WO2021068951A1 - Utilisation médicale d'un composé benzènesulfonamide et d'une composition pharmaceutique associée - Google Patents

Utilisation médicale d'un composé benzènesulfonamide et d'une composition pharmaceutique associée Download PDF

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WO2021068951A1
WO2021068951A1 PCT/CN2020/120280 CN2020120280W WO2021068951A1 WO 2021068951 A1 WO2021068951 A1 WO 2021068951A1 CN 2020120280 W CN2020120280 W CN 2020120280W WO 2021068951 A1 WO2021068951 A1 WO 2021068951A1
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compound
sting
protein
cells
mice
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王琛
洪泽
余文颖
孙宏斌
梅家豪
李晨辉
刘星
孙立
柳军
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China Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • 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.
  • TMEM173 transmembrane protein 173
  • STING Stimulator of interferon gene
  • PRRs pattern recognition receptors
  • PAMPs non-self pathogen-related molecular patterns
  • DAMPs damage-associated molecular patterns 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).
  • 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
  • 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).
  • 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).
  • STING also known as TMEM173, MITA, ERIS or MPYS
  • 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).
  • STING protein can also activate the NF- ⁇ B signaling pathway by recruiting TBK1 and TRAF6 molecules, and promote the expression of inflammatory factors such as TNF- ⁇ and IL-6 (J Virol, 2014, 88(10): 5328-41).
  • AGS syndrome (Aicardi-Goutines 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).
  • 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
  • STING protein N Engl J Med, 2014, 371(6): 507-518
  • 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 Met
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 osteo
  • the compound of the present invention or a pharmaceutically acceptable salt or solvate thereof can be used to prevent or treat autoimmune diseases.
  • autoimmune diseases Including: Aicardi-Goutines 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.
  • Aicardi-Goutines syndrome Aicardi-Goutines 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, psori
  • 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.
  • 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.
  • 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.
  • 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-Goutines syndrome, rheumatoid arthritis, inflammatory bowel disease, STING-related vascular disease (SAVI) or diabetes occurring in infancy complication.
  • SAVI STING-related vascular disease
  • 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.
  • 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, oxa
  • 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.
  • metal including sodium, potassium, calcium, etc.
  • 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.
  • 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
  • the compound of formula V SN-010
  • the half inhibition rate of the compound of formula I on IFN- ⁇ activation in mouse primary bone marrow-differentiated macrophages and human primary foreskin fibroblasts is about 100 nM and 500 nM, respectively.
  • 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- ⁇ B and their nuclear transcription levels are significantly reduced, thereby inhibiting the downstream expression of type I interferon, IL-6 and TNF- ⁇ .
  • 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 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.
  • 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:
  • 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.
  • the compound of formula I can significantly inhibit the expression of cell type I interferons and related ISGs and inflammatory cytokines.
  • 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.
  • 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.
  • the compound of formula I can significantly improve the lipid accumulation and inflammatory infiltration of the liver of mice, and improve liver damage.
  • the compound of formula I can significantly improve psoriasis-like inflammation.
  • 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.
  • the present invention has the following advantages:
  • 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.
  • 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.
  • 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 10 ⁇ M test compounds. After 6 hours of incubation, lipids The cells were transfected with 5 ⁇ g ISD for 6 hours, and the cells were collected to detect the expression of Ifn ⁇ gene;
  • B MEF cells were incubated with 10 ⁇ M test compound, and the cells were collected after 6 hours of incubation to detect the expression of Ifn ⁇ 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 ⁇ 20 ⁇ M compound SN-001 and SN-100 were incubated with L929 cells for 6 hours, liposomes were transfected with HT- DNA 4 ⁇ g stimulation for 6 hours, harvested cells to detect Ifn ⁇ , Ifn ⁇ 4, Cxcl10 and Il6 gene expression; (E ⁇ H)5 ⁇ 20 ⁇ M compound SN-001 and SN-100 were incubated with THP-1 cells for 6 hours, HSV-1 was used 40 ⁇ L of the virus was stimulated for 6 hours, and the cells were collected to detect the expression of IFN ⁇ , IFN ⁇ 4, CXCL10 and IL6 genes; (I) 10 ⁇ M compounds SN-001 and SN-100 were incubated in L929 cells for 6 hours, respectively, liposome transfected with HT-DNA 4 ⁇ g stimulation After 4 hours, the specific antibody immunoblotting method used to collect the protein from the cells was used to detect the expression
  • Figure 3 is a graph showing the in vitro biological activity effects of compound SN-011 (compound of formula I) and related compounds:
  • A 10 ⁇ M SN-001 and related compounds SN-005 ⁇ SN-011 were incubated with L929 cells for 6 hours, and liposome-transformed Stimulate with HT-DNA 4 ⁇ g for 6 hours, collect cells to detect the expression of IFN- ⁇ gene;
  • B Compound structure of compound SN-001 and related compounds SN-005 ⁇ SN-011;
  • C ⁇ E 1 ⁇ M compound SN-011 After incubating the MEF cells for 6 hours, use the classical agonist ISD upstream of STING 5 ⁇ g, HT-DNA 4 ⁇ g, HSV-1 40 ⁇ l, c-di-GMP 1 ⁇ g and 2'3'-cGAMP 1 ⁇ g to stimulate, and then detect the cell Ifn ⁇ 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
  • 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) ⁇ 5 ⁇ M) 6 hours later, use 2'3'-cGAMP 1 ⁇ g to stimulate for 3 hours.
  • 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.
  • (C) HFF cells were incubated with 1 ⁇ 10 ⁇ M SN-011 for 12 or 24 hours.
  • 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) 1 ⁇ M SN-011 or SN-100 after incubating HFF cells overnight, use 2'3'- cGAMP 1 ⁇ g stimulated for 1 hour.
  • the nucleus position was indicated by DAPI staining.
  • the length of the ruler was 25 ⁇ m;
  • the Golgi apparatus position is indicated by GM130 staining, and the length of the ruler is 25 ⁇ m;
  • 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
  • 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 5 ⁇ g each.
  • SN-011 (10 ⁇ M) was added 12 hours after transfection to continue incubating for 12 hours. After the incubation was completed, the cells were collected to detect the IFN ⁇ gene. Calculate the inhibition rate according to the gene expression value; (H ⁇ I) Transfect 5 ⁇ g 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 (10 ⁇ M) and continue to incubate for 9 hours.
  • 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 Ifn ⁇ , 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
  • 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 Ifn ⁇ , 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.
  • 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;
  • BUN 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.
  • 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 5 ⁇ g, after 12 hours of transfection, incubate compound SN-011 (10 ⁇ M), after compound incubation for 12 hours, collect cells to detect the expression of IFN- ⁇ , CXCL10 and TNF- ⁇ genes
  • D HEK293T cells were transfected with 5 ⁇ g plasmids expressing Flag-hSTING and SAVI-related point mutants V155M, G166E, C206Y, R281Q and R284G, 6 hours after transfection, incubate compound SN-011 (10 ⁇ M), and incubate the compound After 12 hours, the cells were
  • the compound SN-011 (10 ⁇ M) 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 Ifn ⁇ , Ifn ⁇ 4, Cxcl10, Mcp1, Tnf ⁇ and Il6 genes; rats were injected into the abdominal cavity at the time of modeling and 12 hours after modeling.
  • 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.
  • high-dose SN-011 (2mg/kg) 24 hours later, take the cerebral cortex tissues to detect the expression of Mcp-1, Il6 and Tnf ⁇ genes;
  • B The mice were intraperitoneally injected with low-dose SN immediately after the middle cerebral artery was blocked.
  • mice 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;
  • 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 Ifn ⁇ , Cxcl10, Mcp-1, Tnf ⁇ , 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
  • 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 21 is a schematic diagram of the mechanism of compound SN-011 (compound of formula I) inhibiting the activation of the STING signaling pathway.
  • 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).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • 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).
  • 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, Glasgow Pharmaceutical, Aladdin, Anaiji and other companies.
  • 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 ⁇ g/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.
  • 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.
  • 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.
  • 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. .
  • mice 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.
  • NAFLD non-alcoholic fatty liver
  • HFD high-fat diet
  • male C57BL6J mice aged 4-6 weeks were fed with high-fat diet (research diets, 60% Kcal High-Fat Diets, D12492) ).
  • high-fat diet search diets, 60% Kcal High-Fat Diets, D12492
  • 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.
  • mice 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.
  • 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).
  • the thickness of the slice is 5 ⁇ m; 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.
  • 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.
  • TG triglyceride
  • TC total cholesterol
  • 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.
  • 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 20 ⁇ l of Fc blocking to each tube, block at 4°C 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
  • ANA HEp-22 antigen substrate slide kit
  • MBL-BION serum antinuclear antibodies
  • 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.
  • 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.
  • 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 ⁇ l 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.
  • 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.
  • CDNs activate the intracellular STING signaling pathway
  • 100mM KCl 100mM KCl
  • 3mM MgCl 2 3mM MgCl 2
  • 0.1mM DTT 85mM sucrose
  • BSA
  • 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 10 ⁇ M ⁇ IC 50 test concentration: 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.039 ⁇ M) overnight, the blank control group only added DMSO.
  • the cells are treated with a digoxigenin solution containing a 2'3'-cGAMP stimulant (final concentration of 2'3'-cGAMP: 1 ⁇ g/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 ⁇ M 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.
  • 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.
  • 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.
  • protein lysis buffer (0.5% TritonX-100, 1mM EDTA, 1% Cocktail dissolved in TBS buffer
  • 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.
  • Co-IP co-immunoprecipitation
  • 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°C 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 .
  • 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
  • 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) 1 ⁇ g plasmid is transformed, transferred, and used at 18°C with a final concentration of 0.5mM IPTG induces a large amount of protein expression in E.
  • STING-CTD 15mg/ml of STING-CTD (149-379) protein was dissolved in 25mM Tris-HCl, pH 7.5, 150mM NaCl solution, and 1 ⁇ L of SN-011(50mM) was added to 100 ⁇ L 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 ⁇ L of protein solution and 1 ⁇ L 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.
  • SPR Surface plasmon resonance technology
  • 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.
  • TRIzol Invitrogen
  • RNA is mixed with the primer Oligo dT and transcribed into cDNA under the action of the reverse transcriptase kit.
  • DNA polymerase and the fluorescent dye FastStart Universal SYBR GREEN MASTER MIX are mixed, they are amplified and detected in the ABI QuantStudio 3 instrument.
  • GAPDH is the internal reference for gene expression, and the 2- ⁇ CT 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:
  • 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.
  • 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.
  • Example 2 The compound I (200 mg, 0.43 mmol) prepared in Example 1 was dissolved in THF (5 mL), pyridine (52 ⁇ L, 0.65 mmol) was added, acetyl chloride (37 ⁇ L, 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.
  • 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.
  • ZINC15 http://zinc15.docking.org
  • the compound SN-100 (CAS:1384744-19-7) does not affect the expression of IFN- ⁇ gene in MEF cells induced by ISD. Therefore, this compound was selected as a negative control compound for subsequent experiments.
  • SN-001 significantly inhibited the phosphorylation expression of STING, TBK1, IRF3, P65, I ⁇ B ⁇ 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.
  • 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).
  • IC 50 half inhibition rate of SN-011 (compound of formula I) in different cells.
  • MEF mouse embryonic fibroblasts
  • BMDM mouse bone marrow primary macrophages
  • HFF human primary foreskin fibroblasts
  • SN-011 also has good safety in vitro.
  • SN-011 (compound of formula I) inhibits the activation of STING protein and its downstream signal transduction mediated by it
  • the STING protein 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).
  • STING and its ligand CDNs molecules such as 2',3'-cGAMP
  • 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.
  • 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.
  • 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).
  • 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 I ⁇ B ⁇ 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).
  • the negative control compound SN-100 did not affect the changes in the above-mentioned protein levels.
  • 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).
  • 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).
  • 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 ⁇ - ⁇ 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 hSTING-Y167A and hSTING-E260A mutants cannot activate downstream IFN- ⁇ 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 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.
  • 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.
  • 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).
  • the present invention evaluates the potential pharmacodynamic activity of SN-011 on systemic inflammatory damage in Trex1-/- mice.
  • BMDMs bone marrow-derived macrophages
  • 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.
  • 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 (Ifn ⁇ , 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 (Ifn ⁇ , Cxcl10, Isg15) and inflammatory factor (Il6) gene expression in the heart, stomach, tongue and muscle of Trex 1 -/- mice ( Figures 10A-10D).
  • 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.
  • SN-011 (compound of formula I) inhibits the expression of inflammatory cytokines induced by SAVI-related STING point mutations
  • 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.
  • SAVI STING-associated vasculopathy with onset 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 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.
  • SAVI STING-related vascular disease
  • 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.
  • DMAP damage-related molecular patterns
  • 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).
  • MCAO middle artery occlusion
  • SN-011 (compound of formula I) improves cerebral ischemic injury in mice
  • mice 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.
  • SN-011 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).
  • NASH non-alcoholic steatohepatitis
  • 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.
  • HFD high-fat diet
  • 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).
  • SN-011 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.
  • 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.
  • 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.
  • TBK1 promotes IRF3 and NF- ⁇ B 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.
  • 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.

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Abstract

L'invention concerne l'utilisation médicale d'un composé benzènesulfonamide et d'une composition pharmaceutique associée. En particulier, la présente invention concerne un composé représenté par l'une des formules I à V ou un sel ou solvate pharmaceutiquement acceptable de celui-ci. La présente invention peut être utilisée pour préparer un inhibiteur de STING ou un médicament pour inhiber l'activation de la voie de signalisation de STING et pour préparer un médicament pour prévenir ou traiter une maladie médiée par STING.
PCT/CN2020/120280 2019-10-12 2020-10-12 Utilisation médicale d'un composé benzènesulfonamide et d'une composition pharmaceutique associée Ceased WO2021068951A1 (fr)

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CN116850292A (zh) * 2023-08-24 2023-10-10 武汉大学 Sting作为作用靶点在制备防治腹膜炎症和纤维化的药物中的应用
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CN117682973B (zh) * 2022-09-26 2026-03-20 中国药科大学 磺酰胺类化合物及其医药用途
CN118908937B (zh) * 2024-07-10 2025-07-15 中山大学中山眼科中心 一种具有sting蛋白降解作用的化合物及其制备方法和用途
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CN116850292A (zh) * 2023-08-24 2023-10-10 武汉大学 Sting作为作用靶点在制备防治腹膜炎症和纤维化的药物中的应用

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