibit the activation of the STING signal pathway.

Example 10

SN-011 (compound of formula I) improves systemic inflammatory damage in ^{TREX1 -/- autoimmune disease mice}

TREX1 acts as an exonuclease in the cytoplasm to degrade abnormal ssDNA in the cytoplasm. When its gene mutation causes the protein to lose its normal enzyme function, the DNA accumulated in the cytoplasm can activate the cytoplasmic cGAS-STING signaling pathway and induce The expression of type I IFNs promotes the occurrence of cell inflammation. It has been found in animal models that knocking out the expression of mouse cGAS or STING protein can significantly improve the degree of inflammation in various tissues and organs induced by TREX1 mutation and the lethality of the disease (Nat Genet, 2007, 39(9): 1065-7; Cell, 2008, 134(4):587-98). Based on this, the present invention evaluates the potential pharmacodynamic activity of SN-011 on systemic inflammatory damage ^{in Trex1-/- mice.} First, after ^{incubating compound SN-011 in Trex1 -/-} mouse bone marrow-derived macrophages (BMDMs), RNA-Seq technology was used to analyze the intracellular mRNA transcripts. The experimental results showed that: (1) in the wild In WT mouse BMDMs, incubation of SN-011 did not significantly affect the expression of ISGs in cells at rest; (2) Compared with WT BMDMs cells, the expression level of ISGs in ^{Trex1 -/- BMDMs cells increased significantly.} High, and can significantly inhibit the expression of related ISGs genes after incubating with SN-011; (3) Further analysis of the changes in cell transcript gene expression found that SN-011 mainly affects the expression of ISGs genes in the cell, which explains from the side The specificity of the compound's action (Figure 9A). Subsequently, qPCR technology was used to detect the expression of Ifnb, Cxcl10, Isg15 and IL-6 genes in the cells. The test results also further verified the ^{inhibitory effect of compound SN-011 on the expression of ISGs in Trex1 -/-} BMDMs cells (Figure 9B～ 9E).

On the basis of in vitro studies, SN-011 (5mg/kg) was intraperitoneally injected into Trex1 ^-/- mice, and the inflammatory infiltration of the mouse heart, stomach, tongue and muscle was detected after the administration for 1 month. The results show that: (1) Compared with wild-type (WT) mice, Trex 1 ^-/- mice have significant immune (Ifnβ, Cxcl10, Isg15) and inflammatory factor (Il6) genes in the heart, stomach, tongue and muscles (2) Administration of compound SN-011 in wild-type mice does not affect the expression of immune and inflammatory factor genes in the above-mentioned tissues; (3) Compared with the autoimmune disease model group of ^{Trex 1 -/-, administration of compound} SN-011 can significantly reduce the immune (Ifnβ, Cxcl10, Isg15) and inflammatory factor (Il6) gene expression in the heart, stomach, tongue and muscle of ^{Trex 1 -/- mice (Figures 10A-10D).} Further pathological sections were performed on the above-mentioned tissues and organs to evaluate the inflammatory damage of the tissues, and the results showed that SN-011 can significantly improve the inflammatory damage and immune cell infiltration in ^{Trex 1 -/- mouse tissues (Figure 10E).} The spleens of mice in the experimental group were taken to disperse spleen cells and stained with T cell antibodies. Flow cytometry was used to detect the content of T cell subtypes. The test results showed that compared with the control Trex1 ^-/- mice, intraperitoneal injection of compound SN-011 can significantly reduce activated CD8T (CD69 ⁺ ) cells, memory CD4T (CD62L ^low CD44 ^high ) cells and CD8 (CD62L ^low CD44) ^{The content of high} T cells in the ^{spleen of Trex1 -/-} mice does not affect the content of activated CD4T (CD69 ⁺ ) cells (Figures 11A to 11B). In addition, the administration of SN-011 Trex 1 ^{- / -} mouse serum anti-nuclear antibody is significantly reduced (FIG. 11D), 1 month and continuous administration significantly prolonged Trex1 ^{- / -} survival (FIG. 11C mice ). The above research results show that SN-011 can improve ^{the systemic inflammatory damage of TREX1 -/-} autoimmune disease mice and prevent the progression of the disease. In addition, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006) and the compound of formula V (SN-010) all have similarities with the compound of formula I (SN-011). The effect.

In summary, the compounds of formula I to V of the present invention can be used to prepare drugs for preventing or treating STING-mediated autoimmune diseases and inflammatory diseases.

Example 11

Study on the pharmacokinetics of SN-011 (compound of formula I) in mice

After a single intraperitoneal injection of compound SN-011 (5mg/kg) in male C57BL6J mice, blood was collected at different time points within 24 hours, and then the blood drug concentration in plasma was detected by LC-MS/MS and calculated Related pharmacokinetic parameters. _{The experimental results show that the half-life T 1/2} of the drug in the plasma after intraperitoneal injection of SN-011 in mice is 1.03 hours, the maximum blood concentration Cmax is 837.02 ng/mL, and the time required to reach the peak concentration of the drug T _max is 0.25 hours , The area under the plasma concentration-time curve, AUC, was 850 ng/mL (Figures 12A-12B).

Example 12

Toxicology evaluation of SN-011 (compound of formula I) in mice

In order to evaluate the potential toxicity of compound SN-011 after long-term administration in mice, we injected SN-011 (1mg/kg) into the abdominal cavity of male C57BL6J mice three times a week for 10 weeks, and the administration was completed Afterwards, the changes of organs and serum biochemical indexes in mice were detected. The experimental results showed that the body weight of the mice in the control group and the administration group did not change significantly within 10 weeks of administration (Figure 13A), and the ALT and AST indicators in the mouse serum that reflect liver injury did not increase significantly after the administration (Figure 13A). 13B). In addition, serum indexes BUN and CREA, which reflect kidney damage, did not change significantly after administration (Figure 13C). Pathological sections were made on the heart, kidney, stomach and liver of mice, and the results showed that none of the above-mentioned organs had obvious substantial damage after 10 weeks of administration (Figure 13D). The above results show that the compound SN-011 has good safety in mice, and it does not show toxic side effects in mice after long-term administration.

Example 13

SN-011 (compound of formula I) inhibits the expression of inflammatory cytokines induced by SAVI-related STING point mutations

The gene encoding STING protein TMEM173 is a type of autoimmune disease induced by mutations in the human body, that is, STING-associated vasculopathy with onset in infancy (SAVI) occurs in infancy. The clinical manifestations of SAVI patients are mainly early onset in infancy, skin rash, shortness of breath, fever and other systemic inflammation, peripheral vascular disease, lung inflammation, and the presence of autoimmune antibodies in the blood. Subsequent gene sequencing results found that multiple sites of the TMEM173 gene of such patients were mutated to cause self-activation of the protein. Mutants include N154S, V155M, G166E, C206Y, R281Q and R284G (J Allergy Clin Immunol, 2017, 140 (2): 543-552; Ann Rheum Dis, 2017, 76(2): 468-472). The present invention constructs the plasmid of the above-mentioned point mutant by cloning method, overexpresses the above-mentioned mutant in HEK293T cells (without STING protein expression), and then studies the effect of compound SN-011 on the activation of STING signal pathway induced by the above-mentioned mutant . The experimental results show that pre-incubating cells with SN-011 can significantly inhibit the expression of Ifnb, Cxcl10 and Tnfa genes induced by the above mutants (Figures 14A-14C). Pre-incubating SN-011 at the protein level can also attenuate the recruitment of over-expressed STING point mutants to downstream target proteins TBK1 and IRF3 and reduce the expression levels of P-TBK1 and P-IRF3 (Figure 14D-14E). Recent studies have shown that SAVI-related STING point mutants can release the self-inhibitory morphology of STING dimers to activate protein multimerization independent of ligand binding (Nature, 2019, 567(7748): 389-393 ), based on this, the effect of compound SN-011 on the multimerization of the above-mentioned point mutants was further tested, and the results showed that SN-011 can significantly inhibit the multimerization expression of the STING mutant (Figure 14F). In summary, SN-011 can reduce the expression of downstream inflammatory cytokines by inhibiting the multimerization activation of SAVI-related STING point mutations, thereby reducing tissue or cell inflammatory damage. In addition, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006) and the compound of formula V (SN-010) all have similarities with the compound of formula I (SN-011). The effect.

Therefore, the compounds of formula I to V of the present invention can be used to prepare drugs for the prevention or treatment of STING-related vascular disease (SAVI) occurring in infancy.

Example 14

SN-011 (compound of formula I) improves cerebral ischemic injury in rats

Tissue ischemic damage is accompanied by the death of a large number of parenchymal cells, and the subsequent release of damage-related molecular patterns (DMAP) further promotes the activation of the immune system at the injury site, and the released inflammatory factors aggravate the tissue damage. Recent studies have found that the cell debris produced by damaged cardiomyocytes in the mouse myocardial ischemia model is taken up by macrophages in the heart tissue, which activates the cGAS-STING-mediated innate immune signaling pathway in macrophages and promotes ISGs. The expression of, and knocking out cGAS or STING gene can significantly improve myocardial ischemia leading to cardiac inflammation damage, improve cardiac function and prolong the survival rate of diseased mice (Nature Medicine, 2017, 23(12): 1481-1487; Circulation, 2018,137(24):2613-2634). Accordingly, the present inventors evaluated the potential therapeutic effect of compound SN-011 in a rat cerebral ischemia model. The experimental results showed that after 24 hours of middle artery occlusion (MCAO), the cerebral infarction area of the model group rats increased significantly, and low-dose (1mg/kg) and high-dose (3mg/kg) SN-011 were given Both can significantly improve the area of cerebral infarction (Figure 15A). The exercise capacity of the rats was evaluated 6 hours and 24 hours after the operation, and administration of SN-011 can significantly improve the motor dysfunction of the rats after stroke (Figure 15B-15C). Further detection of the expression of inflammatory genes in rat brain cortex tissues showed that the expression of Ifnb, Ifna4, Cxcl10, Mcp-1, Tnf-a and Il-6 genes in the cerebral cortex of rats was significantly increased after the middle artery was blocked. In contrast, SN-011 can significantly inhibit the expression of the above-mentioned genes in cerebral cortex tissue induced by ischemia injury (Figure 15D-15I). The above research results show that the compound SN-011 can significantly improve the ischemia-induced parenchymal organ damage. In addition, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006) and the compound of formula V (SN-010) all have similarities with the compound of formula I (SN-011). The effect.

This suggests that the compounds of formula I to V of the present invention can be used to prepare drugs for preventing or treating STING-mediated ischemic cardiovascular and cerebrovascular diseases.

Example 15

SN-011 (compound of formula I) improves cerebral ischemic injury in mice

Stroke can quickly lead to the activation of microglia and the infiltration of peripheral immune cells, and the content of DNA in the cerebrospinal fluid of stroke patients increases significantly. Recent studies have shown that the cGAS-STING pathway is activated after stroke and mediates the damage after stroke (EMBO Molecular Medicine, 2020, 7; 12(4)). The experimental results showed that the mice were intraperitoneally injected with low-dose SN-011 (1mg/kg) and high-dose SN-011 (2mg/kg) immediately after the middle artery was blocked. After 24 hours, the compound SN-011 significantly inhibited Mcp-1 , The expression of Il-6 and Tnf-α genes (Figure 16A); mice were intraperitoneally injected with low-dose SN-011 (1mg/kg) and high-dose SN-011 (2mg/kg) immediately after the middle artery was blocked, 72 After hours, compound SN-011 significantly inhibited the expression of Mcp-1, Il-6 and Cxcl10 genes (Figure 16B); mice were injected intraperitoneally with SN-011 (2mg/kg) immediately or 24 hours after the middle cerebral artery was blocked. Continuous administration to the seventh day after stroke, compound SN-011 administered immediately or delayed for 24 hours can significantly improve the motor coordination of stroke mice (Figure 16C); immediately or 24 hours after the middle cerebral artery is blocked in mice Then SN-011 (2mg/kg) was injected intraperitoneally and continuously administered to the seventh day after the stroke. The immediate administration of compound SN-011 or the delay of 24 hours administration can significantly improve the motor coordination of the stroke mice (Figure 16D). );

Example 16

SN-011 (compound of formula I) improves liver injury and lipid accumulation in mice induced by high-fat diet. Non-alcoholic fatty liver (NAFLD) is mainly manifested as fatty accumulation in the liver induces hepatic steatosis, which is excessive when it is not effectively controlled. The accumulated fat can induce liver inflammation damage and fibrosis, and the disease progresses to non-alcoholic steatohepatitis (NASH). Epidemiological studies have shown that NASH is the most common cause of liver cirrhosis and liver cancer, and there is currently no effective drug for the treatment of NASH. Recent studies have found that the expression of STING in the liver of NAFLD patients is significantly increased, mainly concentrated in the non-parenchymal cells of the liver such as macrophages and Kupffer cells, and knocking out the expression of STING protein in bone marrow cells in animal models can Significantly improve NAFLD disease progression in mice induced by high-fat diet (Gastroenterology, 2018, 155(6): 1971-1984; Journal of Clinical Investigation, 2018, 129(2): 546-555). Accordingly, the present inventors evaluated the efficacy of SN-011 in the NAFLD model induced by a high-fat diet. C57BL6J male mice aged 4-6 weeks were fed with high-fat diet (HFD) for 10 weeks and then started to be given low-dose (1mg/kg) and high-dose (2mg/kg) SN-011 for intervention, continuously administered for 10 weeks After testing the mouse serum biochemical indicators. The results showed that compared with mice fed with a normal diet, the levels of ALT, AST, TC and TG in the serum of HFD mice were significantly increased, and the above indicators in the serum of mice were significantly reduced after administration, indicating that SN-011 can improve HFD diet induced liver damage and reduced serum total cholesterol (TC) and triglyceride (TG) content (Figure 17A-17D). After administration of SN-011, the weight of mice, liver weight and perirenal visceral fat weight were significantly reduced (Figure 17E～17G), and the content of total cholesterol and triglycerides in the liver was also significantly reduced after administration (Figure 17H～17I). The above results confirmed the regulation of SN-011 on lipid metabolism in mice. Further detection of the immune and inflammatory cytokines activated by STING in the liver and the expression levels of lipid metabolism protein genes in the liver showed that the up-regulation of liver Ifnb, Cxcl10, Mcp-1 and Tnfa genes induced by high-fat diet was after the administration of SN-011 Both are significantly improved (Figure 17J-17M), which shows that SN-011 can improve liver inflammation, and can also significantly inhibit HFD-induced liver lipid synthesis protein fatty acid synthase (FAS) and sterol regulatory element binding protein ( SREBP-1c) gene up-regulation (Figure 17N ~ 17O). The liver of the experimental group was further subjected to pathological sectioning to evaluate tissue inflammatory damage and lipid vacuoles. The results showed that SN-011 can significantly improve the infiltration of inflammatory cells and the formation of large vesicles formed by lipid aggregation in the liver tissues of HFD mice. Steatosis (Figure 17P). In addition, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006) and the compound of formula V (SN-010) all have similarities with the compound of formula I (SN-011). The effect.

The above results suggest that the compounds of formula I to V of the present invention can be used to prepare drugs for preventing or treating STING-mediated non-alcoholic fatty liver disease (including NASH).

Example 17

SN-011 (compound of formula I) improves imiquimod-induced psoriasis-like inflammation. BALB/c mice were used to establish a model of imiquimod-induced psoriasis-like inflammation to observe whether compound SN-011 can reverse imiquine Mott-induced psoriasis-like inflammation. BALB/c mice were randomly divided into blank control group (Control group), model control group (Model group), SN-011 250 mg/kg group and SN-011 50 mg/kg group. Psoriasis lesion area and severity of disease (PASI) scores were used to observe the disease progression and measure the thickness of the right ear. H&E staining was used to observe the skin epidermis thickness of the right ear and back of the mice in each group, and the Munro microabscess. The experimental results show (Figure 18 and Figure 19) that the ear thickness of the Model group is significantly higher than that of the Control group, and the ear thickness of the compound SN-011 group is lower than that of the Model group, and there is a significant difference. The results of H&E staining showed (Figure 20) that compound SN-011 reduced the thickness of mouse spine skin, and there was a significant difference. In addition, the compound of formula II (SN-001), the compound of formula III (SN-005), the compound of formula IV (SN-006) and the compound of formula V (SN-010) all have similarities with the compound of formula I (SN-011). The effect.

The above results suggest that the compound of the present invention can improve psoriasis-like inflammation, and can be used to prepare drugs for preventing or treating STING-mediated psoriasis.

In summary, the compounds of formula I to V of the present invention or their pharmaceutically acceptable salts or solvates can be used in the preparation of drugs for the prevention or treatment of diseases mediated by STING.

The mechanism of action of the compound of the present invention is shown in Figure 21. Taking compound SN-011 (compound of formula I) as an example, under normal circumstances, abnormal DNA in the cytoplasm is recognized by cGAS to produce 2'3'-cGAMP molecules, and 2'3'-cGAMP then interacts with the endoplasmic reticulum regulatory protein STING Binding, induce the conformational change of STING protein and cause multimerization activation, and then the multimerized STING protein transfers to the Golgi apparatus to recruit downstream proteins TBK1 and IRF3. TBK1 promotes IRF3 and NF-κB phosphorylation after autophosphorylation, and then Entering the nucleus promotes the expression of interferon and inflammatory cytokines; after adding SN-011 molecules, SN-011 can specifically bind to the pocket formed by the STING dimer, thereby preventing CDNs molecules from activating the STING protein and blocking the signal Of delivery.

Example 18

tablet

The compound of formula I (50g) prepared in Example 1 (50g), hydroxypropylmethylcellulose E (150g), starch (200g), an appropriate amount of povidone K30 and magnesium stearate (1g) were mixed, granulated and pressed sheet.

In addition, the compounds of formula I to V of the present invention can be given to different pharmaceutical excipients according to the conventional preparation method of the pharmacopoeia 2015 edition to make capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, and ointments. , Suppositories or patches, etc.

Claims

Use of benzenesulfonamide compounds or pharmaceutically acceptable salts or solvates thereof as shown in any one or more of the following formulas I to V in the preparation of drugs for inhibiting activation of the STING signal pathway:
A use of any one or more of benzenesulfonamide compounds of formula I to V according to claim 1 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicine for preventing or treating STING-mediated diseases.
The use according to claim 2, the STING-mediated diseases include infectious diseases, inflammatory diseases, autoimmune diseases, organ fibrotic diseases, ischemic cardiovascular and cerebrovascular diseases, degenerative neurological diseases, brain diseases Trauma, spinal cord injury, cancer, or cancerous syndrome.
The use according to claim 2, wherein the STING-mediated disease is spinal cord injury caused by psoriasis, stroke, traumatic brain injury, trauma or other diseases, Parkinson's disease, Huntington's disease, familial amyotrophic lateral sclerosis Syndrome, myocardial infarction, non-alcoholic steatohepatitis, systemic lupus erythematosus, Aicardi-Goutières syndrome, rheumatoid arthritis, inflammatory bowel disease, STING-related vascular disease (SAVI) or diabetes occurring in infants complication.
The use according to claim 1 or 2, wherein the pharmaceutically acceptable salt of the compound of formula I to V is a salt formed by a metal ion or a pharmaceutically acceptable amine, ammonium ion or choline.
A pharmaceutical composition for preventing or treating STING-mediated diseases, which comprises any one or more benzenesulfonamide compounds of formula I to V or a pharmaceutically acceptable salt or solvent compound thereof as an active ingredient and a pharmaceutically acceptable compound. Accepted excipients.
The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository, or Patches.