WO2022242768A1 - 一种吡咯并嘧啶类化合物的应用 - Google Patents

一种吡咯并嘧啶类化合物的应用 Download PDF

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WO2022242768A1
WO2022242768A1 PCT/CN2022/094256 CN2022094256W WO2022242768A1 WO 2022242768 A1 WO2022242768 A1 WO 2022242768A1 CN 2022094256 W CN2022094256 W CN 2022094256W WO 2022242768 A1 WO2022242768 A1 WO 2022242768A1
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multiple sclerosis
myelitis
pharmaceutically acceptable
formula
autoimmune disease
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PCT/CN2022/094256
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English (en)
French (fr)
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李永国
陈小宁
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广州嘉越医药科技有限公司
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Priority to EP22804087.9A priority Critical patent/EP4353234A1/en
Publication of WO2022242768A1 publication Critical patent/WO2022242768A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the application of a pyrrolopyrimidine compound.
  • Demyelinating disease of the central nervous system is an autoimmune disease characterized by multifocal and inflammatory demyelination of the central nervous system (CNS). Its clinical features mainly include recurrent attacks, multiple remissions and relapses. Clinically more common demyelinating diseases of the central nervous system include multiple sclerosis.
  • MS Multiple sclerosis
  • the most common demyelinating disease of the central nervous system and it is also the most common non-traumatic neurological disease among young adults, with more than two million patients worldwide. More and more attention has been paid to the disease and the improvement of diagnostic techniques.
  • diagnosis rate of MS in China is increasing year by year.
  • the drugs used to treat multiple sclerosis are mostly hormones, but the side effects of hormones are relatively large, which can cause water, salt, sugar, protein and fat metabolism disorders, and may also cause obesity, hirsutism, and weakness.
  • symptoms of MS include numbness or weakness in one or more parts of the body, partial or total loss of vision, prolonged double vision, tingling or pain, Lermi features, tremors, slurred speech, fatigue, dizziness and impaired bowel and bladder function.
  • EAE Experimental autoimmune encephalomyelitis
  • the technical problem to be solved by the present invention is to find a compound that has good preventive and/or therapeutic effects on suppurative myelitis, acute myelitis, encephalomyelitis or autoimmune diseases. For this reason, the present invention provides a Application of pyrrolopyrimidine compounds.
  • the present invention provides the application of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of STAT phosphorylation inhibitors:
  • the present invention also provides a compound represented by formula I or a pharmaceutically acceptable salt thereof used in the preparation for treating and/or preventing suppurative myelitis, acute myelitis or encephalomyelitis characterized by abnormally elevated STAT phosphorylation levels
  • a compound represented by formula I or a pharmaceutically acceptable salt thereof used in the preparation for treating and/or preventing suppurative myelitis, acute myelitis or encephalomyelitis characterized by abnormally elevated STAT phosphorylation levels
  • the present invention also provides the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and/or preventing autoimmune diseases characterized by abnormally elevated STAT phosphorylation levels:
  • the present invention also provides the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and/or preventing suppurative myelitis, acute myelitis or encephalomyelitis:
  • the present invention also provides the use of a compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of medicines for treating and/or preventing autoimmune diseases:
  • the present invention also provides a method for treating and/or preventing a subject's suppurative myelitis, acute myelitis or encephalomyelitis, comprising: administering a therapeutically effective amount of a compound represented by formula I to the subject or a pharmaceutically acceptable salt thereof;
  • the present invention also provides a method for treating and/or preventing an autoimmune disease in a subject, comprising: administering to the subject a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt thereof;
  • the present invention also provides a method for inhibiting STAT phosphorylation in a subject, comprising: administering to the subject a therapeutically effective amount of a compound represented by formula I or a pharmaceutically acceptable salt thereof:
  • the present invention also provides a method for treating and/or preventing suppurative myelitis, acute myelitis or encephalomyelitis drugs characterized by abnormally elevated STAT phosphorylation levels in a subject, comprising: administering to the subject or a therapeutically effective dose of the compound shown in formula I or a pharmaceutically acceptable salt thereof;
  • the present invention also provides a method for treating and/or preventing an autoimmune disease characterized by abnormally elevated STAT phosphorylation levels in a subject, comprising: administering to the subject a therapeutically effective amount of The indicated compound or its pharmaceutically acceptable salt;
  • the STAT phosphorylation may be one or more of STAT1 phosphorylation, STAT3 phosphorylation and STAT5 phosphorylation, such as STAT3 phosphorylation.
  • the autoimmune disease can be an autoimmune disease of the central nervous system or an autoimmune disease of the optic nervous system;
  • the autoimmune disease of the central nervous system can be neuromyelitis optica, multiple sclerosis , such as neuromyelitis optica, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, clinically isolated syndrome, or radiologically isolated syndrome;
  • the optic nerve A systemic autoimmune disease may be neuromyelitis optica.
  • the encephalomyelitis may be acute disseminated encephalomyelitis, subacute necrotizing myelitis, acute necrotizing and hemorrhagic encephalomyelitis, pediatric acute disseminated encephalomyelitis, or tuberculous myelitis.
  • the autoimmune disease characterized by an abnormally elevated STAT phosphorylation level may be an autoimmune disease of the central nervous system or an autoimmune disease of the optic nervous system; the autoimmune disease characterized by an abnormally elevated STAT phosphorylation level
  • Highly featured CNS autoimmune diseases can be neuromyelitis optica, multiple sclerosis, e.g. neuromyelitis optica, relapsing remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive Relapsing multiple sclerosis, clinically isolated syndrome or radiologically isolated syndrome; said autoimmune disease of the optic nervous system may also be neuromyelitis optica.
  • the encephalomyelitis, suppurative myelitis or acute myelitis characterized by abnormally elevated STAT phosphorylation levels wherein encephalomyelitis can be acute disseminated encephalomyelitis, subacute necrotizing myelitis Acute necrotizing and hemorrhagic encephalomyelitis, acute disseminated encephalomyelitis or tuberculous myelitis in children.
  • the compound represented by formula I or a pharmaceutically acceptable salt thereof is one or the only active ingredient of the inhibitor or the drug.
  • a therapeutically effective amount of the compound represented by formula I or a pharmaceutically acceptable salt thereof may be administered to a subject in need.
  • the mode of administration may be any suitable mode, such as oral administration.
  • the compound represented by formula I or a pharmaceutically acceptable salt thereof may further contain pharmaceutically acceptable excipients, for example, tablets, hypodermic injections.
  • the dosage of the compound represented by formula I or a pharmaceutically acceptable salt thereof may be 1-120 mg/kg/d, preferably 3-100 mg/kg/d, for example 3 mg/kg /d, 6mg/kg/d, 10mg/kg/d, 20mg/kg/d, 40mg/kg/d, 60mg/kg/d or 90mg/kg/d.
  • the single administration dose of the compound represented by formula I or a pharmaceutically acceptable salt thereof may be 1-400 mg, such as 3 mg, 6 mg, 9 mg, 100 mg or 400 mg.
  • the administration frequency of the compound represented by formula I or a pharmaceutically acceptable salt thereof can be 1 time/day, 2 times/day or 3 times/day, preferably 1 time/day or 2 times/day /day.
  • the present invention also provides a pharmaceutical composition for treating and/or preventing subjects with suppurative myelitis, acute myelitis, encephalomyelitis or autoimmune diseases, which comprises: the compound shown in formula I or its Pharmaceutically acceptable salts, and pharmaceutical excipients.
  • STAT phosphorylation inhibitor can be used in mammalian organisms; it can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample to provide comparison, or to make a kit according to conventional methods in the art, Provides a rapid assay for the effect of STAT phosphorylation inhibition.
  • autoimmune disease characterized by an abnormally elevated level of STAT phosphorylation means that the autoimmune disease exhibits a higher than normal level of STAT phosphorylation.
  • the compound represented by formula I can prevent and/or treat autoimmune diseases by inhibiting STAT phosphorylation.
  • suppurative myelitis, acute myelitis or encephalomyelitis characterized by an abnormally elevated level of STAT phosphorylation means that the suppurative myelitis, acute myelitis or encephalomyelitis exhibits a higher than normal level of STAT phosphorylation .
  • the compound represented by formula I can prevent and/or treat suppurative myelitis, acute myelitis or encephalomyelitis by inhibiting STAT phosphorylation.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt prepared from a compound of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base.
  • base addition salts can be obtained by contacting the prototype of such compounds with a sufficient amount of a pharmaceutically acceptable base in neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, diethanolamine salts.
  • acid addition salts can be obtained by contacting the prototype of such compounds with a sufficient amount of pharmaceutically acceptable acid in neat solution or in a suitable inert solvent.
  • the pharmaceutically acceptable acid includes inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid and the like.
  • the pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid , fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid , tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (ie 4,4'-methylene-bis( 3-hydroxy-2-naphthoic acid)), amino acids (eg glutamic acid, arginine) and the like.
  • the compounds of the present invention When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts.
  • base addition salts For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19 (1977), or, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).
  • treatment refers to therapeutic therapy.
  • treatment means: (1) amelioration of one or more biological manifestations of the disease or condition, (2) interference with (a) one or more points in the biological cascade leading to or causing the condition or (b ) one or more biological manifestations of the disorder, (3) amelioration of one or more symptoms, effects or side effects associated with the disorder, or one or more symptoms, effects or side effects associated with the disorder or its treatment, Or (4) slowing the development of the disorder or one or more biological manifestations of the disorder.
  • prevention refers to a reduction in the risk of acquiring or developing a disease or disorder.
  • subject refers to any animal that is about to or has received the administration of the compound according to the embodiments of the present invention, preferably a mammal, and most preferably a human.
  • mammal includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being most preferred.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive progress effect of the present invention is: the present invention finds that the compound shown in formula I has the effect of inhibiting STAT phosphorylation, and has good prevention and/or effect on suppurative myelitis, acute myelitis, encephalomyelitis or autoimmune diseases Therapeutic effect.
  • Fig. 1 is the effect of continuous multiple administration of the test substance Compound I on the clinical evaluation of EAE in mice. (Data expressed as mean or mean ⁇ standard error (Mean ⁇ SEM), *p ⁇ 0.05,**p ⁇ 0.01,***p ⁇ 0.001VS G2)
  • Figure 2 is the impact of test compound I on the pathological score of mouse spinal cord (data are represented by mean ⁇ standard error (Mean ⁇ SEM), *p ⁇ 0.05, **p ⁇ 0.01, *** p ⁇ 0.001, ****p ⁇ 0.0001 vs G2).
  • Figure 3 is the impact of test compound I on the phosphorylation level of spinal cord STAT3 for multiple consecutive administrations (data are represented by mean ⁇ standard error (Mean ⁇ SEM), **p ⁇ 0.01, ***p ⁇ 0.001VS G2 ).
  • Figure 4 shows the effect of compound I co-incubated with cells for 24 hours on cell viability in the NMO-IgG injured astrocyte model.
  • Fig. 5 shows the effect of compound I on the release of IL-6 from astrocytes injured by NMO-IgG.
  • Compound I was provided by Guangzhou Jiayue Pharmaceutical Technology Co., Ltd. and prepared according to the preparation method described in patent PCT/CN2020094534
  • Cell culture conditions are: 37°C, 5% CO 2 and 95% humidity.
  • IC 50 value the concentration of inhibitor when 50% inhibitory effect is achieved.
  • BV-2 was purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • Step 1 Collect BV-2 cells in exponential growth phase and count viable cells with Vi-Cell XR cell counter. Adjust the cell suspension to an appropriate concentration with medium. Add 160 ⁇ l of cell suspension to each well in a 96-well cell culture plate, and the final cell concentration is 12000 cells/well. Incubate the plate in a 37°C, 5% CO 2 incubator for 24 hours.
  • Step 2 Use DMSO to dilute the 10mM DMSO compound stock solution at a ratio of 1:4 to prepare a series of 1000X test compound solutions, and then use cell culture medium to dilute the 1000X test compound solution 1:100 to prepare a series of 10X test compound solutions . Finally, the 96-well plate that had been plated the day before was taken out, and 20 ⁇ l of the corresponding 10X solution was added to each well of cells according to the experimental well plate loading design, and 3 replicate wells were made for each drug concentration. The ranges of compound concentrations (ie 1X solution) used in the final test of this experiment were 10, 2.5, 0.625, 0.156, 0.0391, 0.0098, 0.0024 ⁇ M, and the final concentration of DMSO in each well was 0.1%.
  • the 96-well plate was placed in a 37°C, 5% CO 2 incubator for half an hour.
  • Step 3 After 24 hours of compound treatment, centrifuge the culture plate at 1200 rpm for 5 minutes, aspirate the supernatant to another plate, seal it with tinfoil and store it at -20°C, which will be used for ELISA detection and Griess reagent system detection. The cell culture plate after aspirating the supernatant will be used immediately for CTG method to detect cell proliferation.
  • Step 4 Add 100 ⁇ l of PBS to each well of the cell culture plate after absorbing the supernatant, according to the operation instructions of the CTG kit, add 50 ⁇ l of pre-melted and equilibrated CTG solution to each well, and mix with a microplate shaker for 2 minutes , After standing at room temperature for 10 minutes, measure the fluorescence signal value with Envision2104 plate reader.
  • Step 5 use ELISA kit (Mouse CCL5/RANTES DuoSet ELISA; R&D; DY478-05), according to the kit instructions, detect the level of CCL5 in the supernatant; use Griess reagent system (Griess Reagent System; Promega G2930), according to the instructions of the kit, detect the content of nitric oxide (NO) in the supernatant.
  • ELISA kit Mae CCL5/RANTES DuoSet ELISA; R&D; DY478-05
  • Step 2 Plot the logarithm of the compound concentration against the percentage of surviving cells, then use nonlinear regression to calculate the fitting curve, and use the software GraphPad Prism 5 formula log(inhibitor) vs.response--Variable slope to calculate the IC50 value.
  • Step 3 Finally, use the logarithm value of the compound concentration to plot the relative percentage of CCL5 secretion or the relative percentage of NO secretion, use nonlinear regression to calculate the fitting curve, and use the software GraphPad Prism 5 formula log(inhibitor)vs.response--Variable slope IC50 values were calculated.
  • compound I can effectively inhibit the production of cytokines (CCL5) and reactive nitrogen (NO) induced by LPS/IFN ⁇ , in a dose-effect relationship: the maximum inhibition rate for cytokines (CCL5) was 80.29%, and IC50 was 0.863 ⁇ M; the maximum inhibitory rate to reactive nitrogen (NO) was 100.00%, and IC50 was 0.487 ⁇ M.
  • the IC50 of sinimod in this experiment were all greater than 10 ⁇ M.
  • Compound I can significantly inhibit the release of pro-inflammatory factors caused by excessive activation of microglia (BV-2), suggesting that it is one of the important mechanisms for the treatment of neuroinflammatory diseases such as multiple sclerosis.
  • BV-2 microglia
  • EAE Experimental autoimmune encephalomyelitis
  • MOG35-55 was purchased from Jill Biochemical, product number: 51716; 2) IFA was purchased from Sigma-Aldrich, product number: F5506; 3) Mycobacterium tuberculosis H37Ra, Difco, product number: 231141; 4) PTX (Pertussis toxin) was purchased from List Biological Laboratories, Item No.: 180235AIA; 5) Dexamethasone acetate tablets are provided by Chenxin Pharmaceutical Co., Ltd; 7) Compound I was provided by Guangzhou Jiayue Pharmaceutical Technology Co., Ltd.;
  • mice were marked with ear tags, and the backs of the mice were shaved.
  • mice Of 85 mice, 5 were randomly selected as the normal control group, and the other 80 were anesthetized with isoflurane, and subcutaneously injected with 100 ⁇ L immune emulsion (IFA containing 1 mg/ml MOG35-55 and 2 mg/ml Mycobacterium tuberculosis), respectively. Inject about 33 ⁇ L of emulsion at each of the three sites, including the neck and both sides of the groin. The day the animals were injected with the emulsion was defined as day 0. At 0 hours (Day 0) and 48 hours after injection of the emulsion (Day 2), each mouse was intraperitoneally injected with 200 ng (ie 200 ⁇ L) of PTX solution (1 ⁇ g/mL).
  • IFA immune emulsion containing 1 mg/ml MOG35-55 and 2 mg/ml Mycobacterium tuberculosis
  • the day of animal immunization was the 0th day. From the 2nd day to the 10th day, the animal weight was recorded three times a week, and the body weight and clinical score were closely observed and recorded every day from the 11th day.
  • the scoring standards are shown in Table 7 below:
  • the end-point collection of 75 animals used for drug efficacy evaluation was carried out on the 28th day, and the sampling time point was 0.25h after administration in the morning.
  • mice in each group were euthanized and then perfused with normal saline, followed by an equal volume of 10% formalin.
  • the spinal cords of the mice were taken and soaked in 10% formalin solution for tissue sectioning.
  • H&E hematoxylin - Eosin stain
  • LFB fast blue stain
  • inflammation 0 normal 1 pial inflammation, ⁇ 5 foci/section 2 pial inflammation, >5 foci/section 3 Inflammation of the cerebrospinal parenchyma, ⁇ 5 foci/section (involvement from pia mater to parenchyma) 4 parenchymal inflammation, ⁇ 5 foci/section 5 Parenchymal inflammation, >5 foci/section (small parenchymal lesions) 6 parenchymal inflammation, >5 foci/section (large parenchymal lesions) 7 Parenchymal inflammation, large lesions, and tissue cavities Lesion area Leision Area%, the percentage of lesion area involved in the section demyelination 0 no demyelination 1 few, scattered bare axons 2 small foci with exposed axons 3 multifocal exposed axons
  • the experimental data was plotted with Graph Pad Prism 7, expressed as mean ⁇ standard error (Mean ⁇ SEM), and the area under the clinical score curve was analyzed by one-way ANOVA (One-way ANOVA), p ⁇ 0.05 was considered to be compared with the model group There are significant differences.
  • BID 3mg/kg twice a day
  • QD 20mg/kg once a day
  • the inhibition rates of compound I administration groups relative to the vehicle control group clinical score AUC were 44% (3mg/kg twice a day (BID)), 77% (10mg/kg twice a day (BID)), 63% (20mg/kg /kg twice a day (BID)) and 60% (20 mg/kg once a day (QD)) (Figure 1, B). Therefore, compound I can significantly improve the clinical symptoms of EAE model, and the effective dose is 3 mg/kg twice a day (BID), and there is a certain dose-effect relationship.
  • the therapeutic effect of the 10mg/kg twice a day (BID) group was better than that of dexamethasone and fingolimod, and the inhibition rates of these two positive drugs on clinical score AUC were 65% and 49%, respectively.
  • mice were collected, including 2 mice in the normal control group and 5 mice in each of the other groups.
  • the spinal cord was fixed in formalin, embedded in paraffin and sectioned. Two sections were taken from each mouse.
  • the spinal cord was observed pathologically by H&E and FLB staining, and the cell infiltration, lesion area and demyelination were scored.
  • the model vehicle control (Veh) group showed significant pathological damage, with inflammatory cell infiltration and multifocal exposed axons in the white matter of the spinal cord. Specifically, as shown in FIG. 2 , the cell infiltration score of the model group reached 4 points, the lesion area reached 15%, and the demyelination score reached 3.2.
  • the results show that the mouse experimental autoimmune encephalomyelitis (EAE) model has a better therapeutic effect, and the present invention provides a new drug for the treatment of multiple sclerosis and encephalomyelitis, which has a greater therapeutic effect clinical application value.
  • EAE mouse experimental autoimmune encephalomyelitis
  • Example 3 Compound I in each dose group significantly reduced the phosphorylation level of STAT3 in the spinal cord
  • MOG35-55 was purchased from Jill Biochemical, item number: 51716;
  • IFA was purchased from Sigma-Aldrich, item number: F5506;
  • PTX Pertussis toxin
  • Dexamethasone acetate tablets are provided by Chenxin Pharmaceutical Co., Ltd., approved by the National Medicines Agency: H37021898;
  • dexamethasone acetate tablet take dexamethasone acetate tablet and dissolve in normal saline.
  • Compound I was provided by Guangzhou Jiayue Pharmaceutical Technology Co., Ltd.;
  • mice were marked with ear tags, and the backs of the mice were shaved.
  • mice Of 85 mice, 5 were randomly selected as the normal control group, and the other 80 were anesthetized with isoflurane, and subcutaneously injected with 100 ⁇ L immune emulsion (IFA containing 1 mg/ml MOG35-55 and 2 mg/ml Mycobacterium tuberculosis). Inject about 33 ⁇ L of emulsion at each of the three sites, including the neck and both sides of the groin. The day the animals were injected with the emulsion was defined as day 0. At 0 hours (Day 0) and 48 hours after injection of the emulsion (Day 2), each mouse was intraperitoneally injected with 200 ng (ie 200 ⁇ L) of PTX solution (1 ⁇ g/mL).
  • mice were used as the normal control group, and 70 mice were selected from the 80 model mice for the drug efficacy test) were collected at the end point on the 28th day. Dose for 0.25h in the morning. A total of the following samples were collected:
  • the spinal cord was collected and stored directly at -80°C for Western blot experiments to detect the protein expression levels of STAT3, pSTAT3 and GAPDH.
  • the phosphorylation level of STAT3 (p-STAT3/STAT3) reflects the level of myelitis cell infiltration and inflammatory response.
  • pSTAT1 refers to phosphorylation of STAT1
  • pSTAT5 refers to phosphorylation of STAT5.
  • the cells were centrifuged, washed once with staining buffer (DPBS+2% bovine serum albumin), and then stained with antibody 4 blood for 30 minutes (CD4 antibody was added to IL-6 and IL-2 stimulation).
  • staining buffer DPBS+2% bovine serum albumin
  • Calculate Response% 100 weight (MFI value of drug-dosed sample well-MFI mean value of Control well)/(MFI mean value of DMSO well-MFI mean value of Control well);
  • Compound I had inhibitory activity on both IL-6-induced pSTAT1 levels and IL-2-induced pSTAT5 levels in hPBMCs, with IC50 of 73.1 and 36.3 nM, respectively (Table 14).
  • IL-2 is interleukin 2
  • IL-6 is interleukin 6.
  • Compound I has inhibitory activity on the levels of hPBMC pSTAT1 and pSTAT5.
  • Wistar rats (pregnant rats) were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.
  • the concentration range of compound I used in the final test solution (ie 1X solution) of this experiment is 20, 2.222, 0.247, 0.027 ⁇ M; the concentration of methylprednisone compound (ie 1X solution) is 30 ⁇ M; the concentration of BAY 11–7082 (ie 1X solution) 5 ⁇ M; the final concentration of DMSO in each well is 0.1%. Blank control wells were added DMSO without compound, the final concentration was 0.1%.
  • the isolated astrocytes can be cultured in a 5% carbon dioxide incubator at 37° C., and the medium is changed every 3 to 4 days. Astrocytes can also be digested with trypsin (0.25%) and frozen in liquid nitrogen for future use.
  • Human NMO-IgG was isolated from a sterile-filtered serum pool (AQP4-IgG positive NMOSD) using a HiTrap protein G-HP (GE Healthcare) column.
  • the serum sample was diluted 1:1 with the binding buffer, and after filling the purification column with the binding buffer, the serum sample was added, and then the column was washed again with the binding buffer, and the antibody was eluted according to the instructions. Finally, antibodies were pooled on an Amicon Ultra-4 centrifuge (Milliwell) with a cutoff of 10,000 MW.
  • the concentrated immunoglobulins were filtered through a 0.22 ⁇ m membrane filter. The protein concentration of the concentrate was determined by the BCA method and stored at -80°C.
  • the total IgG extracted from the serum of NMOSD patients was NMO-IgG (including AQP4-IgG), and the cells were stimulated with a protein concentration of 250 ⁇ g/ml during the experiment.
  • Control-IgG (Jackson ImmunoResearch, 009-000-002) is a commercial reagent, the concentration of the stock solution is 11.5 mg/ml, and the working concentration is 250 ⁇ g/ml during the experiment.
  • Astrocytes were cultured or cultured after recovery until the second day and seeded in 24-well plates at a cell density (1.5 ⁇ 10 5 cells/well). Cells were pre-incubated for 30 min. Then each group continued to stimulate the cells with NMO-IgG (working final concentration of 250 ⁇ g/ml) for 24 hours to induce astrocyte injury. Normal human Control-IgG (CON-IgG, final working concentration: 250 ⁇ g/ml) was stimulated as a negative control.
  • the absorbance (OD value) of each group of samples was measured, and calculated by standard curve fitting The concentration of IL-6 in each experimental group.
  • the cell culture plate after aspirating the supernatant was detected by the CCK8 method to detect the cell viability of each group.
  • the absorbance (OD value) of the cells in each group was measured according to the operation steps in the instructions of the CCK8 kit (Beiren Chemical Technology Co., Ltd., #CK04), and the cell survival rate of each experimental group after the incubation was completed was calculated.
  • IL-6 inhibition rate % ( The absolute concentration of the blank control group - the absolute concentration of the test concentration hole) / (the absolute concentration of the blank control group - the absolute concentration of the negative control hole) * 100.
  • IL-6 ELISA The results of IL-6 ELISA showed that Compound I had a significant inhibitory effect on the release of IL-6 from astrocytes injured by NMO-IgG ( Figure 5 and Table 17), and the inhibition rate was about 98.8% at a test concentration of 20 ⁇ M. Methylprednisone inhibits IL-6 by about 74.8% at a test concentration of 30 ⁇ M. The inhibition rate of BAY compound on IL-6 is about 104.2% at the test concentration of 5 ⁇ M.

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Abstract

提供了一种吡咯并嘧啶类化合物的应用。该吡咯并嘧啶类化合物的结构如式I所示，其具有抑制STAT磷酸化的作用，对化脓性脊髓炎、急性脊髓炎、脑脊髓炎或自身免疫性疾病具有良好的预防或治疗作用。

Description

一种吡咯并嘧啶类化合物的应用
本申请要求申请日为2021/5/21的中国专利申请2021105572488和申请日为2022/5/9的中国专利申请2022105035016的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种吡咯并嘧啶类化合物的应用。
背景技术
中枢神经系统脱髓鞘疾病是以中枢神经系统(CNS)多灶性以及炎性脱髓鞘为主的一种自身免疫系统疾病，其临床特征主要包括反复发作、多次缓解及复发。临床较为常见的中枢神经系统脱髓鞘疾病包括多发性硬化症。
多发性硬化(multiple sclerosis，MS)是最常见的一种中枢神经系统脱髓鞘疾病，也是在青壮年中最常见的非创伤性的神经系统疾病，全球现有两百多万患者，随着对该病认识的越来越重视和诊断技术的提高，目前国内MS的诊断率逐年提高。但是，由于神经系统疾病较为复杂，多发性硬化病因不明，还未有多发性硬化的有效预防和治疗方法。目前，用于治疗多发性硬化的药物多为激素类，但是激素类的副作用较大，会引起水、盐、糖、蛋白质及脂肪代谢紊乱，而且还可能会出现肥胖、多毛、无力等。在一些情况下，MS的症状包括身体一个或多个部分的麻木或虚弱、视力的部分或全部丧失、长期复视、麻刺或疼痛、莱尔米特征、震颤、言语不清、疲劳、头晕和肠及膀胱功能受损。
实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)从最初建立至今大概50年(Kabat et al.,1947)。EAE通过激活自身反应性T细胞对CNS髓鞘产生的免疫应答，它可以有效研究免 疫系统在CNS炎症性脱髓鞘病变中所起的作用。其典型的病理特征表现为：(1)血管周围的炎性细胞(主要是淋巴细胞、巨噬细胞、激活的小胶质细胞)浸润；(2)病灶区域的脱髓鞘，特点是髓鞘破坏、轴突保存(Rao and Segal,2004)。它的免疫炎症反应、临床特征和组织病理学改变与人类MS疾病有惊人的相似(Sosa andForsthuber,2011)。因此，它是目前MS研究中应用最广泛的动物模型(Kipp et al.,2012)。MS和EAE两者的CNS白质均出现T细胞、巨噬细胞和B细胞浸润的脱髓鞘病灶。其中，病灶内出现吞噬了疏水的髓磷脂形成的泡沫样巨噬细胞被认为是活动病灶。寡克隆IgG在EAE和MS患者脑脊液(Cerebrospinal fluid,CSF)中均可见到。两者的差异之处在于后者发病机制中还有基因、表观遗传和环境因素(包括感染和免疫系统之间的相互作用)的参与(Becher etal.,2016)。
因此，研究开发能够安全、有效的预防和治疗多发性硬化的药物，以消除患者体内的神经免疫炎症的异常，对于临床治疗多发性硬化具有重要指导意义。
发明内容
本发明所要解决的技术问题是寻找一种对化脓性脊髓炎、急性脊髓炎、脑脊髓炎或自身免疫性疾病具有良好的预防和/或治疗作用的化合物，为此，本发明提供了一种吡咯并嘧啶类化合物的应用。
本发明提供了一种如式Ⅰ所示化合物或其药学上可接受的盐在制备STAT磷酸化抑制剂中的应用：
Figure PCTCN2022094256-appb-000001
本发明还提供了一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治 疗和/或预防具有STAT磷酸化水平异常升高特征的化脓性脊髓炎、急性脊髓炎或脑脊髓炎的药物中的应用：
Figure PCTCN2022094256-appb-000002
本发明还提供了一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防具有STAT磷酸化水平异常升高特征的自身免疫性疾病的药物中的应用：
Figure PCTCN2022094256-appb-000003
本发明还提供了一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防化脓性脊髓炎、急性脊髓炎或脑脊髓炎的药物中的应用：
Figure PCTCN2022094256-appb-000004
本发明还提供了一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防自身免疫性疾病的药物中的应用：
Figure PCTCN2022094256-appb-000005
本发明还提供了一种治疗和/或预防受试者化脓性脊髓炎、急性脊髓炎或脑脊髓炎疾病的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
Figure PCTCN2022094256-appb-000006
本发明还提供了一种治疗和/或预防受试者自身免疫性疾病的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
Figure PCTCN2022094256-appb-000007
本发明还提供了一种抑制受试者中STAT磷酸化的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐：
Figure PCTCN2022094256-appb-000008
本发明还提供了一种治疗和/或预防受试者中具有STAT磷酸化水平异常升高特征的化脓性脊髓炎、急性脊髓炎或脑脊髓炎药物的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
Figure PCTCN2022094256-appb-000009
本发明还提供了一种治疗和/或预防受试者中具有STAT磷酸化水平异常升高特征的自身免疫性疾病的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
Figure PCTCN2022094256-appb-000010
在一些实施方案中，所述的STAT磷酸化可为STAT1磷酸化、STAT3磷酸化和STAT5磷酸化中的一种或多种，例如STAT3磷酸化。
在一些实施方案中，所述自身免疫性疾病可为中枢神经系统的自身免疫性疾病或视神经系统的自身免疫性疾病；所述中枢神经系统的自身免疫性疾病可以为视神经脊髓炎、多发性硬化，例如视神经脊髓炎、复发缓解多发性硬化、继发进行性多发性硬化、原发进行性多发性硬化、进行性复发性多发性硬化、临床孤立综合征或放射学孤立综合征；所述视神经系统的自身免疫性疾病可为视神经脊髓炎。
在一些实施方案中，所述脑脊髓炎可以为急性播散性脑脊髓炎、亚急性坏死性脊髓炎、急性坏死出血性脑脊髓炎、小儿急性播散性脑脊髓炎或结核性脊髓炎。
在一些实施方案中，所述具有STAT磷酸化水平异常升高特征的自身免疫性疾病可为中枢神经系统的自身免疫性疾病或视神经系统的自身免疫性疾病；所述具有STAT磷酸化水平异常升高特征的中枢神经系统的自身免疫性疾病可以为视神经脊髓炎、多发性硬化，例如视神经脊髓炎、复发缓解多 发性硬化、继发进行性多发性硬化、原发进行性多发性硬化、进行性复发性多发性硬化、临床孤立综合征或放射学孤立综合征；所述视神经系统的自身免疫性疾病还可以为视神经脊髓炎。
在一些实施方案中，所述具有STAT磷酸化水平异常升高特征的脑脊髓炎、化脓性脊髓炎或急性脊髓炎，其中脑脊髓炎可以为急性播散性脑脊髓炎、亚急性坏死性脊髓炎、急性坏死出血性脑脊髓炎、小儿急性播散性脑脊髓炎或结核性脊髓炎。
在一些实施方案中，所述的如式Ⅰ所示化合物或其药学上可接受的盐为所述抑制剂或所述药物的有效成分之一或者唯一有效成分。
在一些实施方案中，向有需要的受试者可施用治疗有效量的所述如式Ⅰ所示化合物或其药学上可接受的盐。所述施用的方式可为任何合适的方式，例如口服。
在一些实施方案中，所述的如式Ⅰ所示化合物或其药学上可接受的盐还可以进一步包含药学上可接受的赋形剂，例如，片剂、皮下注射剂。
在一些实施方案中，所述如式Ⅰ所示化合物或其药学上可接受的盐的施用剂量可为1～120mg/kg/d，优选为3～100mg/kg/d，例如为3mg/kg/d、6mg/kg/d、10mg/kg/d、20mg/kg/d、40mg/kg/d、60mg/kg/d或90mg/kg/d。
在一些实施方案中，所述如式Ⅰ所示化合物或其药学上可接受的盐的单次施用剂量可为1-400mg，例如3mg、6mg、9mg、100mg或400mg。
在一些实施方案中，所述如式Ⅰ所示化合物或其药学上可接受的盐的施用频率可为1次/日、2次/日或3次/日，优选1次/日或2次/日。
本发明还提供了一种用于治疗和/或预防受试者化脓性脊髓炎、急性脊髓炎、脑脊髓炎或自身免疫性疾病的药物组合物，其包括：如式I所示化合物或其药学上可接受的盐，以及药用辅料。
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清 楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语在“STAT磷酸化抑制剂”可用于哺乳动物生物体内；也可用于生物体外，主要作为实验用途，例如：作为标准样或对照样提供比对，或按照本领域常规方法制成试剂盒，为STAT磷酸化抑制效果提供快速检测。
术语“具有STAT磷酸化水平异常升高特征的自身免疫性疾病”是指该自身免疫性疾病表现出高于正常水平的STAT磷酸化。本发明中，如式I所示化合物可通过抑制STAT磷酸化对自身免疫性疾病具有预防和/或治疗作用。
术语“具有STAT磷酸化水平异常升高特征的化脓性脊髓炎、急性脊髓炎或脑脊髓炎”是指该化脓性脊髓炎、急性脊髓炎或脑脊髓炎表现出高于正常水平的STAT磷酸化。本发明中，如式I所示化合物可通过抑制STAT磷酸化对化脓性脊髓炎、急性脊髓炎或脑脊髓炎具有预防和/或治疗作用。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物与相对无毒的、药学上可接受的酸或碱制备得到的盐。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的碱与这类化合物的原型接触的方式获得碱加成盐。药学上可接受的碱加成盐包括但不限于：锂盐、钠盐、钾盐、钙盐、铝盐、镁盐、锌盐、铋盐、铵盐、二乙醇胺盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的酸与这类化合物的原型接触的方式获得酸加成盐。所述的药学上可接受的酸包括无机酸，所述无机酸包括但不限于：盐酸、氢溴酸、氢碘酸、硝酸、碳酸、磷酸、亚磷酸、硫酸等。所述的药学上可接受的酸包括有机酸，所述有机酸包括但不限于： 乙酸、丙酸、草酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、水杨酸、酒石酸、甲磺酸、异烟酸、酸式柠檬酸、油酸、单宁酸、泛酸、酒石酸氢、抗坏血酸、龙胆酸、富马酸、葡糖酸、糖酸、甲酸、乙磺酸、双羟萘酸(即4,4’-亚甲基-双(3-羟基-2-萘甲酸))、氨基酸(例如谷氨酸、精氨酸)等。当本发明的化合物中含有相对酸性和相对碱性的官能团时，可以被转换成碱加成盐或酸加成盐。具体可参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977)、或、Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl and Camille G.Wermuth,ed.,Wiley-VCH,2002)。
术语“治疗”指治疗性疗法。涉及具体病症时，治疗指：(1)缓解疾病或者病症的一种或多种生物学表现，(2)干扰(a)导致或引起病症的生物级联中的一个或多个点或(b)病症的一种或多种生物学表现，(3)改善与病症相关的一种或多种症状、影响或副作用，或者与病症或其治疗相关的一种或多种症状、影响或副作用，或(4)减缓病症或者病症的一种或多种生物学表现发展。
术语“预防”是指获得或发生疾病或障碍的风险降低。
术语“受试者”是指根据本发明的实施例，即将或已经接受了该化合物给药的任何动物，哺乳动物为优，人类最优。术语“哺乳动物”包括任何哺乳动物。哺乳动物的实例包括但不限于牛、马、羊、猪、猫、狗、小鼠、大鼠、家兔、豚鼠、猴、人等，以人类为最优。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明发现如式I所示化合物具有抑制STAT磷酸化的作用，对化脓性脊髓炎、急性脊髓炎、脑脊髓炎或自身免疫性疾病具有良好的预防和/或治疗作用。
附图说明
图1为受试物化合物Ⅰ连续多次给药对小鼠EAE临床评价的影响。(数据以均值或均值±标准误(Mean±SEM)表示，*p<0.05,**p<0.01,***p<0.001VS G2)
图2为受试物化合物Ⅰ连续多次给药对小鼠脊髓病理评分的影响(数据以均数±标准误(Mean±SEM)表示，*p<0.05,**p<0.01,***p<0.001,****p<0.0001VS G2)。
图3为受试物化合物Ⅰ连续多次给药对脊髓STAT3磷酸化水平的影响(数据以均数±标准误(Mean±SEM)表示，**p<0.01,***p<0.001VS G2)。
图4为在NMO-IgG损伤星型胶质细胞模型中，化合物Ⅰ与细胞共孵育24小时，对细胞活力的影响。
图5为化合物Ⅰ对NMO-IgG损伤星型胶质细胞所致IL-6释放的影响。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。本发明所使用的溶剂和试剂均可经市售获得。
化合物Ⅰ由广州嘉越医药科技有限公司提供，按照专利PCT/CN2020094534记载的制备方法制备得到
Figure PCTCN2022094256-appb-000011
实施例1测试化合物Ⅰ对BV-2细胞系分泌CCL5和NO的抑制作用研 究
1、实验材料和方法
(1)细胞系
表1细胞系
细胞名称 组织类型 培养特性 培养基 每孔铺板数
BV-2 小鼠神经胶质瘤 贴壁型 RPMI 1640+10％FBS 12000
备注：1.细胞培养条件为：37℃,5％CO 2和95％湿度。
2.IC 50值：达到50％抑制效果时抑制剂的浓度。
3.BV-2购买于南京科佰生物科技有限公司。
4.表1中10％FBS中的百分数为体积百分数。
(2)试剂
1)RPMI1640培养基(购于Gibco，产品号：C22400500BT)；2)FBS(胎牛血清)(购于ExCell，产品号：FND500)；3)DMSO(购于Sigma，产品号：D2650)；4)LPS脂多糖(购于Sigma，产品号：L4391)；5)Mouse IFN-gamma Protein(购于R&D，产品号：485-MI-100)；6)CellTiter-Glo(CTG)(购于Promega，产品号：G7572)；7)Mouse CCL5/RANTES DuoSet ELISA(R&D，产品号：DY478-05)；8)Griess Reagent System(Promega，产品号：G2930)。
(3)供试品和阳性对照品
表2供试品和阳性对照品信息
Figure PCTCN2022094256-appb-000012
称取化合物化合物Ⅰ及西尼莫德，分别加入DMSO(二甲亚砜)溶解成10mM储液，放入-20℃储存待用。
(4)CTG方法测定化合物细胞增殖、上清CCL5和NO检测
具体步骤如下：
步骤1：收集处于指数生长期的BV-2细胞，并用Vi-Cell XR细胞计数 仪进行活细胞计数。用培养基将细胞悬液调整到适当浓度。每孔加160μl细胞悬液于96-孔细胞培养板，最终细胞浓度为12000细胞/孔。将培养板37℃,5％CO 2孵箱中培养24小时。
步骤2：使用DMSO将10mM DMSO化合物储存液按1:4比例梯度稀释，配制系列1000X供试化合物溶液，然后用细胞培养基将1000X供试化合物溶液1:100稀释后配制系列10X供试化合物溶液。最后，取出前一天已铺板的96-孔板，按照实验孔板加样设计，每孔细胞分别加入20μl相应的10X溶液，每个药物浓度各3个复孔。本实验最终测试所用化合物浓度(即1X溶液)范围分别为10,2.5,0.625,0.156,0.0391,0.0098,0.0024μM，每孔DMSO终浓度为0.1％。
化合物加样完毕后，将96孔板置于37℃，5％CO 2孵箱中培养半小时。
预孵育结束之后，每孔分别加入20μl含有100ng/ml LPS和100ng/ml IFN-γ的培养基(最终浓度为10ng/ml LPS和10ng/ml IFN-γ)，用以诱导激活BV-2细胞。然后将培养板置于37℃,5％CO 2孵箱中培养24小时。
步骤3：化合物处理24小时后，将培养板离心1200转5分钟，吸上清至另外板中，锡纸密封于-20℃保存，将用于ELISA检测和Griess试剂系统检测。吸完上清的细胞培养板将立即用于CTG法检测细胞增殖。
步骤4：在吸完上清的细胞培养板中每孔加入100μl PBS，按照CTG试剂盒操作说明，每孔加入50μl预先融化并平衡到室温的CTG溶液，用微孔板震荡器混匀2分钟，于室温放置10分钟后用Envision2104读板仪测定萤光信号值。
步骤5(NO和CCL5检测)：使用ELISA试剂盒(Mouse CCL5/RANTES DuoSet ELISA；R&D；DY478-05)，按照试剂盒说明书，检测上清中CCL5水平；使用Griess试剂系统(Griess Reagent System；Promega；G2930)，按照试剂盒说明书，检测上清中一氧化氮(NO)的含量。
(5)数据分析
1)CTG数据分析
步骤1：通过CTG荧光信号值，计算各测试浓度相对于空白(DMSO)组的存活细胞百分数(surviving cells％)，计算公式为：surviving cells％＝待测浓度组荧光值/空白组荧光值。
步骤2：以化合物浓度的对数值对存活细胞百分数作图，然后采用非线性回归算出拟和曲线，利用软件GraphPad Prism 5公式log(inhibitor)vs.response--Variable slope计算得到IC50值。
步骤3：抑制率的计算公式为：inh.(％)＝{空白组荧光值-待测浓度组荧光值}/空白(DMSO)组荧光值。
2)CCL5和NO数据分析
步骤1：首先通过ELISA和Griess的光密度值，绘制标准曲线并计算出各样品的绝对浓度。然后使用各样品所对应的CTG荧光值进行校准，得到各样品的校准浓度，计算公式为：校准浓度＝绝对浓度/同一样本CTG荧光值。
步骤2：根据各样品校准浓度计算各测试浓度相对于空白(DMSO)组分泌CCL5或NO的相对百分比，即CCL5production(％)或NO production(％)，计算公式为：相对百分比％＝待测浓度组校准浓度/空白组校准浓度。
步骤3：最后，以化合物浓度的对数值对CCL5分泌相对百分比或NO分泌相对百分比作图，采用非线性回归算出拟和曲线，利用软件GraphPad Prism 5公式log(inhibitor)vs.response--Variable slope计算得到IC50值。
步骤4：抑制率的计算公式为：inh.(％)＝{空白组校准浓度-待测浓度组校准浓度}/空白组校准浓度。
(6)实验结果
表3化合物Ⅰ和西尼莫德对BV-2细胞增殖的IC50和最大抑制率
Figure PCTCN2022094256-appb-000013
结论：化合物Ⅰ与BV-2细胞共孵育24小时，对细胞增殖无抑制作用(表3)，最高测试浓度为10μM。此外，化合物Ⅰ浓度在>0.1μM时，细胞数目较空白(DMSO)孔有所增加，最高细胞数目增长率为46.39％(表3)，接近未加LPS/IFNγ条件下的细胞数目，化合物Ⅰ可能提高活化的BV-2细胞的存活能力。化合物西尼莫德在所有测试浓度下，对细胞增殖亦无明显抑制，最大抑制率为4.44％。
表4化合物Ⅰ和西尼莫德对BV-2细胞系分泌Mouse CCL5的IC50和最大抑制率
Figure PCTCN2022094256-appb-000014
结论：CCL5 ELISA结果显示，化合物Ⅰ与BV-2共孵育24小时后，IC50为0.863μM，最大抑制率为80.29％(表4)。同样条件下，化合物西尼莫德最大抑制率为35.16％，IC50为>10μM(表4)。
表5化合物Ⅰ和西尼莫德对BV-2细胞系分泌NO的IC50和最大抑制率
Figure PCTCN2022094256-appb-000015
结论：Griess试剂系统检测结果显示，化合物Ⅰ与BV-2共孵育24小时后，IC50为0.487μM，最大抑制率为100％(表5)。同样条件下，化合物西尼莫德IC50为>10μM,最大抑制率为7.71％(表5)。
综上所述，化合物Ⅰ能够有效抑制LPS/IFNγ诱导产生的细胞因子(CCL5)及活性氮(NO)，并呈量效关系：对细胞因子(CCL5)的最大抑制率为80.29％， IC50为0.863μM；对活性氮(NO)的最大抑制率为100.00％，IC50为0.487μM。西尼莫德在本实验中的IC50均大于10μM。
化合物Ⅰ可显著抑制小胶质细胞(BV-2)过度激活导致的促炎症因子释放，提示其为治疗多发性硬化等神经炎症疾病的重要机制之一。
实施例2研究化合物I对MOG35-55诱导的实验性自身免疫性脑脊髓炎(EAE)小鼠模型的体内药效学研究
实验性自身免疫性脑脊髓炎(EAE)小鼠模型是研究多发性硬化的理想动物模型。
实验目的：研究化合物I在MOG35-55诱导的小鼠实验性自身免疫性脑脊髓炎模型中的药效
实验设计：
(1)动物：C57BL/6小鼠，雌性，6-8周龄，无特定病原体(SPF)。由上海斯莱克实验动物技术有限公司提供。
(2)受试化合物和试剂：
1)MOG35-55购于吉尔生化，货号：51716；2)IFA购于Sigma-Aldrich，货号：F5506；3)结核分枝杆菌H37Ra，Difco，货号：231141；4)PTX(Pertussis toxin)购于List Biological Laboratories,货号：180235AIA；5)醋酸地塞米松片由辰欣药业提供，国药准字：H37021898；6)醋酸地塞米松片的配制方法：取醋酸地塞米松片溶于生理盐水；7)化合物Ⅰ由广州嘉越医药科技有限公司提供；
化合物Ⅰ的配制方法：
称取适量化合物Ⅰ直接溶于溶媒(0.5％(w/v)羟丙甲基纤维素(HPMC)E5/0.5％(w/v)聚乙烯吡咯烷酮PVP/0.2％(w/v)十二烷基硫酸钠SDS水溶液)，在室温下超声、搅拌约1小时，得到均匀、不透明的细颗粒悬浮液。每三天一配，4℃保存。
(3)EAE造模方法：
1)动物适应期过后打耳标标记小鼠，将小鼠背部剃毛。提前1-2天准备无菌的玻璃瓶3个和匀浆机分散刀头；
2)配制免疫乳剂(含1mg/ml MOG35-55和2mg/ml结核分枝杆菌)和PTX溶液配制(PTX粉末每支50μg，加入PBS 1mL，配成50μg/mL母液，用前使用PBS稀释至1μg/mL，现配现用)；
3)动物免疫
85只小鼠，随机挑出5只作为正常对照组，另外80只异氟烷麻醉，在皮下注射100μL免疫乳剂(含1mg/ml MOG35-55和2mg/ml结核分枝杆菌的IFA)，分别在三个位点，包括颈部和腹股沟两侧，每点各注射约33μL乳剂。动物注射乳剂当天定义为第0天。于注射乳剂后0小时(第0天)和48小时后(第2天)，每只小鼠腹腔注射200ng(即200μL)PTX溶液(1μg/mL)。
(4)分组与给药
表6分组与剂量设计
Figure PCTCN2022094256-appb-000016
Figure PCTCN2022094256-appb-000017
(5)临床指征测定
动物免疫当天为第0天，从第2天至第10天每周三次记录动物体重，第11天开始每天密切观察记录体重和临床评分，评分标准见下表7：
表7.EAE临床评分标准
分值 临床症状
0 正常表现，没有明显的疾病征兆
1 尾巴下垂无力，后肢单侧无力
2 尾巴下垂无力，双后肢均无力步态蹒跚
3 单侧后肢无力麻痹瘫痪
4 双后肢均无力麻痹瘫痪
5 死亡
(6)样品收集
用于药效评价的75只动物终点收样在第28天进行，收样时间点为上午给药0.25h。
每组5只小鼠，安乐死后生理盐水心脏灌流，再等体积的10％福尔马林灌流，取小鼠脊髓，用10％福尔马林溶液浸泡后进行组织切片，H&E(苏木精-伊红染色)和LFB(固蓝染色)染色和病理评分。病理评分标准见表8。
表8.脊髓H&E和LFB病理评分标准
炎症
0正常
1软膜炎症，<5灶/切片
2软膜炎症，>5灶/切片
3开始有脑脊髓实质的炎症，<5灶/切片(从软膜向实质累及)
4实质炎症，<5灶/切片
5实质炎症，>5灶/切片(实质病灶较小)
6实质炎症，>5灶/切片(实质病灶较大)
7实质炎症，有大的病灶，组织出现空洞
病灶面积
Leision Area％,病灶累及面积占截面的百分比
脱髓鞘
0无脱髓鞘
1少量，散在的裸露的轴索
2小灶裸露的轴索
3多灶性裸露的轴索
4融合的脱髓鞘病灶
5广泛的脱髓鞘
(7)统计学处理
实验数据用Graph Pad Prism 7作图，用平均数±标准误表示(Mean±SEM)表示，临床评分曲线下面积分析用单因素方差分析(One-way ANOVA)，p<0.05认为和模型组对比有显著性差异。
(8)实验结果
1)临床评分和发病率
表9化合物Ⅰ连续给药多次给药对小鼠EAE临床评分的影响
(给药14天，第13天-第27天)
Figure PCTCN2022094256-appb-000018
Figure PCTCN2022094256-appb-000019
小鼠免疫后第12天，小鼠开始出现临床症状。第13天分组后开始给药。如图1的A和表9所示，溶媒对照组的平均临床评分逐渐升高，至第19天达到峰值，平均分值约为3.0分，证明本实验EAE模型成功建立(图1的A和表9)。与溶媒对照(Veh control)组相比，阳性药地塞米松(DEX)对疾病的发展有着明显的抑制作用，临床评分自第15天至第27天均与溶媒对照组有显著性差异，临床评分曲线下面积AUC下降率为65％(图1的B)。阳性药芬戈莫德组的临床评分自第17天至第23天均与溶媒对照组呈显著性差异，临床评分AUC下降率为49％(图1的B)。
受试物化合物Ⅰ各剂量组均显著降低EAE小鼠的临床评分，其中3mg/kg每天2次(BID)组于第15天至第21天、10mg/kg每天2次(BID)组于第15天至第27天、20mg/kg每天2次(BID)组于第16天至第27天、20mg/kg每天1次(QD)组于第17天至第27天分别与溶媒对照组呈显著性差异。化合物Ⅰ各给药组相对溶媒对照组临床评分AUC的抑制率分别为44％(3mg/kg每天2次(BID))、77％(10mg/kg每天2次(BID))、63％(20mg/kg每天2次(BID))和60％(20mg/kg每天1次(QD))(图1的B)。因此，化合物Ⅰ能显著改善EAE模型临床症状，起效剂量为3mg/kg每天2次(BID)，并呈一定的量效关系。10mg/kg每天2次(BID)组的治疗效果优于地塞米松和芬戈莫德，这两个阳性药对临床评分AUC的抑制率分别为65％和49％。
表10连续给药多次给药对EAE小鼠发病率的影响(％)
(给药14天，第13天-第27天)
Figure PCTCN2022094256-appb-000020
Figure PCTCN2022094256-appb-000021
此外，发病率结果显示(表10)，溶媒对照组于第17天发病率为100％，阳性药地塞米松和芬戈莫德组发病率均有所降低(图1的C)。化合物Ⅰ给药组均表现出延后发病和发病率降低，其中10mg/kg每天2次(BID)组发病率最低；20mg/kg每天2次(BID)组在发病后期展现了持续抑制发病率的优势(图1的C)。
2)脊髓病理分析
实验终点收集小鼠的脊髓，其中正常对照组2只，其余各组每组5只。脊髓固定于福尔马林，进行石蜡包埋和切片，每只小鼠取2张切片，通过H&E和FLB染色对脊髓进行病理观察并对细胞浸润，病灶区和脱髓鞘进行评分。实验表明，模型溶媒对照(Veh)组呈现出显著的病理损伤，脊髓白质出现炎性细胞的浸润和多灶性裸露的轴索。具体地，如图2所示，模型组的细胞浸润评分达4分，病灶区达15％，脱髓鞘评分达3.2。与EAE临床评分结果相一致，不同剂量的受试物化合物Ⅰ都显著抑制了EAE建模对脊髓的损伤，其中10mg/kg每天2次(BID)组治疗效果在细胞浸润(图2的A)、病灶区范围(图2的B)和脱髓鞘(图2的C)三方面都显著优于阳性药芬戈莫德组(图2)。其中，小鼠脊髓H&E染色后对脊髓切片中细胞浸润(A)和病灶区面积(B)进行统计。LFB染色后对脊髓切片中脱髓鞘程度评分(C)。数据以均数±标准误(Mean±SEM)表示。*p<0.05,**p<0.01,***p<0.001,****p<0.0001,vs.溶媒对照组(Vehicle),单因素方差分析(One-way ANOVA)。
病理分析结果表明，化合物Ⅰ各剂量组均能改善EAE模型引起的病理改变，其中化合物Ⅰ10mg/kg每天2次(BID)组对炎细胞浸润、病灶区范围和脱髓鞘三方面的改善都优于阳性药芬戈莫德组。
本发明中，结果表明对小鼠实验性自身免疫性脑脊髓炎(EAE)模型有较好的治疗作用，本发明为治疗多发性硬化症和脑脊髓炎提供了新的药物，有较大的临床应用价值。
实施例3化合物Ⅰ各剂量组均显著降低脊髓STAT3磷酸化水平
(1)动物：C57BL/6小鼠，雌性，6-8周龄，无特定病原体(SPF)。由上海斯莱克实验动物技术有限公司提供。
(2)受试化合物和试剂：
MOG35-55购于吉尔生化，货号：51716；
IFA购于Sigma-Aldrich，货号：F5506；
结核分枝杆菌H37Ra，Difco，货号：231141；
PTX(Pertussis toxin)购于List Biological Laboratories,货号：180235AIA；
醋酸地塞米松片由辰欣药业提供，国药准字：H37021898；
醋酸地塞米松片的配制方法：取醋酸地塞米松片溶于生理盐水。
化合物Ⅰ由广州嘉越医药科技有限公司提供；
化合物Ⅰ的配制方法：
称取适量化合物Ⅰ直接溶于溶媒(0.5％(w/v)羟丙甲基纤维素(HPMC)E5/0.5％(w/v)聚乙烯吡咯烷酮PVP/0.2％(w/v)十二烷基硫酸钠SDS水溶液)，在室温下超声、搅拌约1小时，得到均匀、不透明的细颗粒悬浮液。每三天一配，4℃保存。
(3)EAE造模方法：
1)动物适应期过后打耳标标记小鼠，将小鼠背部剃毛。提前1-2天准备无菌的玻璃瓶3个和匀浆机分散刀头；
2)配制免疫乳剂(含1mg/ml MOG35-55和2mg/ml结核分枝杆菌)和 PTX溶液配制(PTX粉末每支50μg，加入PBS 1mL，配成50μg/mL母液，用前使用PBS稀释至1μg/mL，现配现用)；
3)动物免疫
85只小鼠，随机挑出5只作为正常对照组，另外80只异氟烷麻醉，在皮下注射100μL免疫乳剂(含1mg/ml MOG35-55和2mg/ml结核分枝杆菌的IFA)，分别在三个位点，包括颈部和腹股沟两侧，每点各注射约33μL乳剂。动物注射乳剂当天定义为第0天。于注射乳剂后0小时(第0天)和48小时后(第2天)，每只小鼠腹腔注射200ng(即200μL)PTX溶液(1μg/mL)。
(4)分组与给药
表11分组与剂量设计
Figure PCTCN2022094256-appb-000022
(5)样本采集和检测
用于药效评价的75只动物(其中，5只作为正常对照组，80只造模小鼠中挑选出70只小鼠进行药效试验)终点收样在第28天进行，收样时间点 为上午给药0.25h。共收集如下样本：
每组5只动物，异氟烷麻醉后心脏取血，不进行灌流，安乐死后取样。
取脊髓，直接-80℃保存，用于Western blot实验，检测STAT3,pSTAT3和GAPDH的蛋白表达水平。
(6)实验结果
实验第28天上午给药0.25h后终点收样，收集小鼠脊髓，包括正常对照组(n＝3)、溶媒对照组(n＝5)、阳性对照芬戈莫德组(n＝5)和所有化合物Ⅰ给药组(每组n＝5)，通过Western blot检测STAT3、pSTAT3和GAPDH的蛋白水平并使用GE Healthcare 1D quant densitometry software对条带进行定量。STAT3的磷酸化水平(p-STAT3/STAT3)反应了脊髓炎细胞浸润和炎症反应的水平。与正常组相比，EAE模型溶媒对照组脊髓样品STAT3磷酸化显著提高(图3的A、B)。如图3的A、B所示，阳性药芬戈莫德可以显著抑制STAT3磷酸化至正常动物水平。与临床评分一致，受试物化合物Ⅰ各剂量组均显著抑制脊髓STAT3磷酸化水平，其中10mg/kg每天2次(BID)和20mg/kg每天2次(BID)抑制效果最好，抑制率达86％(图3的C)。其中，数据以均数±标准误(Mean±SEM)表示。**p<0.01,***p<0.001,vs.溶媒对照组(Vehicle),单因素方差分析(One-way ANOVA)。
结论：化合物Ⅰ各剂量组均显著降低脊髓STAT3磷酸化水平，其中10mg/kg每天2次(BID)和20mg/kg每天2次(BID)抑制效果最好，抑制率约86％。
实施例4化合物Ⅰ对hPBMC pSTAT1、pSTAT5抑制的IC50值(nM)
1.细胞
表12细胞
名称 供应商 货号或编号 批号
hPBMC stemcell 70025.3 2009428002
2.主要试剂
表13主要试剂
名称 供应商 货号或编号 储存条件
Human IL-6 absin abs00808 -807
Human IL-2 absin abs00804 -807
人pSTAT1抗体 BD 612564 41
人pSTAT5抗体 BD 562077 42
固定液 BD 558049 43
破膜液 BD 558050 -202
其中，pSTAT1指的是STAT1磷酸化、pSTAT5指的是STAT5磷酸化。
3.实验方法
1.药物孵育及细胞因子刺激
1)配制培养液：1640培养基+10％胎牛血清+1％青/链霉素+1％非必需氨基酸(百分比皆为体积比)
2)细胞37℃水浴解冻后离心，然后用培养液将细胞浓度调整为5×10 5个/mL。
3)将细胞悬液200μL/孔接种到96孔圆底板，37℃孵育90min。
4)加入药物，37℃孵育30min。
5)分别加入10ng/mL IL-6、4ng/mL IL-2刺激细胞，37℃孵育15min。
4.细胞染色及检测
1)将刺激结束后的细胞离心，用染色缓冲液(DPBS+2％牛血清蛋白)洗涤一次后加入抗体4血染色30min(IL-6、IL-2刺激的加CD4抗体)。
2)染色缓冲液洗涤两次后，加入固定液固定细胞，4色固定20min。
3)染色缓冲液洗涤一次后，加入-20冲预冷的破膜液，4冷破膜30min。
4)染色缓冲液洗涤两次后，加入抗体4色染色30min(IL-6、IL-2)
5)用染色缓冲液洗涤细胞两次并重悬，用流式细胞仪检测CD4或CD33阳性细胞中的pSTAT1或pSTAT5 MFI值。
5.数据处理
1)计算Response％＝100重(加药样品孔MFI值-Control孔MFI均值)/(DMSO孔MFI均值-Control孔MFI均值)；
2)将Response％值带入Graphpad prism 8软件，使用log(inhibitor)vs. response--Variable slope(four parameters)方法绘制抑制曲线，并得到样品对应的IC50值。
5.实验结果
化合物Ⅰ对hPBMC中IL-6诱导的pSTAT1水平和IL-2诱导的pSTAT5水平均有抑制活性，其抑制IC50分别为73.1和36.3nM(表14)。
表14药物对pSTAT1、pSTAT5抑制的IC50值(nM)
组别   IC50值(nM)
01 IL-6诱导的pSTAT1 73.1
02 IL-2诱导的pSTAT5 36.3
其中，IL-2为白介素2；IL-6为白介素6。
结论：化合物Ⅰ对hPBMCpSTAT1、pSTAT5水平均有抑制活性。
实施例5
实验目的：化合物Ⅰ对NMO-IgG所致星型胶质细胞损伤模型分泌IL-6的抑制作用研究
实验设计
1.动物：Wistar大鼠(孕鼠)购自北京维通利华实验动物有限公司
2.供试品和对照品信息
表15供试品和阳性对照品信息
Figure PCTCN2022094256-appb-000023
将化合物溶于DMSO配制化合物储存液，使用DMSO将化合物储存液按比例梯度稀释，配制系列1000X供试化合物溶液，然后用细胞培养基将1000X供试化合物溶液1:100稀释后配制系列10X供试化合物溶液。最后，按照实验孔板加样设计，每孔细胞分别加入相应体积的10X溶液，每个药物浓度各2个复孔。本实验最终测试溶液(即1X溶液)所用化合物Ⅰ浓度范围 分别为20,2.222,0.247,0.027μM；甲泼尼松化合物浓度(即1X溶液)为30μM；BAY 11–7082浓度(即1X溶液)为5μM；每孔DMSO终浓度为0.1％。空白对照孔加入不含化合物的DMSO,终浓度为0.1％。
3.实验步骤：
(1)大鼠原代星型胶质细胞提取分离与培养
1)原代星型胶质细胞的提取分离：取1日龄新生大鼠大脑皮层制备混合胶质细胞培养液。新生大鼠处死后浸于酒精，剪皮、取脑、分离大脑皮层、研磨，去除结缔组织，将混合后的胶质细胞通过70μm细胞过滤器过筛，接种于经多聚赖氨酸包被的细胞培养板上，培养基含10％热灭活胎牛血清、100U/mL青霉素和100mg/mL链霉素的DMEM，体外培养约12天后细胞达到融合，通过振摇将小胶质细胞、内皮细胞和少突胶质细胞从混合胶质细胞中去除。随后通过两次胰蛋白酶消化和种板，提升星形胶质细胞纯度，细胞纯度>95％。
2)星型胶质细胞的培养：分离得到的星型胶质细胞可在37℃、5％二氧化碳培养箱中培养，每3～4天换液一次。星型胶质细胞也可经胰酶(0.25％)消化，冻存于液氮中备用。
(2)NMO-IgG的获得与分离纯化
1)收集符合2015修订后的NMOSD国际最新诊断标准的AQP4-IgG阳性NMOSD患者血清样本12例(伦理批号：KY 2021-069-01)，符合NMOSD诊断标准的所有患者血清AQP4-IgG抗体检测方法均采用目前国际认可的间接免疫荧光染色法(即cell based assay，CBA法)判定阴阳。血清样品经0.22μm无菌过滤，保存于-80℃，纯化前经56℃灭活30min。
2)血清中抗体(IgG)纯化
用HiTrap蛋白G-HP(GE Healthcare)柱从无菌滤过的血清池(AQP4-IgG阳性的NMOSD)中分离人源NMO-IgG。血清样品以结合缓冲液按1：1稀释，以结合缓冲液填充纯化柱之后，加入血清样品，随后用结合缓冲液再次 洗涤柱子，并按照说明书要求洗脱抗体。最终，抗体集中在截止量为10,000MW的Amicon Ultra-4离心机(微孔)上。浓缩的免疫球蛋白经0.22μm滤膜过滤。用BCA法测定浓缩物的蛋白浓度，储存在-80℃。由NMOSD患者血清提取的总IgG为NMO-IgG(含AQP4-IgG)，实验时以250μg/ml的蛋白浓度刺激细胞。
3)Control-IgG：
Control-IgG(Jackson ImmunoResearch，009-000-002)为商品化试剂，储存液浓度为11.5mg/ml，实验时工作浓度250μg/ml。
(3)药物孵育和样品检测
1)药物孵育
星型胶质细胞培养或复苏后培养至第2天接种于24孔板，细胞密度(1.5×10 5个/孔)，受试化合物化合物Ⅰ、甲泼尼松、BAY11-7082按照上述浓度与细胞预先孵育30min。然后各组继续以NMO-IgG(工作终浓度为250μg/ml)刺激细胞24小时以引发星型胶质细胞损伤。正常人源Control-IgG(CON-IgG，工作终浓度为250μg/ml)刺激作为阴性对照。
2)样品收集检测
NMO-IgG刺激细胞处理24小时后，收集上清。应用酶联免疫吸附法(ELISA)，按照IL-6ELISA试剂盒说明书(美国R&D，#RD6000B)进行IL-6检测，测定各组样本的吸光度(OD值)，通过标准曲线拟合，计算得出各实验组IL-6浓度。吸完上清的细胞培养板使用CCK8法检测各组细胞存活率。按CCK8试剂盒(北仁化工科技有限公司，#CK04)说明书操作步骤，测定各组细胞的吸光度(OD值)，计算得出各实验组在孵育结束后细胞存活率。
(5)数据分析
1)细胞活力百分率：通过CCK8光密度值(OD值)，计算各测试浓度相对于空白对照组(NMO-IgG+DMSO)的细胞活力百分率％，计算公式为： 细胞活力百分率％＝(待测浓度孔OD值-空板背景值)/(空白对照组OD值-空板背景值)*100。
2)IL-6抑制率：
首先通过ELISA光密度值，绘制标准曲线并计算出各样品的绝对浓度。
根据各样品绝对浓度计算各测试浓度相对于空白对照组(NMO-IgG+DMSO)分泌IL-6的相对抑制百分比，即IL-6抑制率％，计算公式为：IL-6抑制率％＝(空白对照组绝对浓度-待测浓度孔绝对浓度)/(空白对照组绝对浓度-阴性对照孔绝对浓度)*100。
(6)实验结果
CCK8实验结果显示，在NMO-IgG损伤星型胶质细胞模型中，供试化合物、化合物Ⅰ与细胞共孵育24小时，对细胞活力无抑制作用(图4和表16)，最高测试浓度为20μM。甲泼尼松在测试浓度30μM下，对细胞活力有抑制趋势。BAY化合物在测试浓度5μM下，细胞活力百分率显著降低至39.5％。
表16化合物Ⅰ和对照化合物对星形胶质细胞活力的影响
Figure PCTCN2022094256-appb-000024
IL-6ELISA结果显示，化合物Ⅰ对NMO-IgG损伤星型胶质细胞所致IL-6释放具有明显抑制作用(图5和表17)，在测试浓度20μM下，抑制率约为98.8％。甲泼尼松在测试浓度30μM下，对IL-6抑制率约为74.8％。BAY化合物在测试浓度5μM下，对IL-6抑制率约为104.2％。
表17化合物Ⅰ和对照化合物对星形胶质分泌IL-6的影响
Figure PCTCN2022094256-appb-000025
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不违背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (13)

  1. 一种如式Ⅰ所示化合物或其药学上可接受的盐在制备STAT磷酸化抑制剂中的应用：
    Figure PCTCN2022094256-appb-100001
  2. 一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防化脓性脊髓炎、急性脊髓炎或脑脊髓炎的药物中的应用：
    Figure PCTCN2022094256-appb-100002
    所述脑脊髓炎可以为急性播散性脑脊髓炎、亚急性坏死性脊髓炎、急性坏死出血性脑脊髓炎、小儿急性播散性脑脊髓炎或结核性脊髓炎。
  3. 一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防自身免疫性疾病的药物中的应用：
    Figure PCTCN2022094256-appb-100003
    所述自身免疫性疾病可为中枢神经系统的自身免疫性疾病或视神经系统的自身免疫性疾病；所述中枢神经系统的自身免疫性疾病可为视神经脊髓炎或多发性硬化，例如视神经脊髓炎、复发缓解多发性硬化、继发进行 性多发性硬化、原发进行性多发性硬化、进行性复发性多发性硬化、临床孤立综合征或放射学孤立综合征；所述视神经系统的自身免疫性疾病可为视神经脊髓炎。
  4. 一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防具有STAT磷酸化水平异常升高特征的化脓性脊髓炎、急性脊髓炎或脑脊髓炎的药物中的应用：
    Figure PCTCN2022094256-appb-100004
    所述具有STAT磷酸化水平异常升高特征的脑脊髓炎可以为急性播散性脑脊髓炎、亚急性坏死性脊髓炎、急性坏死出血性脑脊髓炎、小儿急性播散性脑脊髓炎或结核性脊髓炎。
  5. 一种如式Ⅰ所示化合物或其药学上可接受的盐在制备治疗和/或预防具有STAT磷酸化水平异常升高特征的自身免疫性疾病的药物中的应用：
    Figure PCTCN2022094256-appb-100005
    所述具有STAT磷酸化水平异常升高特征的自身免疫性疾病可为中枢神经系统的自身免疫性疾病或视神经系统的自身免疫性疾病；所述具有STAT磷酸化水平异常升高特征的中枢神经系统的自身免疫性疾病可为视神经脊髓炎或多发性硬化，例如视神经脊髓炎、复发缓解多发性硬化、继发进行性多发性硬化、原发进行性多发性硬化、进行性复发性多发性硬化、临床孤立综合征或放射学孤立综合征；所述视神经系统的自身免疫性疾病可为视神经脊髓炎。
  6. 一种治疗和/或预防受试者化脓性脊髓炎、急性脊髓炎或脑脊髓炎的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
    Figure PCTCN2022094256-appb-100006
    所述脑脊髓炎可以为急性播散性脑脊髓炎、亚急性坏死性脊髓炎、急性坏死出血性脑脊髓炎、小儿急性播散性脑脊髓炎或结核性脊髓炎。
  7. 一种治疗和/或预防受试者自身免疫性疾病的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
    Figure PCTCN2022094256-appb-100007
    其中，所述自身免疫性疾病可为中枢神经系统的自身免疫性疾病或视神经系统的自身免疫性疾病；所述中枢神经系统的自身免疫性疾病可为视神经脊髓炎、多发性硬化，例如视神经脊髓炎、复发缓解多发性硬化、继发进行性多发性硬化、原发进行性多发性硬化、进行性复发性多发性硬化、临床孤立综合征或放射学孤立综合征；所述视神经系统的自身免疫性疾病可为视神经脊髓炎。
  8. 一种抑制受试者中STAT磷酸化的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐：所述的STAT磷酸化可以为STAT1磷酸化、STAT3磷酸化和STAT5磷酸化中的一种或多种；
    Figure PCTCN2022094256-appb-100008
  9. 一种治疗和/或预防受试者中具有STAT磷酸化水平异常升高特征的化脓性脊髓炎、急性脊髓炎或脑脊髓炎的自身免疫性疾病的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
    Figure PCTCN2022094256-appb-100009
    所述具有STAT磷酸化水平异常升高特征的脑脊髓炎可以为急性播散性脑脊髓炎、亚急性坏死性脊髓炎、急性坏死出血性脑脊髓炎、小儿急性播散性脑脊髓炎或结核性脊髓炎。
  10. 一种治疗和/或预防受试者中具有STAT磷酸化水平异常升高特征的自身免疫性疾病的方法，其包括:给予所述受试者治疗有效量的如式I所示化合物或其药学上可接受的盐；
    Figure PCTCN2022094256-appb-100010
    其中，所述具有STAT磷酸化水平异常升高特征的自身免疫性疾病可为中枢神经系统的自身免疫性疾病或视神经系统的自身免疫性疾病；所述具有STAT磷酸化水平异常升高特征的中枢神经系统的自身免疫性疾病可以为视神经脊髓炎、多发性硬化，例如视神经脊髓炎、复发缓解多发性硬化、继发 进行性多发性硬化、原发进行性多发性硬化、进行性复发性多发性硬化、临床孤立综合征或放射学孤立综合征；所述视神经系统的自身免疫性疾病可为视神经脊髓炎。
  11. 如权利要求1、4和5至少一项所述的应用、或如权利要求8、9和10至少一项所述的方法，其特征在于，所述的STAT磷酸化为STAT1磷酸化、STAT3磷酸化和STAT5磷酸化中的一种或多种。
  12. 如权利要求1～5至少一项所述的应用或6～10至少一项所述的方法，其特征在于，其满足下述条件的一种或多种：
    (1)所述的如式Ⅰ所示化合物或其药学上可接受的盐为如权利要求1所述抑制剂或如权利要求2～5任一项所述药物的有效成分之一或者唯一有效成分；
    (2)所述如式I所示化合物或其药学上可接受的盐通过口服给予受试者；
    (3)所述的如式Ⅰ所示化合物或其药学上可接受的盐包含药学上可接受的赋形剂；
    (4)所述如式Ⅰ所示化合物或其药学上可接受的盐的施用剂量为1～120mg/kg/d，例如为3～100mg/kg/d，又例如为3mg/kg/d、6mg/kg/d、10mg/kg/d、20mg/kg/d、40mg/kg/d、60mg/kg/d或90mg/kg/d；
    (5)所述如式I所示化合物或其药学上可接受的盐的单次施用剂量为1-400mg；例如为3 3mg、6mg、9mg、100mg或400mg；
    (6)所述如式I所示化合物或其药学上可接受的盐的施用频率为1次/日、2次/日或3次/日，又例如为1次/日或2次/日；
    (7)所述如式I所示化合物或其药学上可接受的盐制成的剂型为片剂或皮下注射剂。
  13. 一种用于治疗和/或预防受试者化脓性脊髓炎、急性脊髓炎、脑脊髓炎或自身免疫性疾病的药物组合物，其包括：如式I所示化合物或其药学上 可接受的盐，以及药用辅料；
    Figure PCTCN2022094256-appb-100011
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