WO2021068951A1 - Medical use of benzenesulfonamide compound and pharmaceutical composition thereof - Google Patents
Medical use of benzenesulfonamide compound and pharmaceutical composition thereof Download PDFInfo
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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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- HUOFEOPPWVHBBM-UHFFFAOYSA-N CC(Oc(cc1)ccc1-c(cc1)ccc1C(Nc(cc1)cc(NS(c(cc2)ccc2F)(=O)=O)c1OC)=O)=O Chemical compound CC(Oc(cc1)ccc1-c(cc1)ccc1C(Nc(cc1)cc(NS(c(cc2)ccc2F)(=O)=O)c1OC)=O)=O HUOFEOPPWVHBBM-UHFFFAOYSA-N 0.000 description 1
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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
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
本发明属于生物医药领域,具体涉及苯磺酰胺类化合物或其药物上可接受的盐或溶剂化物的医药用途及其药物组合物,其可作用于跨膜蛋白173(TMEM173),也称STING(干扰素基因的刺激因子),并抑制其信号通路。因此,该类化合物可用于制备预防或治疗STING介导的疾病的药物。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.
机体的固有免疫系统的激活由表达在细胞膜上和细胞质中的模式识别受体(pattern recognition receptors,PRRs)识别非自身的病原体相关的分子模式(pathogen-associated molecular patterns,PAMPs)所介导。PRRs在识别PAMPs后,激活下游免疫信号的传导,促进炎症和免疫细胞因子的释放,进一步激活机体的适应性免疫系统协同杀伤并清除入侵的病原微生物(Cold Spring Harbor Symposia on Quantitative Biology,1989,54:1-13.)。相似的,细胞在异常状态下(应激、受损、衰老或死亡)分泌的损伤相关的分子模式(damage-associated molecular patterns,DAMPs)也会被模式识别受体识别,进而激活免疫系统,促进细胞的再生以及损伤后的修复(Science,2002,296(5566):301-305)。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).
细胞质中的DNA作为经典的PAMP或DAMP分子在固有免疫系统的激活中发挥重要的作用(Curr Opin Immunol,2018,55:31-37.)。细胞质中的DNA受体蛋白cGAS(Cyclic AMP-GMP synthase)能够识别异常暴露的胞浆DNA分子,并以ATP和GTP为底物催化合成2’,3’-cGAMP分子(2’-3’/3’-5’cyclic GMP-AMP)。2’,3’-cGAMP与细菌代谢中形成的信号分子c-di-AMP和c-di-GMP被称为环状二核苷酸类物质(cyclic dinucleotides,CDNs)。这些CDNs分子能特异性的结合到内质网上调控蛋白STING(也被称为TMEM173,MITA,ERIS或MPYS)二聚体形成的“V”形口袋内(Nature,2008,455(7213):674-678;Nature,2011,478(7370):515-518),进而诱导STING蛋白的多聚化激活(Cell,2013,154(4):748-62)。多聚化的STING蛋白由内质网向高尔基体室转移,并在此过程中招募下游的激酶蛋白TBK1和转录因子IRF3,TBK1在自磷酸化激活后催化STING和IRF3的磷酸化(Nature,2019,567(7748):394-398;Nature,2019,569(7758):718-722)。磷酸化的IRF3进一步二聚化入核,促进I型干扰素(type I interferons)及其相关免疫因子的表达(interferon stimulated genes,ISGs)。此外,STING蛋白也能通过招募TBK1和TRAF6分子,激活NF-κB信号通路,促进TNF-α和IL-6等炎症因子的表达(J Virol,2014,88(10):5328-41)。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).
尽管STING介导的固有免疫信号通路的激活在机体抵抗病原微生物的入侵 过程中发挥重要的作用,持续的STING通路的激活会导致多种自身免疫疾病和炎症疾病的发生和发展(Nature Immunology,2017,18(7):716-724)。AGS综合症(Aicardi-Goutières syndrome)是一种由TREX1,RNASE H2,SAMHD1,ADAR或IFIH1基因突变引起的全身性的罕见的自身免疫疾病(Am J Med Genet A,2015,167A(2):296-312)。动物模型中研究发现核酸代谢酶TREX1,RNASE H2和SAMHD1的基因突变导致胞浆DNA聚集进而激活cGAS-STING信号通路,且进一步在疾病小鼠体内敲除STING蛋白的表达能显著改善疾病的进展(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)。在系统性红斑狼疮(systemic lupus erythematosus,SLE)病人中也发现了TREX1基因的突变,且50%~60%的SLE病人血清中I型干扰素高表达(Nat Rev Rheumatol,2018,14(4):214-228)。在Bloom综合症病人中发现,BLM蛋白突变导致微核的形成,进而激活STING信号通路诱导病人的血清中IFNs和ISGs的高表达(J Exp Med,2019,216(5):1199-1213)。SAVI(STING-associated vasculopathy with onset in infancy)疾病是由STING蛋白中个别氨基酸的突变导致其异常激活诱导的全身性的自身免疫疾病(N Engl J Med,2014,371(6):507-518)。此外,在炎症相关的疾病模型中,如皮肤癌的诱发(Nat Commun,2014,5:5166)、肿瘤转移(Nature,2018,553(7689):467-472)、早衰症(Nature,2017,550(7676):402-406)、脓毒症(Shock,2017,47(5):621-631)、急性胰腺炎(Gastroenterology,2018,154(6):1822-1835)、非酒精性脂肪肝及肝纤维化(Gastroenterology,2018,155(6):1971-1984;Proceedings of the National Academy of Sciences,2017,114(46):12196-12201)、肺炎(Nature Communications,2018,9(1))、慢性肾炎及肾纤维化(Cell Metabolism,2019,DOI:10.1016/j.cmet.2019.08.003)、缺血性再灌注损伤(Nature Medicine,2017,23(12):1481-1487;EMBO Molecular Medicine,2020,7;12(4):e11002)、帕金森病(Nature,2018,561(7722):258-262)、脑外伤(Journal of Neuroscience,2020,40(2):424-446)、外伤或其他疾病诱导的脊髓损伤(Cell,2020,183:1–14;Biochemical and Biophysical Research Communications,2019,517(4):741-748)和亨廷顿病(Journal of Clinical Investigation,2020,130(6):3124-3136;Proceedings of the National Academy of Sciences,2020,117(27):15989-15999)、家族性肌萎缩侧索硬化症(Nature,2020,585(7823):96-101)等,而抑制STING信号通路的激活能显著改善上述疾病的发生发展。总之,靶向STING蛋白的抑制剂的开发具有广泛的临床应用前景。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.
目前文献已报道的STING小分子抑制剂屈指可数,活性较弱且副作用较大(Cell Reports 2018,25,3405–3421;ACS Med.Chem.Lett,2019,10(1),92-97)。因 此,研制新型的STING小分子抑制剂具有迫切的临床需求。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.
发明内容Summary of the invention
发明目的:针对现有技术存在的问题,本发明提供如下式I~V任意一种或者多种所示的苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物在制备抑制STING信号通路激活的药物中的用途以及在制备预防或治疗STING介导的疾病的药物中的用途。本发明通过虚拟筛选、生物活性评价及作用机制研究,发现如下式I~V所示的苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物能特异性抑制STING信号通路的激活。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.
本发明还提供一种预防和治疗STING介导的疾病的药物组合物。The invention also provides a pharmaceutical composition for preventing and treating STING-mediated diseases.
技术方案:为了实现上述目的,如本发明所述的式I~V任意一种或者多种所示的苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物在制备抑制STING信号通路的药物中的用途: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:
本发明所述的式I~V任意一种或者多种苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物在制备预防或治疗STING介导的疾病的药物中的用途。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.
进一步地,所述STING介导的疾病包括感染性疾病、炎性疾病、自身免疫性疾病、器官纤维化疾病、缺血性心脑血管疾病、神经退行性疾病、脑外伤、脊髓损伤、癌症或癌期综合征中的一种或几种。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.
在某些实施方案中,本发明化合物或其药学上可接受的盐或溶剂化物可以用于预防或治疗感染性疾病,包括:结核分枝杆菌感染、衣原体感染、疱疹病毒(单纯疱疹病毒)感染、腺病毒感染、乙肝病毒感染、正粘病毒感染和冠状病毒感染。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.
在某些实施方案中,本发明化合物或其药学上可接受的盐或溶剂化物可以用 于预防或治疗炎性疾病,包括:代谢性炎症相关疾病(如胰岛素抵抗、代谢综合征、1型或2型糖尿病、高脂血症、肥胖症、动脉粥样硬化、心肌缺血、心肌梗死、心律失常、冠心病、高血压、心衰、心肌肥大、心肌炎、缺血性脑病、脑卒中、出血性脑病、脑溢血、脑水肿、糖尿病心肌病、糖尿病肾病、糖尿病视网膜病变、糖尿病神经病变和糖尿病溃疡、非酒精性脂肪肝、非酒精性脂肪性肝炎、酒精性脂肪肝、肝硬化、痛风、中风或脑梗死等),肌肉骨骼肌炎症(手、腕、肘、肩、颈、膝盖、踝和脚关节炎症,例如骨关节炎、类风湿性关节炎、强直性脊柱炎、急性和慢性感染性关节炎等),眼部炎症(角膜炎、巩膜炎、结膜炎等),消化系统炎症(结肠炎、肝炎、胆管炎、胆囊炎、胰腺炎、胃炎、肠炎、炎症性肠病、直肠炎),神经系统炎症(脑膜炎、神经性肌强直、多发性硬化、CNS血管炎),退行性神经系统疾病(帕金森病、亨廷顿病、家族性肌萎缩侧索硬化症),脉管系统或者淋巴系统炎症(血管炎、淋巴管炎、静脉炎),生殖系统炎症(宫颈炎、子宫内膜炎、附睾炎、睾丸炎、尿道炎),呼吸系统炎症(肺炎、哮喘、慢性阻塞性肺病、慢性支气管炎、肺气肿、闭塞性细支气管炎、特发性肺纤维化、囊性纤维化肺病),器官纤维化疾病(肝纤维化、肾纤维化、肺纤维化、心肌纤维化),其他炎性病症包括阑尾炎、心肌炎、腮腺炎、牙龈炎、前列腺炎、腹膜炎、胸膜炎、血管炎、静脉炎、浮肿、肾炎,脊髓损伤包括外伤或其他疾病引起的脊髓损伤,脑外伤。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.
在某些实施方案中,本发明化合物或其药学上可接受的盐或溶剂化物可以用于预防或治疗自身免疫性疾病。包括:Aicardi-Goutières综合征(AGS)、婴儿期发病的STING相关血管炎(SAVI)、伴有脑蛋白营养不良的视网膜血管病变(RCVL)、系统性红斑狼疮(SLE)、家族性冻疮狼疮(CHBL)、贝切特氏病、查加斯病、银屑病、多发性硬化症、硬皮症和白塞氏病等。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.
在某些实施方案中,本发明化合物或其药学上可接受的盐或溶剂化物可以用于预防或治疗具有炎性组分的T细胞介导的超敏反应,包括荨麻疹、皮肤过敏、过敏性鼻炎、接触性皮炎和呼吸道过敏等。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.
在某些实施方案中,本发明化合物或其药学上可接受的盐或溶剂化物可以用于预防或治疗身体各组织器官的癌症及肿瘤的转移,包括但不限于肺、骨、胰腺、肝、肾、头、子宫、卵巢、胃、结肠、食道、小肠、内分泌系统、前列腺、膀胱、宫颈、阴道的癌症。例如肝癌、肾癌、宫颈癌、肺癌、皮肤癌、子宫癌、腺癌、前列腺癌、肉瘤、骨肉瘤、甲状腺癌、非小细胞肺癌、食管癌、慢性髓细胞性白血病、慢性淋巴细胞性白血病、急性髓细胞性白血病、急性淋巴细胞性白血病、多发性骨髓瘤、恶性淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、神经母细胞瘤。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.
在某些优选的实施方案中,所述STING介导的疾病是银屑病、脑卒中、心肌梗死、脑外伤、外伤或其他疾病诱导的脊髓损伤、帕金森病、亨廷顿病、家族性肌萎缩侧索硬化症、非酒精性脂肪性肝炎、系统性红斑狼疮、Aicardi-Goutières综合征、类风湿性关节炎、炎症性肠病、婴儿期发生的STING相关的血管疾病(SAVI)或糖尿病及其并发症。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.
所述式I~V化合物其药学上可接受的盐为金属离子或药学上可接受的胺、铵离子或胆碱形成的盐。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.
本发明化合物或其药学上可接受的盐或溶剂化物可单独使用或可与其他治疗剂组合使用。作为免疫调节剂,本发明的化合物可用于单一治疗或者与其它治疗剂组合使用以治疗与STING蛋白功能异常紊乱相关的疾病,包括感染性疾病、炎性疾病、自身免疫性疾病、器官纤维化疾病、缺血性心脑血管疾病、脑外伤、脊髓损伤、神经退行性疾病、癌症或癌前期综合征。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.
本发明的化合物可作为药用盐使用。该盐可为下列酸中的至少一种的酸盐:半乳糖二酸、D-葡糖醛酸、甘油磷酸、马尿酸、羟乙磺酸、乳糖酸、马来酸、1,5-萘二磺酸、萘-2-磺酸、新戊酸、对苯二甲酸、硫氰酸、胆酸、正十二烷基硫酸、苯磺酸、柠檬酸、D-葡萄糖,乙醇酸、乳酸、苹果酸、丙二酸、扁桃酸、磷酸、丙酸、盐酸、硫酸、酒石酸、琥珀酸、甲酸、氢碘酸、氢溴酸、甲烷磺酸、烟酸、硝酸、乳清酸、草酸、苦味酸、L-焦谷氨酸、糖精酸、水杨酸、龙胆酸、对甲苯磺酸、戊酸、棕榈酸、葵二酸、硬脂酸、月桂酸、乙酸、己二酸、碳酸、苯磺酸、乙烷二磺酸、乙基琥珀酸、富马酸、3-羟基萘-2-甲酸、1-羟基萘-2-甲酸、油酸、十一碳烯酸、抗坏血酸、樟脑酸、樟脑磺酸、二氯乙酸、乙烷磺酸。另一方面,该盐也可以是本发明的化合物与金属(包括钠、钾、钙等)离子或药学上可接受的胺(包括乙二胺、氨丁三醇等)、铵离子或胆碱形成的盐。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.
本发明包括本发明化合物的各种氘代形式。与碳原子相连的每个可用氢原子可以独立的被氘原子取代。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.
本发明所述的预防或治疗STING介导的疾病的药物组合物,其包含式I~V任意一种苯磺酰胺类化合物或其药学上可接受的盐或溶剂化合物作为活性成分和药学上可接受的辅料。可任意混合的辅料根据剂型、给药形式等可以改变。辅料的例子包括赋形剂、粘合剂、崩解剂、润滑剂、矫味剂、香味剂、着色剂或甜味剂等。所述药物组合物可以是胶囊剂、散剂、片剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、软膏剂、栓剂或贴剂等制剂学上常规的制剂形式。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.
本发明化合物可以从EnamineStore公司购买得到,也可参照实施例中描述的方法或其改进的方法来制备。所有化合物的HPLC纯度在95%以上。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%.
本发明通过计算机辅助的虚拟筛选的方法筛选可能与STING蛋白结合的化 合物,并进一步在细胞水平上对化合物进行生物活性验证。结果发现,式I化合物(SN-011,CAS:2249435-90-1)、式II化合物(SN-001,CAS:727699-84-5)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)能高效、特异性且低毒性地抑制STING信号通路。例如,式I化合物在小鼠原代骨髓分化的巨噬细胞和人原代包皮成纤维细胞中对IFN-β激活的半数抑制率分别为100nM和500nM左右。进一步的机制研究发现,式I化合物能显著细胞质中DNA诱导的STING蛋白的多聚化激活,抑制其由内质网向高尔基体的转移,抑制对下游接头蛋白TBK1和IRF3的招募,导致转录因子IRF3和NF-κB的磷酸化及其核转录水平的显著降低,进而抑制下游I型干扰素、IL-6和TNF-α的表达。此外,本发明获得并解析了人源STING蛋白和式I化合物的共结晶结构,结果表明,STING蛋白中多个保守的氨基酸位点与式I化合物存在结合作用,包括Tyr167、Ser241、Ser243、Glu260和Thr263。值得注意的是,这些位点均参与STING蛋白与其内源配体分子2’3’-cGAMP的结合。且进一步的竞争性Pulldown实验证实,式I化合物与STING蛋白以非共价的可逆方式结合。表面离子共振分子互作仪(SPR)检测SN-011与人源STING蛋白间的亲和力为4.03nM,而相比之下2’3’-cGAMP与STING蛋白间的亲和力为9.23nM。这说明相比于2’3’-cGAMP,式I化合物对STING蛋白具有更强的结合能力。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.
据此,本发明提供了式I~V所示的化合物或其药学上可接受的盐或溶剂化物在制备抑制STING信号通路激活的药物中的用途。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.
本发明选取了小鼠Trex1基因敲除的自身免疫疾病模型、SAVI的体外细胞模型、大脑中动脉栓塞(MCAO)诱导的大鼠和小鼠脑卒中模型、高脂饮食诱导的小鼠非酒精性脂肪肝模型和咪喹莫特诱导的小鼠银屑病模型用于本发明化合物的药效学评价。以式I化合物为例进行的研究结果表明: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.式I化合物能显著改善Trex1敲除小鼠自发性的多组织器官的炎症损伤并抑制机体自反应适应性免疫系统的激活,延长疾病小鼠的长期生存率。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.在SAVI相关的STING点突变体过表达的细胞中,式I化合物能显著抑制细胞I型干扰素及其相关的ISGs和炎症细胞因子的表达。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.在MCAO制备的大鼠脑卒中模型中,式I化合物能显著下调缺血区脑组织中I型干扰素及其相关的ISGs和炎症细胞因子的表达,并显著改善脑卒中动物的神经行为学功能。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.在MCAO制备的小鼠脑卒中模型中,式I化合物立即给药能显著下调缺血区脑组织中炎症细胞因子的表达,并显著改善小鼠记忆功能和运动功能;式I化合物于卒中后24小时给药仍然能显著改善卒中小鼠的运动功能。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.在长期高脂饮食诱导的小鼠非酒精性脂肪肝模型中,式I化合物能显著改 善小鼠肝脏的脂质聚集和炎症浸润,改善肝脏的损伤。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.在BALB/c小鼠建立咪喹莫特诱导的银屑病样炎症模型中,式I化合物可以显著改善银屑病样炎症。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.
基于以上研究结果,本发明提供了式I~V化合物或其药学上可接受的盐或溶剂化物在制备预防或治疗STING介导的疾病的药物中的用途。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.
有益效果:与现有技术相比,本发明具有如下优点:Beneficial effects: Compared with the prior art, the present invention has the following advantages:
(1)本发明首次发现式I~V任意一种苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物可以作为靶向STING的全新类型小分子抑制剂,能高效、特异性且低毒性地抑制STING信号通路的激活,因而可用于制备抑制STING信号通路激活的药物。(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)本发明的化合物作用机制明确,其通过直接结合STING蛋白并维持其静息状态下的二聚体构象而抑制STING信号通路的激活。尤其重要的是,本发明的化合物在炎性疾病、自身免疫性疾病、器官纤维化疾病和缺血性心脑血管疾病等疾病模型上具有显著的体内疗效,因而有望用于制备预防或治疗上述STING介导的疾病的药物。(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.
图1是通过虚拟筛选得到的含有苯磺胺酰胺结构的小分子化合物对STING信号通路的抑制效应图及部分化合物的结构:(A)MEF细胞中孵育10μM受试化合物,孵育6小时后,脂质体转染5μg ISD刺激6小时,收取细胞检测Ifnβ基因的表达;(B)MEF细胞中孵育10μM受试化合物,孵育6小时后收取细胞检测Ifnβ基因的表达;(C)化合物SN-001~SN-004和阴性对照化合物SN-100的化学结构;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,P<0.05;**,P<0.01;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;
图2是化合物SN-001(式II化合物)的体外生物活性效应图:(A~D)5~20μM化合物SN-001和SN-100分别孵育L929细胞6小时后,脂质体转染HT-DNA 4μg刺激6小时,收取细胞检测Ifnβ,Ifnα4,Cxcl10和Il6基因的表达;(E~H)5~20μM化合物SN-001和SN-100分别孵育THP-1细胞6小时后,使用HSV-1病毒40μL刺激6小时,收取细胞检测IFNβ,IFNα4,CXCL10和IL6基因的表达;(I)10μM化合物SN-001和SN-100分别孵育L929细胞6小时后,脂质体转染HT-DNA 4μg刺激4小时,收取细胞用蛋白的特异性抗体免疫印迹法检测对应蛋白的表达水平;(J~K)10μM化合物SN-001和SN-100分别孵育L929细胞6小时后,脂质体转染HT-DNA 8μg刺激4小时,免疫荧光发标记转录因子IRF3和P65的入核,细胞核位置用DAPI染色指示,标尺的长度为25μm;(L~N)MEF,L929和THP-1细胞中分别孵育5~20μM化合物SN-001 12或24小时后,用MTS试剂检测 SN-001对细胞活力的影响;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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;
图3是化合物SN-011(式I化合物)及相关化合物的体外生物活性效应图:(A)10μM SN-001及相关化合物SN-005~SN-011分别孵育L929细胞6小时,脂质体转染HT-DNA 4μg刺激6小时,收取细胞检测IFN-β基因的表达;(B)化合物SN-001及相关化合物SN-005~SN-011的化合物结构;(C~E)1μM化合物SN-011孵育MEF细胞6小时后,分别使用STING上游的经典激动剂ISD 5μg,HT-DNA 4μg,HSV-1 40μl,c-di-GMP 1μg和2’3’-cGAMP 1μg刺激,刺激完成后检测细胞Ifnβ,Cxcl10和Il6基因的表达;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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;
图4是化合物SN-011(式I化合物)在MEF,BMDM和HFF细胞中的IC
50测试效应图:(A~C)MEF,BMDM和HFF细胞中分别孵育梯度浓度的化合物SN-011(5nM~5μM)6小时后,使用2’3’-cGAMP 1μg刺激3小时,刺激完成后检测IFN-β基因的表达,并以基因表达的抑制率作图,拟合出IC
50值;(D~F)MEF,BMDM和HFF细胞中分别孵育1.25~20μM化合物SN-011 24或48小时后,用MTS试剂检测SN-011对细胞活力的影响;n.s.,无显著性差异;*,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;
图5是化合物SN-011(式I化合物)特异性地抑制STING信号通路激活的效应图:(A~B)在STING敲除的MEF细胞中孵育SN-011 6小时后,分别使用在细胞中外加LPS 10μg,poly(I:C)100μg,Sendai virus 10μl,IFN-β100U刺激,或脂质体转染CpG-DNA 5μg,poly(I:C)5μg,刺激完成后检测Ifnβ,Il6,Tnfα,Isg15和Isg56基因的表达;(C)HFF细胞分别孵育1~10μM SN-011 12或24小时,孵育完成后分别检测cGAS,STING,TBK1,IRF3蛋白的表达;(D~E)HFF细胞分别孵育1~10μM SN-011 12或24小时,孵育完成后分别检测cGAS(MB21D1)和STING(TMEM173)基因的表达;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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;
图6是化合物SN-011(式I化合物)抑制STING及其介导的下游信号通路激活的效应图:(A)1μM SN-011或SN-100孵育HFF细胞过夜后,使用2’3’-cGAMP 1μg刺激1小时,刺激完成后收取细胞,用免疫印迹法检测STING蛋白的磷酸化和多聚化激活;(B)1μM SN-011或SN-100孵育HFF细胞过夜后,HSV-1病毒40μl感染细胞4小时,刺激完成后收取细胞,用免疫共沉淀法检测STING与其下游蛋白TBK1和IRF3的相互作用;(C)1μM SN-011或SN-100孵育HFF细胞过夜后,使用2’3’-cGAMP 1μg刺激2小时,收取细胞,用蛋白的特异性抗体免疫印迹法检测对应蛋白的表达水平;(D)1μM SN-011或SN-100 孵育HFF细胞过夜后,使用2’3’-cGAMP 1μg刺激2小时后,固定细胞,用免疫荧光法检测IRF3蛋白入核,细胞核位置用DAPI染色指示,标尺的长度为25μm;(E)1μM SN-011孵育Hela细胞过夜后,HSV-1病毒40μl感染细胞3小时,刺激完成后用免疫荧光法检测STING蛋白向高尔基体的转移,高尔基体位置用GM130染色指示,标尺的长度为25μm;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;
图7是化合物SN-011(式I化合物)与hSTING-CTD(149-379)蛋白的共结晶结构图:(A)SN-011与hSTING-CTD(149-379)蛋白的共结晶结构示意图,STING蛋白的二聚体结构中的两个单体分子分别用绿色和蓝色表示,二聚体形成的口袋内的SN-011分子以棒状骨架结构表示;(B)SN-011/hSTING-CTD(149-379)共结晶结构(紫色)与apo-hSTING-CTD(149-379)的结晶结构(4EMU)(黄色)比对示意图;(C)SN-011/hSTING-CTD(149-379)共结晶结构(紫色)与2’3’-cGAMP/hSTING-CTD(149-379)的结晶结构(4LOH)(黄色)比对示意图;(D)SN-011/hSTING-CTD(149-379)共结晶结果在晶格中堆积的方式;(E)hSTING-CTD(149-379)蛋白与SN-011结合后,蛋白上的氨基酸与小分子的相互作用示意图;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;
图8是化合物SN-011(式I化合物)与STING蛋白结合的亲和力及结合位点的验证图:(A~E)SPR法检测小分子SN-011,SN-100和2’3’-cGAMP与hSTING-CTD(149-379)蛋白间的结合曲线,同时检测突变关键结合位点Ser241A,Ser243A和Thr263A突变后蛋白与SN-011分子间亲和力的变化;(F~G)将表达Flag-hSTING及其点突变体Tyr167,Ser241A,Ser243A,Glu260A和Thr263A的质粒各5μg转染至HEK293T细胞中,转染12小时后加入SN-011(10μM)继续孵育12小时,孵育完成后收取细胞检测IFNβ基因的表达,并根据基因表达数值计算出抑制率;(H~I)将表达Flag-hSTING及其点突变体Tyr167,Ser241A,Ser243A,Glu260A和Thr263A的质粒各5μg转染至HEK293T细胞中,转染12小时后加入SN-011(10μM)继续孵育9小时,孵育完成后加入2’3’-cGAMP 1μg刺激3小时,收取细胞检测IFNβ基因的表达,并根据基因表达数值计算出抑制率;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,P<0.05;**,P<0.01;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;
图9是化合物SN-011(式I化合物)抑制Trex1
-/-小鼠原代BMDM细胞中高表达的IFNs和ISGs的效应图:(A)热图分析SN-011(500nM)孵育12小时后,野生型(WT)和Trex1
-/-小鼠BMDM细胞基因表达的RNA-Sequencing测序结果;共72个ISG基因在热图上展示,每个处理组选取两个独立重复实验;(B~E)Trex1
-/-BMDM细胞中分别孵育DMSO,SN-011(500nM)或SN-100(500nM),孵育12h过夜后收取细胞检测Ifnβ,Cxcl10,Isg15和Il6基因的表达;以WT BMDM 的基因表达为对照组计算Trex1
-/-BMDM细胞中相关基因表达的增长倍数;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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;
图10是化合物SN-011(式I化合物)减轻Trex1
-/-小鼠多组织器官炎症反应的效应图:(A~D)WT和Trex1
-/-小鼠分别腹腔注射PBS或SN-011(5mg/kg),每周注射3次连续注射1个月后,分别取小鼠心脏、胃、舌头和肌肉组织,检测组织中Ifnβ,Cxcl10,Isg15和Il6基因的表达;(E)取上述组织,固定后进行石蜡切片和H&E染色,观察各组织的炎症细胞浸润,每组至少对6只小鼠的数据进行统计,实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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;
图11是化合物SN-011(式I化合物)改善Trex1
-/-小鼠系统性的自身免疫反应症状的效应图:(A)取注射SN-011(5mg/kg)1个月后的WT和Trex1
-/-小鼠的脾脏,分离脾脏细胞后,流式细胞仪检测脾脏中激活T细胞(CD4
+CD69
+,CD8
+CD69
+)和记忆T细胞(CD4
+CD44
highCD62L
low,CD8
+CD44
highCD62L
low)的含量;(B)对实验组小鼠脾脏中不同亚型的T细胞含量的统计分析图;(C)注射SN-011(5mg/kg)1个月后的WT和Trex1
-/-小鼠的生存率的统计分析图;(D)取注射SN-011 1个月后的WT和Trex1
-/-小鼠的血清,使用抗核抗体检测试剂盒免疫荧光法检测小鼠血清中抗核抗体的含量,每组至少对6只小鼠的数据进行统计,实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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 ;
图12是化合物SN-011(式I化合物)在小鼠体内的药物代谢动力学评价效应图:(A)单次腹腔注射SN-011(5mg/kg)后24小时内C57BL6小鼠血浆中的药物浓度;(B)药代动力学参数统计表,每个时间点内取3只小鼠的血药浓度进行统计;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;
图13是化合物SN-011(式I化合物)在小鼠体内的毒理学评价效应图:(A)WT小鼠腹腔注射PBS或SN-011(1mg/kg),每周注射3次连续注射10周后体重统计分析图;(B)上述小鼠血清中丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST)统计分析图;(C)上述小鼠血清中血尿素氮(BUN),血清肌酸酐(CREA)统计分析图;(D)上述小鼠分别取小鼠心脏、肾、胃和肝,固定后进行石蜡切片和H&E染色,观察各组织的病理学变化,每组至少对6只小鼠的数据进行统计,实验取平均值并作方差为误差线:n.s.,无显著性差异;*,P<0.05;**,P<0.01;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;
图14是化合物SN-011(式I化合物)抑制SAVI相关的STING点突变体诱导的炎症基因表达的效应图:(A~C)HEK293T细胞中转染表达Flag-hSTING及SAVI相关的点突变体V155M,N154S,G166E,C206Y,R281Q和R284G的质粒各5μg,转染12小时后孵育化合物SN-011(10μM),化合物孵育12小时后收取细 胞检测IFN-β,CXCL10和TNF-α基因的表达;(D)HEK293T细胞中转染表达Flag-hSTING及SAVI相关的点突变体V155M,G166E,C206Y,R281Q和R284G的质粒各5μg,转染6小时后孵育化合物SN-011(10μM),化合物孵育12小时后收取细胞,用免疫共沉淀法检测STING对下游靶蛋白TBK1和IRF3的招募;(E)HEK293T细胞中转染表达Flag-hSTING及SAVI相关的点突变体V155M,G166E,C206Y,R281Q和R284G的质粒各5μg,转染6小时后孵育化合物SN-011(10μM),化合物孵育12小时后收取细胞,用免疫印迹法检测TBK1和IRF3蛋白的磷酸化表达;(F)HEK293T细胞中转染表达Flag-hSTING及SAVI相关的点突变体V155M,G166E,C206Y,R281Q和R284G的质粒各5μg,转染6小时后孵育化合物SN-011(10μM),化合物孵育12小时后收取细胞用非变性凝胶电泳的方法检测STING多聚化表达;所有数据均为三次平行实验取平均值并作方差为误差线:n.s.,无显著性差异;*,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;
图15是化合物SN-011(式I化合物)改善急性脑缺血诱导的大鼠脑损伤的效应图:(A)大脑中动脉阻塞(MCAO)诱导大鼠急性脑缺血24小时后,用TCC染色法检测大脑的梗死面积正常区域为红色,梗死区域为白色;(B~C)大鼠大脑中动脉阻塞6小时和24小时分别对大鼠的行为学进行评分统计;(D~I)大脑中动脉阻塞诱导大鼠急性脑缺血24小时后,取大脑皮层区域组织检测Ifnβ,Ifnα4,Cxcl10,Mcp1,Tnfα和Il6基因的表达;大鼠在造模时和造模12小时后腹腔注射低剂量SN-011(1mg/kg)和高剂量的SN-011(3mg/kg),每组4只大鼠的实验结果进行统计分析,n.s.,无显著性差异;*,P<0.05;**,P<0.01,给药组与MCAO组比较;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;
图16化合物SN-011(式I化合物)是改善急性脑缺血诱导的小鼠脑损伤的效应图:(A)小鼠大脑中动脉阻塞后立即腹腔注射低剂量SN-011(1mg/kg)和高剂量的SN-011(2mg/kg),24小时后,取大脑皮层区域组织检测Mcp-1,Il6和Tnfα基因的表达;(B)小鼠大脑中动脉阻塞后立即腹腔注射低剂量SN-011(1mg/kg)和高剂量的SN-011(2mg/kg),72小时后,取大脑皮层区域组织检测Mcp-1,Il6和Cxcl10基因的表达;(C)小鼠大脑中动脉阻塞后立即或24小时后再腹腔注射SN-011(2mg/kg),连续给药至卒中后第七天,使用转棒实验测定小鼠运动功能的改变;(D)小鼠大脑中动脉阻塞后立即或24小时后再腹腔注射SN-011(2mg/kg),连续给药至卒中后第七天,使用Morris水迷宫实验测定小鼠记忆功能的改变。每组不少于5只小鼠的实验结果进行统计分析,n.s.,无显著性差异;*,P<0.05;**,P<0.01,给药组与MCAO后给与Vehicle组比较;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;
图17是化合物SN-011(式I化合物)改善高脂饮食(HFD)诱导的小鼠非酒精性脂肪肝的进展的效应图:(A~B)高脂饮食诱导的小鼠NAFLD模型及给药后 各组小鼠血清中ALT和AST含量的变化;(C~D)各组小鼠血清中TC和TG的含量;(E)各组小鼠体重的变化;(F)各组小鼠肝脏重量的变化;(G)各组小鼠内脏脂肪重量(肾周脂肪)的变化;(H~I)各组小鼠肝脏中TC和TG的含量;(J~O)各组小鼠肝脏中Ifnβ,Cxcl10,Mcp-1,Tnfα,Fas和Srebp-1c基因表达的变化;(P)各组小鼠肝脏病理切片的H&E染色结果分析;C57BL6小鼠高脂饮食诱导20周,从第10周开始腹腔注射低剂量SN-011(1mg/kg)和高剂量的SN-011(2mg/kg)进行干预,每组至少8只小鼠的实验结果进行统计分析,n.s.,无显著性差异;*,P<0.05;**,P<0.01,给药组与HFD组比较;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;
图18是化合物SN-011(式I化合物)对咪喹莫特诱导银屑病样炎症小鼠右耳及背部的影响图:对照组、模型组、SN-011 250mg/kg组和SN-011 50mg/kg组小鼠的外观表现;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;
图19是化合物SN-011(式I化合物)对咪喹莫特诱导银屑病样炎症小鼠耳厚度的影响图:对照组、模型组、SN-011 250mg/kg组和SN-011 50mg/kg组小鼠右耳厚度,*P<0.05,**P<0.01,***P<0.001,n=8;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;
图20是化合物SN-011(式I化合物)对咪喹莫特诱导银屑病样炎症小鼠右耳及背部的效应图:A.小鼠耳部皮肤H&E染色图;B.小鼠背部皮肤H&E染色图,比例尺50μm(x200);C.取小鼠耳部皮肤进行H&E染色,对棘皮厚度进行分析,比例尺20μm(x400);D.取小鼠背部皮肤进行H&E染色,对棘皮厚度进行分析,比例尺20μm(x400),*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;
图21是化合物SN-011(式I化合物)抑制STING信号通路激活的作用机制示意图。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.
以下结合附图和实施例对本发明作进一步说明。本发明不受到这些实施例的限制。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。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、化学合成试剂、材料1. Chemical synthesis reagents and materials
本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售获得。The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained from the market.
化合物的结构是通过核磁共振(NMR)或(和)质谱(MS)来确定的。NMR的测定是用(Bruker)核磁仪,测定溶剂为氘代二甲基亚砜(DMSO-d
6),氘代氯仿(CDCl
3),内标为四甲基硅烷(TMS)。
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).
柱层析一般使用青岛海洋化工厂分厂硅胶200-300目硅胶为载体。Column chromatography generally uses 200-300 mesh silica gel from Qingdao Ocean Chemical Plant Branch as the carrier.
本发明的已知起始原料可以采用或按照本领域已知的方法合成,或可购买于乐研、毕得医药、阿拉丁、安耐吉等公司。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、细胞及细胞培养2. Cells and cell culture
本发明所使用的细胞系包括人肾胚细胞HEK293T,小鼠胚胎成纤维细胞MEF和L929,人宫颈癌细胞Hela,人原代包皮成纤维细胞HFF等。除特别指出外,所有的细胞使用含有10%胎牛血清(Gibco)及50U/mL盘尼西林和50μg/mL的链霉素(Gibco)的DMEM培养基(Gibco)培养,L929使用1640培养基培养。骨髓来源的巨噬细胞BMDM由小鼠股骨造血干细胞分化得到。分化时间一般是7天,用含L929上清的条件培养基培养,分化完成后即可进行相应的刺激。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、小鼠品系及体内药效学、药动学及毒理学评价3. Evaluation of mouse strains and in vivo pharmacodynamics, pharmacokinetics and toxicology
6~8周的C57BL/6J小鼠购自南京大学模式动物研究所,Trex1杂合小鼠由Dr.Nan Yan(University of Texas Southwestern Medical Center)惠赠。纯合基因敲除小鼠由杂合小鼠杂交得到(Cell Reports 2018,25,3405–3421)。BALB/c小鼠(用于抗银屑病药效实验),雌性,日龄56-62天,由浙江维通利华实验动物有限公司提供。实验中大鼠和小鼠在中国药科大学药物安全评价中心的SPF级动物房饲养。动物实验严格按照中国药科大学动物管理委员会所制定的操作守则执行。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.
在Trex1
-/-小鼠自发的自身免疫疾病模型中,小鼠从第4周开始腹腔注射待测化合物SN-011,给药剂量是5mg/kg小鼠体重,用终浓度2%的Tween20和2%的DMSO促进化合物溶解,溶解液为PBS,每周注射三次,连续给药30天。
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.
评价SN-011在C57BL6J小鼠体内的药物代谢动力学实验中,腹腔注射5mg/kg SN-011后,分别在0.083,0.25,0.5,1,2,4,8,24小时取小时的血浆用于血药浓度的检测。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.
化合物SN-011在小鼠体内的毒理学评价实验使用C57BL6J小鼠腹腔注射1mg/kg SN-011每周三次,连续注射10周,注射完成后检测小鼠血清生化指标及组织器官的病理学变化。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. .
在大鼠中动脉阻塞诱导的急性脑缺血模型中,取体重280-300g的雄性SD大鼠,分别在手术前和手术后12小时给予化合物SN-011,低剂量为1mg/kg,高剂量为3mg/kg。手术后24小时进行相关指标的检测。分别在手术后6小时和24小时对实验组大鼠进行行为学评分。评分规则如下:抓起尾巴只向左侧弯曲为1分。在地上抓住尾巴只向左侧转圈为2分,放开尾巴也只向左侧转圈为3分。左半身瘫痪为4分。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.
小鼠中动脉阻塞诱导的急性脑缺血模型中,取体重25-30g的雄性C57/6J小鼠,在手术后立即给药或手术后24小时给予化合物SN-011,每天给药1次持续至实验终结,低剂量为1mg/kg,高剂量为2mg/kg。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.
在高脂饮食(HFD)诱导的小鼠非酒精性脂肪肝(NAFLD)模型中,取4~6周龄的雄性C57BL6J小鼠饲养高脂饲料(research diets,60%Kcal High-Fat Diets, D12492)。高脂饲料喂养10周后,开始腹腔注射化合物SN-011,低剂量为1mg/kg,高剂量为2mg/kg。每周三次给药,连续给药10周,模型组给予含2%的Tween20和2%的DMSO的PBS溶液。第20周给药完成后进行相关指标的检测。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.
在化合物改善咪喹莫特诱导的银屑病样炎症药效学实验中,BALB/c小鼠,雌性,日龄56-62天,共32只。于普通环境下饲养,自由摄食饮水。化合物SN-011药物软膏的配制:250mg/kg组:1)将560mg化合物SN-011溶于1ml的DMSO中;2)将羊毛脂(11.2g)和凡士林油(11.2g)加入1mL乙醚,在34℃下加热溶解;3)将1)的溶液加入到2)的混合物中,再加入3ml乙醇搅拌均匀,加热若干分钟;4)将溶于0.4ml乙醚的尼泊金甲酯0.05g和尼泊金丙酯0.02g加入到3)的混合物中,用200l乙醚刷洗1-2次再加入混合物中;5)将4)的混合物在88℃下加热60分钟,蒸除乙醇和乙醚,即制得软膏。50mg/kg组:1)将112mg化合物SN-011溶于500l的DMSO中;2)将羊毛脂(11.2g)和凡士林油(11.2g)加入1mL乙醚,在34℃下加热溶解;3)将1)的溶液加入到2)的混合物中,再加入3ml乙醇搅拌均匀,加热若干分钟;4)将溶于0.4ml乙醚的尼泊金甲酯0.05g和尼泊金丙酯0.02g加入到3)的混合物中,用200 l乙醚刷洗1-2次再加入混合物中;5)将4)的混合物在88℃下加热60分钟,蒸除乙醇和乙醚,即制得软膏。将32只BALB/c小鼠按体重随机分为空白对照组(Control组)、模型对照组(Model组)、SN-011 250mg/kg组和SN-011 50mg/kg组,每组8只,用剃毛器去除背部毛发,露出2cm×3cm的皮肤区域。5%咪喹莫特(Imiquimod,IMQ)乳膏(局部剂量62.5mg)每天对右耳及背部给药。实验第一天至第五天,上午造模下午给药,实验第六天至第七天,上午下午均给药,中午造模,实验周期为7天。从咪喹莫特乳膏涂抹造模开始,每天对小鼠进行拍照并观察小鼠右耳及背部情况。每天测量每只小鼠的右耳厚度。取小鼠背部及右耳皮肤标本浸泡于4%多聚甲醛中,石蜡包埋。用苏木精和伊红染色(H&E)。银屑病皮损面积和疾病严重程度(PASI)评分、右耳厚度等均采用graphpad prism 5软件进行数据处理,多组间采用单因素方差分析(One-Way ANOVA)处理。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、小鼠组织切片及相关病理学评价4. Evaluation of mouse tissue sections and related pathology
取小鼠组织切片,H&E染色评价组织的损伤及炎症细胞浸润。具体实验步骤如下:1)小鼠给药周期完成后,取小鼠的各个脏器组织,用4%PFA固定过夜备用;2)取材:将固定后的组织按观察部位的需要对组织横截面进行相应的修整,放入包埋框内,梯度脱水;3)脱水完成后,用石蜡包埋组织,保存组织样本的蜡块;4)修片:修出完整的组织切面后即可切片,切片的厚度为5μm;5)将切片放在水上展片后,用玻片捞起,56℃烘片使样本上的石蜡溶解;6)梯度复水后H&E染色,梯度脱水,封片,保存;7)正置显微镜下观察组织的 病理变化,拍照记录即可。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、血清生化指标的检测5. Detection of serum biochemical indicators
小鼠血清的生化指标包括ALT,AST,TC,TG,GLU,BUN和CREA,上述指标检测均在中国药科大学药物安全评价中心使用Dimention X-Pand plus生化分析仪检测。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、小鼠肝脏甘油三酯(TG)和总胆固醇(TC)含量的检测6. Detection of triglyceride (TG) and total cholesterol (TC) content in mouse liver
小鼠肝脏中的TC和TG的含量使用TG含量检测试剂盒(solarbio,BC0625)和TC含量检测试剂盒(solarbio,BC1985)按照说明书要求进行检测。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、脾脏T细胞含量的检测7. Detection of T cell content in spleen
流式细胞术检测SN-011对Trex1
-/-小鼠脾脏中CD4
+和CD8
+T细胞含量的影响。实验步骤如下:1)脾细胞过200目的滤网上后用10ml的PBS冲洗滤网上的细胞至离心管内,重悬细胞,1200rpm离心5分钟;2)用4ml的红细胞裂解液裂解细胞;3)封闭,每管加入20μl的Fc blocking,4℃封闭10分钟,1200rpm离心后,PBS洗一次,PBSA(1%BSA in PBS)重悬细胞;4)抗体染色,抗体1:100稀释到PBSA内混匀后与细胞混合,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)抗体加入后混匀,4℃避光孵育30分钟后加入PBS洗一遍,用500μl的PBS重悬即可上机(Attune NxT cytometer,Thermo Fisher scientific)检测相应T细胞亚型含量。
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、小鼠血清中自身免疫抗体的检测8. Detection of autoimmune antibodies in mouse serum
使用ANA(HEp-2)antigen substrate slide kit(MBL-BION)检测试剂盒检测血清抗核抗体(ANA)方法如下:1)从试剂盒里取出铺满HEp-2细胞的基质片,室温下平衡;2)用2%BSA in PBS 1:50稀释血清后用其孵育HEp-2细胞,室温反应30分钟后用PBS洗去血清;3)用吸水纸将孔径周围的水吸干,加上FITC标记anti-mouse IgG二抗,室温反应30分钟后用PBS洗去二抗;4)用防荧光猝灭的封片剂Dako封片,镜检。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、分子对接法筛选结合STING蛋白的小分子化合物9. Molecular docking method to screen small molecule compounds that bind to STING protein
hSTING-CTD结晶结构PDB:4EF5用于分子对接筛选,分子对接的软件使用DOCK3.7,虚拟筛选使用小分子虚拟数据库ZINC15(http://zinc15.docking.org)。本实验所使用柔性对接程序计算小分子配体和蛋白受体间的分子能量状态energy score,即通过打分的方式评价小分子与受体间的范德华能和静电能量。实验过程如下:1)蛋白晶体结构:从PDB数据库中选取hSTING-CTD结构文件4KSY,删除蛋白上存在的配体后,在Dock Pre框架下去除结构中的金属离子、溶剂分子并在结构上加上氢原子和电荷完成受体蛋白分子的准备;2)配体小分 子的准备:从ZINC15中下载包含小分子结构的数据后,用Marvin软件在小分子结构中加氢后用Corina软件将小分子转化为3D空间结构,小分子单体能量状态用AMSOL软件计算;3)将准备好的配体和蛋白晶体文件上传到计算机服务器上;4)在2’,3’-cGAMP与蛋白结合的口袋处产生Grid用于评价该空间区域内的能量状态;5)计算格点能量状态:AMBER用于计算范德华能,QNIFFT用于计算静电能量;6)虚拟筛选:将数据库中的小分子结构在受体格点的口袋内进行分子对接,查看对接结果,判断蛋白和化合物结合的可能性。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、细胞活力的评价10. Evaluation of cell viability
检测化合物在细胞中的毒性作用,使用Promega的Cell Proliferation Assay试剂盒检测。实验过程如下:1)根据细胞生长速度的快慢不同,将不同个数的细胞提前一天点至96孔板内;2)待细胞长至合适的密度75%以上时,分别孵育不同浓度的化合物相应的时间;3)化合物孵育完成后每孔加入20μl的MTS工作液反应1小时后,在490nm的波长处检测吸光值并计算化合物对细胞生存率的影响。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、细胞的转染11. Transfection of cells
将目的质粒转染进细胞内的表达方法有两种,氯化钙-HEPES钙转体系和脂质体转染体系,钙转主要针对293T细胞,脂质体转染主要将ISD,HT-DNA等刺激物转入细胞质内。钙转方法如下:待细胞长至密度为70%左右时开始钙转,将目的质粒与CaCl
2混匀后加入HEPES溶液吹打混匀,然后逐滴滴加入细胞内,转染24小时使质粒在细胞内表达。脂质体转染方法如下:将刺激物与lipo 2000以1:1的比例分别加入到Opti-MEM溶液中,静置5分钟后将刺激物加入lipo2000溶液内,混匀加入细胞内,即可完成转染。刺激6小时后,收取细胞即可进行后续实验操作。
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激活细胞内STING信号通路12. CDNs activate the intracellular STING signaling pathway
细胞在培养板中贴壁生长,孵育相应浓度的待测化合物过夜,孵育完成后用含有50mM HEPES(pH=7.0),100mM KCl,3mM MgCl
2,0.1mM DTT,85mM蔗糖的缓冲液,溶解CDNs刺激物,以及(0.2%(m/v)BSA,1mM ATP,0.1mM GTP,10μg/ml Digitonin),配成工作液孵育细胞透膜,使CDNs分子进入细胞质中,进而激活内质网上的STING蛋白,CDNs刺激细胞3小时后收取细胞检测相关基因的表达。
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、检测化合物在细胞中的半数抑制率(IC
50)
13. Test the half inhibition rate (IC 50 ) of the compound in the cell
HFF细胞在培养板中贴壁生长,待细胞长至80%丰度时开始化合物的孵育,分别孵育待测化合物(化合物溶于DMSO中,化合物在培养基中的初筛终浓度:1和10μM。IC
50测试浓度:5,2.5,1.25,0.625,0.3125,0.15625,0.078125, 0.039μM)过夜,空白对照组只加DMSO。孵育完成后用含有2'3'-cGAMP刺激物的洋地黄皂苷溶液(2'3'-cGAMP终浓度:1μg/mL)处理细胞,使2'3'-cGAMP分子进入细胞质中,进而激活内质网上的STING蛋白,2'3'-cGAMP刺激细胞3小时后收取细胞,检测Ifnb基因的表达。化合物在1和10μM浓度下对2'3'-cGAMP刺激后STING信号通路的抑制率依据Ifnb基因表达倍数计算得到:1-[Ifnb(化合物组)/Ifnb(DMSO组)]。IC
50值按照化合物处理组相对DMSO空白对照组抑制Ifnb基因表达的比率曲线拟合得到。
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、质粒构建14. Plasmid construction
STING,TBK1,IRF3,cGAS相关的序列信息在NCBI的Genbank中查询,质粒的cDNA用PCR的方法从胸腺cDNA文库中获得,并克隆到相应的真核表达载体中得到相应的质粒。所有质粒的点突变体均使用QuickChange XL site-directed mutagenesis methods(Stratagene)。质粒的克隆方法如下:1)设计对应质粒片段的克隆引物,保护碱基-酶切序列-目标引物(20-25bp);2)使用DNA聚合酶KOD对目的基因序列进行PCR扩增;3)扩增的DNA产物琼脂糖凝胶回收;4)回收产物和使用的载体进行双酶切和连接;5)连接产物涂板,挑取阳性克隆,抽取质粒测序比对目的基因序列的正确性即完成质粒的克隆。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、免疫印迹法检测目的蛋白的表达15. Detect the expression of the target protein by western blotting
免疫印迹法(western blot)的实验流程如下:1)细胞用蛋白裂解液(0.5%TritonX-100,1mM EDTA,1%Cocktail溶解于TBS缓冲液)裂解后高速离心,取上清蛋白裂解液检测蛋白浓度,利用BCA法检测蛋白浓度试剂盒检测蛋白浓度后,根据蛋白浓度拉平样本的体积和浓度;2)样本中加入5×loading buffer制样,根据目的蛋白分子量大小跑相应浓度的SDS-PAGE凝胶;3)转膜,将凝胶上分离的蛋白湿转至PVDF膜上;4)封闭,普通蛋白用5%脱脂牛奶封闭,磷酸化蛋白用5%BSA封闭;5)一抗孵育,抗体按照相应的浓度稀释后4℃孵育2小时或过夜后用TBST缓冲液洗6遍,每次6分钟,以洗去非特异性吸附;6)二抗孵育,对应属性的二抗按照相应的浓度稀释后室温孵育1小时后,用TBST缓冲液洗6遍,每次6分钟,以洗去非特异性吸附;7)显色,ECL发光液孵育后用Bio-Rad的ChemiDoc检测蛋白的化学发光条带即可反应蛋白的表达。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、非变性聚丙烯酰胺凝胶电泳分析蛋白的二聚化和多聚化表达水平16. Non-denaturing polyacrylamide gel electrophoresis analysis of protein dimerization and multimerization expression levels
实验方法如下:1)配置相应浓度的非变性聚丙烯酰胺凝胶,凝胶中不含SDS;2)将配置好的非变性胶在缓冲液(25mM Tris,192mM Glycine,pH8.4,内槽液中加入0.2%的脱氧胆酸钠)中以40mA预电泳30分钟,用电泳液平衡非变性胶;3)细胞用蛋白裂解液(0.5%TritonX-100,1mM EDTA,1%Cocktail溶解于TBS缓冲液)裂解后,加入5×native的上样缓冲液制样;4)电泳条件为25mA恒流 1-1.5小时,后续按照标准的免疫印迹法检测即可。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、免疫共沉淀法检测蛋白质间的相互作用17. Co-immunoprecipitation method to detect protein-protein interactions
蛋白质的相互作用用免疫共沉淀法(Co-IP)检测方法如下:1)向细胞内加入蛋白裂解液(0.5%TritonX-100,1mM EDTA,1%Cocktail溶解于TBS缓冲液)裂解后超声至细胞裂解液澄清,4℃13000rpm离心15分钟,收取上清蛋白液;2)特异性识别目的蛋白,向蛋白裂解液中加入目的蛋白抗体1μl,4℃摇匀4小时孵育;3)钓取目的蛋白,向蛋白裂解液中加入预制好Protein G Agarose20μl放回4℃继续孵育2小时,使Beads与抗体结合,用缓冲液(0.5%TritonX-100,1mM EDTA)洗去agarose上的非特异性吸附蛋白;5)制样,向管内加入蛋白5×loading buffer,95℃6分钟使蛋白变性,后续按标准的免疫印迹法检测兴趣蛋白的表达,即可判断出目的蛋白和兴趣蛋白有无存在相互作用。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、免疫荧光18. Immunofluorescence
细胞的免疫荧光实验操作如下:1)将细胞铺在盖玻片上生长至合适的密度;2)细胞进行对应的化合物孵育与刺激后加入4%多聚甲醛室温固定1小时,PBS洗两遍后用破膜缓冲液(0.25%Triton,1mM EDTA溶解于PBS)透膜20-30分钟;3)将玻片转移至暗合内,加入封闭液(5%BSA溶解于PBS)封闭至少1小时后孵育上相应检测蛋白的一抗(1:1000稀释于PBST),4℃过夜;4)一抗孵育完成后,加PBS洗去非特异性吸附后加入荧光标记的二抗,室温避光孵育1小时;5)孵育完成后用DAPI染细胞核1分钟;6)核染后,使用防荧光淬灭剂封片,避光保存,激光共聚焦显微镜Carl Zeiss LSM700的63x NA1.4油镜下观察细胞内蛋白的荧光分布。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、His-hSTING-CTD(149-379)蛋白的原核表达和纯化19. Prokaryotic expression and purification of His-hSTING-CTD (149-379) protein
在大肠杆菌表达目的蛋白片段的实验方法如下:1)构建包含目的蛋白片段的表达载体质粒His-STING-149-379;2)1μg质粒经转化,转接,18℃下用终浓度0.5mM的IPTG诱导蛋白在大肠杆菌中大量表达;3)用缓冲液(25mM Tris8.0,150mM NaCl,pH=8.0)细菌收取后,超声破碎细菌15分钟,至菌液稍澄清即可;4)高速离心(18000rpm,30分钟),收取上层蛋白液;5)挂1.5ml Ni柱吸附目的蛋白至Ni柱上,随后洗去非特异性结合;6)过梯度咪唑,80mM,250mM,500mM的梯度咪唑将目的蛋白从Ni-NTA Agarose上洗脱下来;7)跑SDS-PAGE胶,考马斯亮蓝染色,检测目的蛋白的表达;8)收集目的蛋白溶液,用50KD超滤管浓缩蛋白溶液后将浓缩蛋白液过Akata蛋白纯化系统,将杂蛋白除去后收取目标蛋白;9)用于结晶实验的His-STING-149-379蛋白预先用TEV酶切His-tag标签后,过分子排阻色谱柱纯化无标签的目的蛋白后浓缩至15mg/ml的浓度;10)纯化后的目的蛋白浓缩至合适的浓度,分装,液氮速冻后置 于-80℃保存。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、SN-011与hSTING-CTD(149-379)蛋白共结晶及数据的处理与结构解析20. Co-crystallization of SN-011 and hSTING-CTD (149-379) protein and data processing and structure analysis
15mg/ml的STING-CTD(149-379)蛋白溶解在25mM Tris-HCl,pH 7.5,150mM NaCl的溶液中,在100μL的蛋白溶液中加入1μL的SN-011(50mM),孵育1小时后,高速离心取上清蛋白液用于结晶。使用悬滴法将1μL的蛋白液与1μL的池液(0.1M HEPS-NaOH,pH 7.0,0.1M sodium formate,25%PEG3350)混合,在16度环境中使晶体生长,晶体长成后用含30%PEG 3350的冻存液将捞取的晶体在液氮中保存。X射线衍射在上海国家光源中心进行,衍射数据的收取,整合和处理使用HKL3000软件,以apo-STING(PDB:4F5W)为模版使用分子替换的方法进行结构的解析。共结晶结构使用COOT软件进行调整并使用CCP4软件进行结构的精修。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、表面等离子共振技术(SPR)检测SN-011与His-STING-CTD(149-379)蛋白间的亲和力21. Surface plasmon resonance technology (SPR) detects the affinity between SN-011 and His-STING-CTD (149-379) protein
SPR实验温度25℃,实验仪器为Biacore T200SPR instrument(GE Healthcare)。纯化的2μg/ml hSITNG-CTD蛋白及其突变体在ph=5.5条件下,使用Amine Coupling Kit固定在Sensor Chip CM5(carboxymethylated dextran surface)芯片上。SN-01梯度稀释(nM):15.6,7.81,3.91,1.85,0.93和cGAMP梯度稀释(nM):125,62.5,31.3,15.6,7.81,以30μl min
-1的速度流经芯片表面,结合解离时间都是90s。实验中使用的缓冲液都是含有0.05%Tween 20的PBS。所有数据都在相同的条件下重复了两次。最终亲和力数据利用软件Biacore T200 Evaluation software version 3.0(GE Healthcare)中的1:1结合模型拟合得到。
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、转棒试验(Rotarod test)22. Rotarod test
用XR1514型号的大小鼠转棒疲劳测试仪(购自上海欣软信息科技有限公司)进行检测,设置转速为24r/min,最长记录时间为5min,超5min一律登记为5min。在正式实验前先将小鼠训练3次。正式实验为记录小鼠在转棒上的运动时间,测试3次取平均值为最终结果。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水迷宫测试23, Morris water maze test
1)将小鼠头朝池壁放入水中,将平台置于第四象限,从池壁四个起始点的任一点将小鼠面向池壁放入水池。记录动物找到水下平台的时间(s)。在前几次训练中,如果这个时间超过60s,则引导小鼠到平台。让其在平台上停留10s.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)将小鼠移开、擦干。每只小鼠每天从不同象限入水训练,两次训练之间间隔15~20min,连续训练5天。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)最后一次获得性训练结束后的第二天,将平台撤除,开始60s的探查训练。将小鼠由原先平台象限的对侧放入水中。记录小鼠在目标象限(原先放置平 台的象限)所花的时间和和运动路程占总时间和运动路程的比例,以此作为空间记忆的检测指标;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抽提及实时荧光定量PCR技术检测目的基因的表达24. RNA extraction and real-time fluorescence quantitative PCR technology to detect the expression of the target gene
细胞或组织中总RNA的抽提过程如下:1)取适量的细胞和组织用TRIzol(Invitrogen)充分裂解后,1:5加入氯仿萃取裂解液中的RNA,4℃12000g 15分钟离心;2)上层水相溶液中加入相同体积异丙醇将溶液中的RNA沉淀下来;3)RNA沉淀用1ml 75%的乙醇洗去杂质;4)RNA干燥透明后用适量的DEPC水55℃溶解,OD260处检测RNA浓度。总RNA抽提完成后即可用于后续的荧光定量PCR检测目的基因的表达,实验步骤如下:RNA与引物Oligo dT混合后在反转录酶试剂盒的作用下转录成cDNA,cDNA与目的基因的引物以及DNA聚合酶和荧光染料FastStart Universal SYBR GREEN MASTER MIX(Roche)混合后,在ABI QuantStudio 3仪器中进行扩增和检测,GAPDH为基因表达的内参,2
-ΔΔCT法进行相对定量计算目的基因的表达。检测目的基因所使用的引物序列见下表1:
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:
表1、qPCR primer sequencesTable 1. qPCR primer sequences
25、RNA-Sequencing25, RNA-Sequencing
从6~8周WT或Trex1
-/-的小鼠的大腿股骨中分离骨髓细胞后,使用含L929上清的条件培养基将细胞诱导分化至BMDMs。诱导完成后按实验组别设计分别孵育SN-011(500nM)12小时,提取细胞中RNA并按照标准的Illumina RNA-seq protocol将RNA整合至cDNA文库。产生的cDNA文库用Illumina HiSeq2000以1×100bp run进行测序,测序结果处理后与小鼠的基因组进行比对,做热图反应基因表达的差异并进一步聚类分析。
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.
实施例1Example 1
N-(3-((4-氟苯基)磺酰氨基)-4-羟基苯基)-[1,1'-联苯]-4-甲酰胺(化合物I)N-(3-((4-Fluorophenyl)sulfonamido)-4-hydroxyphenyl)-[1,1'-biphenyl]-4-carboxamide (Compound I)
将化合物1(2.00g,13.0mmol)和吡啶(1.54g,19.5mmol,1.57mL)溶解在40mL的二氯甲烷中,在0℃条件下逐滴加入溶解在20mL二氯甲烷中的4-氟苯磺酰氯1A(3.04g,15.68mmol),混合后在25℃搅拌反应12小时。随后用薄层色谱法(TLC)鉴定反应完全,流动相为石油醚:乙酸乙酯=2:1。溶剂在减压下蒸发除去,粗产物用硅胶色谱柱纯化(流动相为石油醚:乙酸乙酯=10:1~2:1, V/V),得淡黄色固体产物化合物2(2.74g,67.6%收率)。
1H 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 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] + .
将化合物2(1.50g,4.80mmol)溶解在15mL的甲醇中,加入10%Pd/C(0.1g),在25℃氢气氛围下反应16小时,反应产物用LC-MS鉴定判断反应完全。反应液用硅藻土填充柱过滤,减压除去溶剂,得1.4g黄色固体产物化合物3。
1H 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=8.4Hz,1H),6.19(dd,J1=2.4Hz,J2=8.4Hz,1H),4.60(br.s,1H),3.17(s,1H).LCMS(m/z):282.05[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] + .
在10mL二氯甲烷中溶解990mg的化合物3,随后加入229mg的咪唑并逐滴滴入溶解在1mL二氯甲烷中的507mg的TBSCl(叔丁基二甲基氯硅烷)。反应在15℃条件下搅拌12小时,LC-MS鉴定反应产物判断反应完全。反应液用水(20mL x 3)洗,无水Na
2SO
4干燥,在减压条件下将溶剂蒸除。粗产物用Al
2O
3填充色谱柱纯化(流动相为二氯甲烷:甲醇=10:1,V/V),得红棕色固体化合物4。
1H 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]
+.
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] + .
在5mL的二氯甲烷中加入185mg的化合物4A,215mg EDCl(1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐),139mg HOBt(1-羟基苯并三氮唑)和181mg的DIPEA(N,N-二异丙基乙胺),最后加入370mg的化合物4。反应在15℃条件下搅拌16小时,LC-MS鉴定反应产物判断反应完全。反应产物用10mL的二氯甲烷稀释,用水(20mL x 3)洗,无水Na
2SO
4干燥,在减压条件下将溶剂蒸除,得400mg红棕色固体粗产物化合物5,不经纯化直接用于后续反应。
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.
在15mL的二氯甲烷中加入630mg的化合物5和176mg的Et
3N.3HF,反应在15℃条件下搅拌3小时,LC-MS鉴定反应产物判断反应完全。向反应液中加入25mL的二氯甲烷稀释,用水(20mL x 2)洗,无水Na
2SO
4干燥,在减压条件下将溶剂蒸除。残余物用硅胶色谱柱法纯化(流动相为石油醚:乙酸乙酯=5:1),得类白色固体化合物I(260mg,51.5%收率)。
1H 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]
+.
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] + .
实施例2Example 2
N-(3-((4-氟苯基)磺酰氨基)-4-甲氧基苯基)-[1,1'-联苯]-4-甲酰胺(化合物II)N-(3-((4-Fluorophenyl)sulfonylamino)-4-methoxyphenyl)-[1,1'-biphenyl]-4-carboxamide (Compound II)
将化合物6(0.54g,3.2mmol),吡啶(0.5mL)加入二氯甲烷(10mL)中,冰水浴下滴加4-氟苯磺酰氯(1A:0.744g,3.84mmol)的二氯甲烷(10mL)溶液,滴毕,室温反应8h。减压蒸除溶剂,加入乙酸乙酯(50mL)和水(20mL),摇匀分液,有机相分别用1N HCl(10mL)、水(20mL)和饱和食盐水(20mL)洗涤,无水Na
2SO
4干燥,过滤,减压蒸除溶剂得残留物,粗产物用硅胶色谱柱纯化(流动相为石油醚:乙酸乙酯=10:1~2:1,V/V),得淡黄色固体产物化合物7(0.74g,71%)。
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%).
将化合物7(0.5g,1.53mmol),10%Pd/C(50mg)加入MeOH(20mL)中,H
2氛围反应12h。滤除Pd/C,用MeOH洗涤滤饼,滤液减压蒸除溶剂,得黄褐色固体产物化合物8(0.43g)。
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.
将化合物4A(119mg,0.60mmol)和DIPEA(0.16mL,0.96mmol)加入THF(4mL)中,加入HATU(O-(7-氮杂苯并三氮唑-1-基)-N,N,N',N'-四甲基脲六氟磷酸酯)(291mg,0.77mmol),室温反应1h后,加入化合物8(237mg,0.80mmol),室温反应5h。加入乙酸乙酯(20mL)和水(10mL),分液,有机相分别用1N HCl(5mL)、水(10mL)和饱和食盐水(10mL)洗涤,无水Na
2SO
4干燥,过滤,减压蒸除溶剂得残留物,经硅胶柱层析纯化(流动相为石油醚:乙酸乙酯=5:1),得类白色固体化合物II(117mg,41%)。
1H 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]
+.
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] + .
实施例3Example 3
4-([1,1'-联苯基]-4-甲酰胺基)-2-((4-氟苯基)磺酰氨基)苯基乙酸酯(化合物V)4-([1,1'-biphenyl]-4-carboxamido)-2-((4-fluorophenyl)sulfonamido)phenyl acetate (Compound V)
将由实施例1制备的化合物I(200mg,0.43mmol)溶于THF(5mL),加入吡啶(52μL,0.65mmol),室温下滴加乙酰氯(37μL,0.52mmol),室温反应10h。用水(10mL)淬灭,EtOAc(30mL)提取,有机相依次用1N HCl(10mL)、水(10mL)和饱和食盐水(10mL)洗涤,无水Na
2SO
4干燥,过滤,减压蒸除溶剂,得白色固体,经硅胶柱层析(DCM/MeOH=10/1)纯化,得白色固体即化合物V(76mg,35%)。
1H 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=7.4Hz,2H),7.62(dd,J=8.8,2.3Hz,1H),7.52(dd,J=7.4,7.4Hz,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
3CO]
-.
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] - .
实施例4Example 4
4'-((3-((4-氟苯基)磺酰氨基)-4-甲氧基苯基)carbamoyl甲酰胺基)-[1,1'-联苯基]-4-基乙酸酯(化合物III)4'-((3-((4-Fluorophenyl)sulfonamido)-4-methoxyphenyl)carbamoylcarboxamido)-[1,1'-biphenyl]-4-ylacetic acid Ester (Compound III)
参照实施例2的方法制得化合物III:ESI-MS:m/z 491.3[M-CH
3CO]
-.
Refer to the method of Example 2 to prepare compound III: ESI-MS: m/z 491.3[M-CH 3 CO] - .
实施例5Example 5
N-(4-甲氧基-3-(苯磺酰胺基)苯基)-[1,1'-联苯基]-4-甲酰胺(化合物IV)N-(4-Methoxy-3-(benzenesulfonamido)phenyl)-[1,1'-biphenyl]-4-carboxamide (Compound IV)
参照实施例2的方法制得化合物IV:ESI-MS:m/z 459.1[M+H]
+.
Refer to the method of Example 2 to prepare compound IV: ESI-MS: m/z 459.1 [M+H] + .
实施例6Example 6
基于STING蛋白晶体结构的虚拟筛选及体外活性评价Virtual screening and in vitro activity evaluation based on the crystal structure of STING protein
本发明基于人源STING蛋白C端结构域晶体结构的解析(hSTING-CTD-139-379,PDB:4EF5),进行了计算机模拟筛选潜在的与STING结合的小分子。使用DOCK3.7软件将ZINC15(http://zinc15.docking.org)虚拟化合 物数据库中小分子与hSTING-CTD结构进行对接筛选,筛选完成后共有152个小分子响应hSTING-CTD的结合。通过分析筛选出的小分子化合物发现有一类含有1-磺胺-3-酰胺-苯环骨架结构的小分子化合物对hSTING-CTD结构具有很好的响应性。在此基础上,从EnamineStore公司购买了具有此特征结构的小分子化合物进行体外生物学活性评价。结果发现,在DNA类似物ISD或HT-DNA等诱导刺激的MEF细胞或L929细胞中,式I化合物(SN-011)、式II化合物(SN-001)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)能够显著抑制Ifnb等免疫炎症因子基因的表达(图1A,图2A~2H,图3A,图3C,图3E),且不影响静息状态下Ifnb基因的表达(图1B)。化合物SN-100(CAS:1384744-19-7)不影响ISD诱导的MEF细胞中IFN-β基因的表达,因此,选择此化合物作为阴性对照化合物用于后续的实验。此外,在HT-DNA刺激的L929细胞中,SN-001显著抑制STING,TBK1,IRF3,P65,IκBα的磷酸化表达和IRF3蛋白的二聚化(图2I),随后转录因子IRF3和P65的入核在孵育化合物后也显著降低(图2J~2K),而阴性对照化合物SN-100均不影响上述蛋白的磷酸化表达与转录因子的入核。为了排除SN-001的细胞毒性对其生物学活性的影响,在MEF,L929和THP-1细胞中用MTS法检测化合物对细胞活力的影响,结果表明,SN-001(5μM~20μM)孵育至24小时均不影响细胞的活力(图2L~2N)。图1C和图3B显示了通过虚拟筛选后所购买的部分化合物的结构。需要说明的是,图1A和1B中的化合物ZINC72311784等化合物均为商业购买的化合物(有些化合物的结构并未显示)。图1和3中部分化合物的CAS号如下: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)。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).
在MEF细胞中,使用STING信号通路的经典激动剂ISD,HT-DNA,HSV-1,c-di-GMP和2’3’-cGAMP进行刺激,预孵育化合物SN-011(式I化合物)能显著下调上述刺激物诱导的Ifnb,Cxcl10和Il-6基因的表达(图3C~3E)。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).
进一步通过评价SN-011(式I化合物)在不同细胞中的半数抑制率(IC
50)研究化合物的体外生物活性。在MEF(小鼠胚胎成纤维细胞),BMDM(小鼠骨髓原代巨噬细胞)和HFF(人原代包皮成纤维细胞)中,SN-011的IC
50数值分别为127.5±5.5nM,121.7±32.2nM和502.8±94.5nM(图4A~4C)。SN-011在体外同样具有很好的安全性,具体表现为在MEF,BMDM和HFF细胞中孵育化合物最高至20μM的浓度至48小时,均不表现出对细胞活力的影响(图4D~4F)。在STING敲除的MEF细胞中,预孵育SN-011并不影响LPS,CpG-DNA, poly(I:C)和Sendai Virus激活的IFR3和NF-κB下游基因Ifnb,Tnf-α和Il-6的表达(图5A~5B)。此外,预孵育SN-011也不影响Ifnβ激活STING敲除MEF细胞中Isg15和Isg56基因的表达(图5B)。
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).
以上结果表明,本发明的式I~V化合物能高效、安全且特异性地抑制cGAS-STING信号通路的激活。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.
实施例7Example 7
SN-011(式I化合物)抑制STING蛋白的激活及其介导的下游信号传导SN-011 (compound of formula I) inhibits the activation of STING protein and its downstream signal transduction mediated by it
静息状态下STING蛋白以同源二聚体的形式以其N端跨膜区(amino acid 1-137)锚定在内质网上,当STING与其配体CDNs分子如2’,3’-cGAMP结合后,STING二聚体的空间构象发生变化,具体表现为C端结构(amino acid 138-379)相对于跨膜区发生180°的旋转后,以并肩排列的方式多个二聚体分子形成四聚体及更高的多聚体形式(Nature,2019,567(7748):389-393)。多聚化的STING蛋白由内质网向高尔基体转移,在此过程中STING多聚体以其C末端区域的PLPLRT/SD序列招募激酶蛋白TBK1并促进TBK1(Ser172)的自身磷酸化激活(Nature,2019,567(7748):394-398;Nature,2019,569(7758):718-722)。激活的TBK1进一步磷酸化STING多聚体pLxIS366序列中的丝氨酸,进而促进转录因子IRF3的招募,IRF3招募到多聚体复合物后进一步被TBK1磷酸化激活(Science,2015,347(6227):aaa2630;Proceedings of the National Academy of Sciences,2016,113(24):E3403-E3412),这就是蛋白水平STING的激活及其介导下游信号传递的过程。以此为依据,本发明发现,预孵育SN-011能显著抑制2’,3’-cGAMP诱导的HFF细胞中STING的多聚化、磷酸化(图6A)及其向高尔基体上的转移(图6E)。此外,SN-011能显著减少STING蛋白对下游TBK1和IRF3的招募(图6B),与之一致的是2’,3’-cGAMP诱导的TBK1,IRF3,P65和IκBα蛋白的磷酸化和IRF3的二聚化水平(图6C)及IRF3的入核在HFF细胞预孵育SN-011后显著降低(图6D)。相比之下,阴性对照化合物SN-100并不影响上述蛋白水平的变化。作为对照,进一步检测SN-011在细胞静息状态下对cGAS,STING,TBK1和IRF3蛋白稳定性的影响,结果表明,SN-011不影响HFF细胞内上述蛋白的基态表达(图5C),以及cGAS基因(MB21D1)和STING基因(TMEM173)的表达(图5D~5E)。这表明SN-011的作用机制是通过直接影响蛋白质的功能而不是影响STING信号通路蛋白的稳定性。以上研究表明,SN-011能抑制细胞内STING蛋白的激活及其介导的下游信号传导。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.
实施例8Example 8
SN-011(式I化合物)与STING-CTD(149-379)蛋白的共结晶结构Co-crystal structure of SN-011 (compound of formula I) and STING-CTD (149-379) protein
为了研究化合物SN-011与STING蛋白结合的结构基础,本发明获得并解析了SN-011与STING-CTD(149-379)蛋白的共结晶结构,分辨率为
C末端区域(amino acid 341-379)在共结晶结构中不可见可能是由于该段结构空间不稳定性。在共结晶结构中每一个对应的结构单元包含两个STING蛋白单体形成的同源二聚体和在蛋白二聚体CDNs结合口袋中的结合的两个SN-011分子(图7A)。从结构上看,SN-011分子反相平行地结合在STING二聚体表面,小分子结构上的联苯环结合在二聚体口袋的底部,4-氟-苯磺胺基团延伸到蛋白二聚体口袋的顶部。与apo-STING-CTD蛋白的结晶结构相似,STING-CTD(149-379)与SN-011结合后仍维持“V”字型的二聚体结构,且在口袋两侧的His185氨基酸的距离为
(图7B)。相比之下,STING-CTD蛋白结合2’3’-cGAMP和c-di-AMP后其口袋两侧的单体会在小分子的牵引下相互接近,使蛋白二聚体口袋变成封闭的“U”型且在口袋两侧的His185氨基酸的距离缩短至为
(图7C)(Cell,2019,178:1-12)。随后,进一步分析了SN-011结合后STING-CTD蛋白在晶格中堆积的方式,共结晶结构以类似“头尾对应”的方式堆积在晶格中(图7D),这种堆积方式也出现在未激活的STING结构中(ACS Med Chem Lett,2019,10(1):92-97),且此堆积方式明显不同于2’3’-cGAMP和c-di-AMP结合后STING-CTD蛋白在晶格中的“并肩排列”的堆积方式(Nature,2019,567(7748):389-393)。综上所述,在与SN-011分子结合后,STING蛋白的空间构象并未发生显著变化,仍维持其在静息状态下的二聚体构形。
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.
进一步的结构解析表明,SN-011通过与STING蛋白二聚体口袋内的特定氨基酸形成氢键和堆积作用而稳定在口袋内。SN-011结构中的联苯环与蛋白Tyr167氨基酸侧链的苯环形成π-π堆积作用。SN-011结构中连接酚羟基的苯环同样能与周围的Glu260和Ser241氨基酸形成共轭或氢键作用而稳定。而SN-011中的酚羟基和磺胺键能进一步与Ser243氨基酸侧链上的羟基形成氢键作用(图7E)。据此,本发明解析的SN-011与STING蛋白的共结晶结构不仅明确了该化合物在蛋白中结合的位置,也提供了二者相互作用的方式和结合位点信息。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.
以上结果表明,本发明的化合物能通过直接结合STING蛋白并维持其静息状态下的二聚体构象而抑制STING信号通路的激活。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.
实施例9Example 9
突变与SN-011(式I化合物)结合的氨基酸位点影响小分子的结合及其发挥的生物活性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
为了进一步确定由共结晶结构分析出的氨基酸位点对SN-011结合的影响,用表面等离子共振(Surface Plasmon Resonance,SPR)检测相关氨基酸位点突变后对小分子与蛋白间亲和力的影响。SPR的检测结果表明,SN-011与 hSTING-CTD蛋白间的亲和力约为4.03nM,优于2’3’-cGAMP与之结合的亲和力(Kd~9.23nM)。值得注意的是,hSTING-CTD结合位点的单突变体S243A和三突变体蛋白S241A,S243A和T263A显著减弱hSTING-CTD与SN-011间的相互作用,Kd分别为176.2nM和1.36μM(图8A~8E)。hSTING-CTD-Y167A和hSTING-CTD-E260A在大肠杆菌中由于溶解性问题无法纯化,所以在此实验体系中无法评价Y167和E260对小分子与蛋白间亲和力的影响。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.
随后,在HEK293T细胞中过表达hSTING蛋白及其相关点突变体蛋白以评价结合位点突变后对SN-011的生物学活性的影响。实验结果表明,相比于野生型hSTING,SN-011对其点突变体S241A,S243A,T263A和3A诱导的IFN-β基因表达的抑制活性显著降低,且在2’3’-cGAMP刺激条件下突变上述氨基酸位点同样能减弱SN-011的抑制活性(图8F~8I)。hSTING-Y167A和hSTING-E260A突变体在HEK293T细胞中过表达后并不能激活下游IFN-β基因表达,也不响应外源2’3’-cGAMP刺激(图8G,8I),所以也无法评价这两个位点突变对SN-011生物活性的影响。以上研究结果表明,本发明化合物通过几个关键氨基酸残基(Tyr167、Glu260、Ser241和Ser243)与STING蛋白结合,进而抑制STING信号通路的激活。因此,本发明式I~V所示的苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物可以用于制备抑制STING信号通路激活的药物。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.
实施例10Example 10
SN-011(式I化合物)改善TREX1
-/-自身免疫疾病小鼠的系统性炎症损伤
SN-011 (compound of formula I) improves systemic inflammatory damage in TREX1 -/- autoimmune disease mice
TREX1在细胞质内发挥核酸外切酶的作用,用于降解细胞质中异常ssDNA,当其基因突变导致蛋白丧失正常酶的功能后,细胞质中聚集的DNA能激活胞浆的cGAS-STING信号通路,诱导I型IFNs的表达,促进细胞炎症的发生。在动物模型上发现,将小鼠cGAS或STING蛋白的表达敲除后能显著改善TREX1突变诱导各组织器官的炎症病变程度和疾病的致死(Nat Genet,2007,39(9):1065-7;Cell,2008,134(4):587-98)。以此为依据,本发明在Trex1
-/-小鼠体内评价SN-011对系统性炎症损伤的潜在药效学活性。首先,在Trex1
-/-小鼠骨髓来源的巨噬细胞(BMDMs)中孵育化合物SN-011后,用RNA-Seq技术检测对细胞内mRNA转录本进行分析,实验结果表明:(1)在野生型(WT)小鼠BMDMs中,孵育SN-011并不能显著影响细胞静息状态下ISGs基因的表达;(2)相比于WT BMDMs细胞,Trex1
-/-BMDMs细胞中的ISGs表达水平显著升高,且在孵育SN-011后能显著抑制相关ISGs基因的表达;(3)进一步分析细胞转录本基因表达的变化发现,SN-011在细胞内主要影响的也是ISGs基因的表达,从侧面说明该化合物作用的特异性(图9A)。随后,使用qPCR技术检测细胞内Ifnb,Cxcl10,Isg15和IL-6基因的表达情况,检测结果也进一步验证了化合物SN-011对 Trex1
-/-BMDMs细胞中ISGs基因表达的抑制作用(图9B~9E)。
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).
在体外研究基础上,将SN-011(5mg/kg)腹腔注射到Trex1
-/-小鼠体内,在持续1个月的给药后检测小鼠心脏、胃、舌头和肌肉的炎症浸润。结果表明:(1)相比于野生型(WT)小鼠,Trex 1
-/-小鼠心脏、胃、舌头和肌肉的免疫(Ifnβ,Cxcl10,Isg15)和炎症因子(Il6)基因都有显著的上升;(2)在野生型小鼠中给予化合物SN-011不影响上述组织中免疫和炎症因子基因的表达;(3)相比于Trex 1
-/-的自身免疫疾病模型组,给予化合物SN-011能显著降低Trex 1
-/-小鼠心脏、胃、舌头和肌肉的免疫(Ifnβ,Cxcl10,Isg15)和炎症因子(Il6)基因的表达(图10A~10D)。进一步对上述组织器官进行病理切片评价组织的炎症损伤,结果表明,SN-011能显著改善Trex 1
-/-小鼠组织的炎症损伤和免疫细胞浸润(图10E)。取实验组小鼠的脾脏分散脾脏细胞后进行T细胞抗体染色,流式细胞仪检测T细胞亚型含量。检测结果表明:相比于对照的Trex1
-/-小鼠,腹腔注射化合物SN-011能显著降低激活的CD8T(CD69
+)细胞和记忆的CD4T(CD62L
lowCD44
high)细胞和CD8(CD62L
lowCD44
high)T细胞在Trex1
-/-小鼠脾脏中的含量,而并不影响激活的CD4T(CD69
+)细胞的含量(图11A~11B)。此外,SN-011给药后Trex 1
-/-小鼠血清中的抗核抗体显著减少(图11D),且持续给药1个月能显著延长Trex1
-/-小鼠的生存率(图11C)。以上研究结果表明,SN-011能改善TREX1
-/-自身免疫疾病小鼠的系统性炎症损伤并阻止疾病的进展。此外,式II化合物(SN-001)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)都具有与式I化合物(SN-011)相类似的效应。
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.
综上所述,本发明的式I~V化合物可用于制备预防或治疗STING介导的自身免疫性疾病和炎症性疾病的药物。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.
实施例11Example 11
SN-011(式I化合物)在小鼠体内的药物代谢动力学研究Study on the pharmacokinetics of SN-011 (compound of formula I) in mice
在单次腹腔注射给予雄性C57BL6J小鼠化合物SN-011(5mg/kg)后,分别在24小时内设置不同的时间点进行采血,随后用LC-MS/MS检测血浆中的血药浓度并计算相关的药代参数。实验结果表明:小鼠腹腔注射注射SN-011后血浆中药物的半衰期T
1/2是1.03小时,最大血药浓度Cmax为837.02ng/mL,达到药峰浓度所需的时间T
max为0.25小时,血药浓度-时间曲线下面积AUC为850ng/mL(图12A~12B)。
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).
实施例12Example 12
SN-011(式I化合物)在小鼠体内的毒理学评价Toxicology evaluation of SN-011 (compound of formula I) in mice
为了评价化合物SN-011在小鼠体内长期给药后的潜在毒性,我们在雄性C57BL6J小鼠内腹腔注射SN-011(1mg/kg)每周给药三次,持续给药10周, 给药完成后检测小鼠体内脏器和血清生化指标的变化。实验结果表明:给药10周内对照组和给药组的小鼠体重无显著变化(图13A),小鼠血清中反应肝脏损伤的ALT和AST指标在给药后也没有显著的增加(图13B)。此外,反应肾脏损伤的血清指标BUN和CREA在给药后也没发生显著变化(图13C)。对小鼠的心脏、肾脏、胃和肝脏做病理切片,结果表明,在10周给药后上述器官均未发生明显的实质性损伤(图13D)。以上结果表明,化合物SN-011在小鼠体内具有很好的安全性,长期给药后在小鼠体内也未表现出毒副作用。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.
实施例13Example 13
SN-011(式I化合物)抑制SAVI相关STING点突变诱导的炎症细胞因子的表达SN-011 (compound of formula I) inhibits the expression of inflammatory cytokines induced by SAVI-related STING point mutations
编码STING蛋白的基因TMEM173在人体内发生突变诱发的一类自身免疫疾病,即在婴儿期发生STING激活相关血管病变(STING-associated vasculopathy with onset in infancy,SAVI)。SAVI病人的临床表征主要为早发于婴儿时期,皮肤红疹和呼吸急促,发热等系统性的炎症,外周血管病变,肺部炎症和血液中存在自身免疫抗体等。随后的基因测序结果发现,此类病人的TMEM173基因的多个位点发生突变而导致蛋白的自激活,突变体包括N154S,V155M,G166E,C206Y,R281Q和R284G(J Allergy Clin Immunol,2017,140(2):543-552;Ann Rheum Dis,2017,76(2):468-472)。本发明通过克隆的方法构建上述点突变体的质粒,在HEK293T细胞(本身无STING蛋白的表达)中过表达上述突变体,进而研究化合物SN-011对上述突变体诱导的STING信号通路激活的影响。实验结果表明,细胞预孵育SN-011能显著抑制上述突变体诱导的Ifnb,Cxcl10和Tnfa基因的表达(图14A~14C)。在蛋白水平预孵育SN-011同样能减弱过表达的STING点突变体对下游靶蛋白TBK1和IRF3的招募及降低P-TBK1和P-IRF3的表达水平(图14D~14E)。近期的研究表明,SAVI相关的STING点突变体能通过解除STING二聚体的自抑制形态而使蛋白发生不依赖与配体结合的多聚化激活(Nature,2019,567(7748):389-393),以此为依据,进一步检测化合物SN-011对上述点突变体的多聚化的影响,结果表明,SN-011能显著抑制STING突变体的多聚化表达(图14F)。综上所述,SN-011能通过抑制SAVI相关的STING点突变的多聚化激活而减少下游炎症细胞因子的表达,进而降低组织或细胞的炎症损伤。此外,式II化合物(SN-001)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)都具有与式I化合物(SN-011)相类似的效应。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.
因此,本发明的式I~V化合物可用于制备预防或治疗婴儿期发生的STING相关的血管疾病(SAVI)的药物。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.
实施例14Example 14
SN-011(式I化合物)改善大鼠脑缺血损伤SN-011 (compound of formula I) improves cerebral ischemic injury in rats
组织的缺血性损伤伴随大量实质细胞的死亡,随之释放的损伤相关的分子模式(DMAP)进一步促进损伤部位免疫系统的激活,释放的炎症因子加重组织的损伤。最近研究发现,在小鼠的心肌缺血模型中受损的心肌细胞产生的细胞碎片被心脏组织中的巨噬细胞摄取,激活巨噬细胞内cGAS-STING介导的固有免疫信号通路,促进ISGs的表达,且基因敲除cGAS或STING后能显著改善心肌缺血导致心脏炎症损伤,改善心脏功能进而延长疾病小鼠的生存率(Nature Medicine,2017,23(12):1481-1487;Circulation,2018,137(24):2613-2634)。据此,本发明人在大鼠的脑缺血模型中评价化合物SN-011的潜在治疗效果。实验结果表明,大鼠在中动脉阻塞(MCAO)24小时后,模型组大鼠的脑梗死区域显著增大,而给予低剂量(1mg/kg)和高剂量(3mg/kg)的SN-011均能显著改善脑梗死面积(图15A)。分别在手术后6小时和24小时后对大鼠的运动能力进行评价,给予SN-011能显著改善脑卒中后的大鼠运动功能障碍(图15B~15C)。进一步检测大鼠脑皮层组织炎症基因的表达,结果发现,大鼠中动脉阻塞后大脑皮层内Ifnb,Ifna4,Cxcl10,Mcp-1,Tnf-a和Il-6的基因表达显著升高,与之相比,SN-011能显著抑制缺血损伤诱导的大脑皮层组织中上述基因的表达(图15D~15I)。以上研究结果表明,化合物SN-011能显著改善缺血诱导的实质器官损伤。此外,式II化合物(SN-001)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)都具有与式I化合物(SN-011)相类似的效应。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.
这提示本发明的式I~V化合物可用于制备预防或治疗STING介导的缺血性心脑脑血管疾病的药物。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.
实施例15Example 15
SN-011(式I化合物)改善小鼠鼠脑缺血损伤SN-011 (compound of formula I) improves cerebral ischemic injury in mice
脑卒中可迅速导致小胶质细胞的激活和外周免疫细胞的浸润,且在卒中病人脑脊液中DNA的含量显著上升。最近的研究表明脑卒中后cGAS-STING通路发生激活,并介导卒中后的损伤(EMBO Molecular Medicine,2020,7;12(4))。实验结果显示:小鼠在中动脉阻塞后立即腹腔注射低剂量SN-011(1mg/kg)和高剂量的SN-011(2mg/kg),24小时后,化合物SN-011显著抑制Mcp-1,Il-6和Tnf-α基因的表达(图16A);小鼠在中动脉阻塞后立即腹腔注射低剂量SN-011(1mg/kg)和高剂量的SN-011(2mg/kg),72小时后,化合物SN-011显著抑制Mcp-1,Il-6和Cxcl10基因的表达(图16B);小鼠大脑中动脉阻塞后立即或24小时后再腹腔注射SN-011(2mg/kg),连续给药至卒中后第七天,化合物SN-011立即给药或延迟24小时给药均可显著改善卒中小鼠的运动协调性(图16C);小鼠大脑中动脉阻塞后立即或24小时后再腹腔注射SN-011(2mg/kg),连续给药至卒中 后第七天,化合物SN-011立即给药或延迟24小时给药均可显著改善卒中小鼠的运动协调性(图16D);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). );
实施例16Example 16
SN-011(式I化合物)改善高脂饮食诱导的小鼠肝损伤及脂质聚集非酒精性脂肪肝(NAFLD)主要表现为肝脏的脂肪聚集诱导肝脏脂肪变性,在未得到有效控制时,过度聚集的脂肪会诱导肝脏炎症损伤和纤维化进而疾病进展为非酒精脂肪肝炎(NASH)。流行病学研究表明NASH是导致肝硬化和肝癌发生的最常见因素,而目前尚无有效的治疗NASH的药物。近期的研究发现,在NAFLD病人的肝脏中STING的表达显著升高,主要集中在肝脏的非实质细胞如巨噬细胞和枯否细胞内,且在动物模型中敲除骨髓细胞STING蛋白的表达能显著改善高脂饮食诱导的小鼠NAFLD疾病进展(Gastroenterology,2018,155(6):1971-1984;Journal of Clinical Investigation,2018,129(2):546-555)。据此,本发明人在高脂饮食诱导的NAFLD模型中评价SN-011的药效。用高脂饲料(HFD)喂养4~6周龄的C57BL6J雄性小鼠10周后开始给予低剂量(1mg/kg)和高剂量(2mg/kg)的SN-011进行干预,连续给药10周后检测小鼠血清生化指标。结果表明,与正常饮食喂养的小鼠相比,HFD小鼠血清中ALT,AST,TC和TG的含量显著升高,且给药后小鼠血清中上述指标显著降低,说明SN-011能改善HFD饮食诱导的肝损伤并减少血清总胆固醇(TC)和甘油三酯(TG)的含量(图17A~17D)。SN-011给药后能显著减少小鼠的体重、肝脏重量和肾周内脏脂肪重量(图17E~17G),且肝脏内的总胆固醇和甘油三酯的含量在给药后也显著降低(图17H~17I)。以上结果证实了SN-011对小鼠体内脂代谢的调控作用。进一步检测肝脏中STING激活的免疫和炎症细胞因子与调控脂代谢蛋白基因的表达水平,结果表明,高脂饮食诱导的肝脏Ifnb,Cxcl10,Mcp-1和Tnfa基因的上调在SN-011给药后都显著得到改善(图17J~17M),这表明SN-011能改善肝脏炎症反应,且也能显著抑制HFD诱导肝脏调控脂质合成的蛋白脂肪酸合酶(FAS)和固醇调节元件结合蛋白(SREBP-1c)基因的上调(图17N~17O)。进一步对实验组小鼠的肝脏进行病理切片以评价组织的炎症损伤和脂质空泡,结果表明,SN-011能显著改善HFD小鼠肝组织的炎症细胞浸润及脂质聚集形成的大泡样脂肪变性(图17P)。此外,式II化合物(SN-001)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)都具有与式I化合物(SN-011)相类似的效应。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.
以上结果提示,本发明的式I~V化合物可用于制备预防或治疗STING介导的非酒精性脂肪性肝病(包括NASH)的药物。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).
实施例17Example 17
SN-011(式I化合物)改善咪喹莫特诱导的银屑病样炎症用BALB/c小鼠建立咪喹莫特诱导的银屑病样炎症模型,观察化合物SN-011是否能逆转咪喹莫特诱导的银屑病样炎症。将BALB/c小鼠随机分为空白对照组(Control组)、模型对照组(Model组),SN-011 250mg/kg组和SN-011 50mg/kg组。采用银屑病皮损面积和疾病严重程度(PASI)评分来观察疾病进展,并测量右耳厚度。H&E染色观察各组小鼠右耳及背部皮肤表皮厚度,Munro微脓肿情况。实验结果表明(图18和图19),Model组小鼠耳厚度明显高于Control组,化合物SN-011组耳厚度低于Model组,且有显著性差异。H&E染色结果表明(图20),化合物SN-011降低了小鼠棘皮厚度,且有显著性差异。此外,式II化合物(SN-001)、式III化合物(SN-005)、式IV化合物(SN-006)和式V化合物(SN-010)都具有与式I化合物(SN-011)相类似的效应。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.
以上结果提示,本发明的化合物可以改善银屑病样炎症,可用于制备预防或治疗STING介导的银屑病的药物。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.
综上所述,本发明式I~V化合物或其药学上可接受的盐或溶剂化物可以应用在制备预防或治疗STING介导的疾病的药物中。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.
本发明化合物的作用机制如图21所示。以化合物SN-011(式I化合物)为例,在正常情况下,细胞质中异常的DNA被cGAS识别后产生2’3’-cGAMP分子,2’3’-cGAMP随后与内质网调控蛋白STING结合,诱导STING蛋白的构象变化进而发生多聚化激活,随后多聚化的STING蛋白转移到高尔基体上招募下游蛋白TBK1和IRF3,TBK1在自磷酸化后促进IRF3和NF-κB磷酸化,随后入核促进干扰素和炎症细胞因子的表达;在加入SN-011分子后,SN-011能特异性结合到STING二聚体形成的口袋内,进而阻止CDNs分子对STING蛋白的激活,阻断信号的传递。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.
实施例18Example 18
片剂tablet
将实施例1中制得的式I化合物(50g)、羟丙甲基纤维素E(150g)、淀粉(200g)、聚维酮K30适量和硬脂酸镁(1g)混合,制粒,压片。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.
此外,可以根据药典2015版常规制剂法,将本发明的式I~V化合物赋予不同的药物辅料制成胶囊剂、散剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、软膏剂、栓剂或贴剂等。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 (7)
- 如下式I~V任意一种或者多种所示的苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物在制备抑制STING信号通路激活的药物中的用途: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:
- 一种权利要求1所述的式I~V任意一种或者多种苯磺酰胺类化合物或其药学上可接受的盐或溶剂化物在制备预防或治疗STING介导的疾病的药物中的用途。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.
- 根据权利要求2所述的用途,所述STING介导的疾病包括感染性疾病、炎性疾病、自身免疫性疾病、器官纤维化疾病、缺血性心脑血管疾病、退行性神经系统疾病、脑外伤、脊髓损伤、癌症或癌期综合征。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.
- 根据权利要求2所述的用途,所述STING介导的疾病是银屑病、脑卒中、脑外伤、外伤或其他疾病引起的脊髓损伤、帕金森病、亨廷顿病、家族性肌萎缩侧索硬化症、心肌梗死、非酒精性脂肪性肝炎、系统性红斑狼疮、Aicardi-Goutières综合征、类风湿性关节炎、炎症性肠病、婴儿期发生的STING相关的血管疾病(SAVI)或糖尿病及其并发症。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.
- 根据权利要求1或2所述的用途,其特征在于,所述式I~V化合物其药学上可接受的盐为金属离子或药学上可接受的胺、铵离子或胆碱形成的盐。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.
- 一种预防或治疗STING介导的疾病的药物组合物,其包含式I~V任意一种或者多种苯磺酰胺类化合物或其药学上可接受的盐或溶剂化合物作为活性成分和药学上可接受的辅料。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.
- 根据权利要求6所述的药物组合物,其特征在于,所述药物组合物为胶囊剂、散剂、片剂、颗粒剂、丸剂、注射剂、糖浆剂、口服液、吸入剂、软膏剂、栓剂或贴剂。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.
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