WO2019001416A1 - 噻唑啉酮杂环化合物、其制备方法、药用组合物及应用 - Google Patents

噻唑啉酮杂环化合物、其制备方法、药用组合物及应用 Download PDF

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WO2019001416A1
WO2019001416A1 PCT/CN2018/092824 CN2018092824W WO2019001416A1 WO 2019001416 A1 WO2019001416 A1 WO 2019001416A1 CN 2018092824 W CN2018092824 W CN 2018092824W WO 2019001416 A1 WO2019001416 A1 WO 2019001416A1
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formyl
substituted
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piperazinyl
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周荣斌
邓贤明
江维
姜华
陈云
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中国科学技术大学
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Definitions

  • the invention relates to the technical field of medicinal chemistry, in particular to a thiazolinone heterocyclic compound, a preparation method thereof, a pharmaceutical composition and application thereof.
  • NLRP3 (NACHT, LRR and PYD domains-containing protein 3) is an important member of the intracellular pattern recognition receptor NOD-like receptors (NLRs), which are subject to exogenous pathogen-associated molecular patterns (eg bacteria, fungi). When stimulated by a risk-related molecular pattern (such as urate crystals, saturated fatty acids), NLRP3 recruits a protein-associated speck-like protein containing a CARD and a caspase-1 precursor to form a multimeric complex. It is the NLRP3 inflammatory body.
  • NLRs intracellular pattern recognition receptor NOD-like receptors
  • NLRP3 inflammatory bodies After assembly of NLRP3 inflammatory bodies, self-shearing occurs, forming caspase-1 with splicing activity, which induces maturation and secretion of IL-1 ⁇ and IL-18, mediates immune response and inflammatory response, and maintains immune homeostasis. .
  • various diseases such as enteritis, hepatitis, silicosis, ultraviolet sun-induced skin sunburn and contact hypersensitivity, sepsis, and tumor. , neurodegenerative diseases, even type 2 diabetes and atherosclerosis. This suggests that NLRP3 inflammatory bodies are potential targets for the treatment of these diseases.
  • the drugs for clinical treatment of NLRP3 inflammatory body-related diseases are mainly concentrated on the target IL-1, including the IL-1 receptor agonist anakinra, IL-1 monoclonal antibody cannanumab (canakinumab) And the IL-1 receptor inducer rilonacept.
  • targets of IL-1 have certain curative effects on some diseases, IL-1 is not the only effector molecule produced by the activation of NLRP3 inflammatory bodies, nor is it the only way to induce NLRP3 inflammatory bodies to induce related diseases. Therefore, the drug of target IL-1 is not effective in treating all NLRP3 inflammatory body related diseases.
  • IL-1 is not only derived from NLRP3 inflammatory bodies, but activation of other inflammatory bodies (such as NLRP1, NLRC4) can also cause IL-1 maturation and secretion. Therefore, interventions targeting NLRP3 inflammatory body activation can fundamentally inhibit diseases mediated by IL-1, IL-18 and HMGB1, compared to the intervention of target IL-1 and its receptors. Good and more specific therapeutic effects, so NLRP3 is more valuable as a potential target, and it is more meaningful to target NLRP3 inflammatory body treatment-related inflammatory diseases.
  • NLRP3 inflammatory bodies such as MCC950, sulforaphane, isoliquiritigenin, ⁇ -hydroxybutyrate, flufenamic acid, mefenamic acid, 3,4-methylenedioxy- ⁇ -nitrostyrene (MNS), parthenolide , Bay 11-7082, etc.
  • MNS 3,4-methylenedioxy- ⁇ -nitrostyrene
  • parthenolide Bay 11-7082, etc.
  • Targeting other protein components of NLRP3 inflammatory vesicles inhibits inflammatory bodies such as AIM2 and NLRC4, thereby inhibiting the body's anti-infective ability; targeting NLRP3 inflammatory body-associated signaling pathways or signaling molecules, such as NEK7, mitochondrial damage, potassium outflow, etc. will inevitably have inevitable non-specific effects, because these signaling molecules or signal events will also affect other life processes. There are no evidences of small molecules that have been reported so far that they directly target NLRP3 itself. Sulforaphane, isoliquiritigenin, beta-hydroxybutyrate, parthenolide and Bay 11-7082 have been reported to inhibit the activation of the NF-kB signaling pathway.
  • MNS 3,4-methylenedioxy- ⁇ -nitrostyrene
  • Src and Syk Flufenamic acid and mefenamic acid have been reported to inhibit the activation of NLRP3 inflammatory bodies by inhibiting chloride efflux.
  • MCC950 does not currently report non-specific effects, the molecule does not directly inhibit the assembly of inflammatory bodies, such as NLRP3 poly- and NLRP3-ASC interactions, suggesting that it may target the upstream signaling pathway of NLRP3 inflammatory bodies.
  • no specific inhibitors capable of targeting NLRP3 itself have been found so far. To find a small molecule chemical that is capable of specifically inhibiting the activation of NLRP3 inflammatory bodies and is safe and highly specific for the treatment of NLRP3 inflammatory body-related diseases is of great significance and imperative.
  • the technical problem to be solved by the present invention is to provide a thiazolinone heterocyclic compound, a preparation method thereof, a pharmaceutical composition and use thereof, and to find a series of compounds having specific inhibitory activity against NLRP3 inflammatory bodies.
  • the present invention provides a use of a thiazolinone heterocyclic compound having a structure represented by Formula I or Formula II or an isomer thereof, a prodrug, or a pharmaceutically acceptable form thereof.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from:
  • R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from:
  • X 1 and X 2 are each independently selected from O or S;
  • n is an arbitrary integer from 0 to 6;
  • Q is C or N
  • Q 1 , Q 2 , Q 3 , Q 4 are independently selected from -CR 1 , -CR 2 , -CR 3 , -CR 4 , -CR 5 , N, -NR 1 , S or O;
  • Ar 2 is a substituted or unsubstituted aryl, heteroaryl or fused ring group
  • m 0 or 1.
  • the broken line indicates that the linkage may be a single bond or a double bond.
  • the above substituted or unsubstituted amino group is preferably an amino group, a C1-C10 alkyl-substituted amino group, a C1-C10 dialkyl-substituted amino group, a C3-C7 cycloalkyl-substituted amino group or an acyl-substituted amino group.
  • the substituent of the above substituted C1-C6 oxyalkyl group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the substituent of the above substituted C1-C6 alkoxy group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the substituent of the above-mentioned substituted C1-C6 alkyl group is preferably any one or more of a halogen, a nitro group and a cyano group.
  • the substituent of the above substituted C3-C7 cycloalkyl group is preferably any one or more of a halogen, a nitro group and a cyano group.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from:
  • the C1-C6 alkyl group is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, an isopentyl group or a neopentyl group.
  • the above C1-C6 alkyl group may be substituted with any one or more of a halogen, a nitro group and a cyano group.
  • the C1-C6 fluoroalkyl group is preferably substituted with any one or more of halogen, nitro, amino and cyano groups, and in certain embodiments of the invention, it is a trifluoromethyl group.
  • the C1-C6 oxyalkyl group is preferably a methoxyethyl group or a methoxyethoxymethyl group.
  • the above C1-C6 oxyalkyl group may be substituted by any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the C1-C6 alkoxy group is preferably a methoxy group or an ethoxy group.
  • the above C1-C6 alkoxy group may be substituted by any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the C1-C6 fluoroalkoxy group is preferably substituted with any one or more of halogen, nitro, amino and cyano groups, and in certain embodiments of the invention, it is a trifluoromethoxy group.
  • the C3-C7 cycloalkyl group is preferably a cyclopropyl group, a cyclobutyl group, a cyclopentyl group or a cyclohexyl group.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from:
  • any three or four of R 1 , R 2 , R 3 , R 4 and R 5 are simultaneously H.
  • R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from:
  • the substituent of the substituted C1-C6 alkyl group is preferably any one or more of a halogen, a nitro group, a cyano group, an ester group and a carboxyl group.
  • the ester group is preferably a methyl formate group.
  • the above substituted C1-C6 alkyl group has the structure:
  • R 11 is preferably a C1-C6 alkyl group.
  • R 11 is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl.
  • the substituent of the substituted C1-C6 oxyalkyl group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group. .
  • the substituent of the substituted C1-C6 alkoxy group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • said R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from:
  • the C1-C10 alkylsulfonyl group is methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl or isobutylsulfonyl.
  • the C1-C10 alkylaminosulfonyl group is N-methylaminosulfonyl, N-ethylaminosulfonyl, N-propylaminosulfonyl, N-isopropylamino Sulfonyl, N-butylaminosulfonyl, N-isobutylaminosulfonyl, N,N-dimethylaminosulfonyl, N,N-diethylaminosulfonyl or N,N-dipropylamino Sulfonyl.
  • the C3 to C10 cycloalkylaminosulfonyl group is N-cyclopropylaminosulfonyl, N-cyclobutylaminosulfonyl, N-cyclopentylaminosulfonyl or N - cyclohexylaminosulfonyl group.
  • the C6-C12 arylaminosulfonyl group is an N-phenylaminosulfonyl group.
  • the C5-C12 heteroarylaminosulfonyl group is a five-membered heteroarylaminosulfonyl group or a six-membered heteroarylaminosulfonyl group, such as N-pyridylaminosulfonyl, N -Thienylaminosulfonyl, N-furylaminosulfonyl or N-pyrrolylaminosulfonyl.
  • the C1-C10 sulfonylamino group is methanesulfonylamino, ethanesulfonylamino, propylsulfonylamino, isopropylsulfonylamino, butylsulfonylamino, isobutylsulfonylamino , sulfonic acid group, aminosulfonyl group.
  • the above C1 to C10 alkylsulfonyl group, C1 to C10 alkylaminosulfonyl group, C3 to C10 cycloalkylaminosulfonyl group, C6 to C12 arylaminosulfonyl group, C5 to C12 heteroarylaminosulfonyl group, Or a C1 to C10 sulfonylamino group may be bonded to any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • any three or four of R 6 , R 7 , R 8 , R 9 , and R 10 are simultaneously H.
  • X 1 and X 2 are each independently selected from O or S.
  • X 1 is O or S and X 2 is O.
  • X 1 is S and X 2 is O.
  • X 1 and X 2 are both O.
  • n is 0, 1, 2, 3, 4, 5 or 6; preferably, n is 0, 1, 2 or 3.
  • Q is C or N.
  • n is preferably 0, 1, 2 or 3.
  • n is preferably 0 or 1.
  • the Formula II has the structure of Formula II-a or Formula II-b:
  • Ar 2 is a substituted or unsubstituted aryl group, a heteroaryl group or a fused ring group.
  • Ar 2 is a substituted or unsubstituted phenyl, five- or six-membered heteroaryl, or a substituted or unsubstituted phenyl with a substituted or unsubstituted aryl, heteroaryl, hetero A fused ring group formed by the condensation of a ring group.
  • the aryl group is preferably a C6-C10 aryl group, and more preferably a phenyl group.
  • the heteroaryl group is preferably a five- or six-membered heteroaryl group, preferably one or more hetero atoms in N, O, and S. Further preferred is a pyridyl group, a pyrimidinyl group, a pyridazinyl group, a pyrazinyl group or a pyranyl group.
  • the heterocyclic group is preferably a C2-C10 heterocyclic group.
  • the Ar 2 is a substituted or unsubstituted phenyl, pyrrolyl, thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, triazolyl, tetra An azolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyranyl group, or a group:
  • the substituent is independently hydrogen, halogen, nitro, hydroxy, cyano, substituted or unsubstituted amino, methylsulfonyl, p-toluenesulfonyl or substituted or unsubstituted C1-C6 oxygen.
  • the substituted or unsubstituted C6-C10 aryl group is a substituted phenyl group.
  • the substituent of the phenyl group is a halogen, a nitro group, an amino group, a hydroxyl group, a cyano group or a C1-C6 alkyl group.
  • the present inventors have found that the above compounds inhibit the activation of NLRP3 inflammatory bodies, thereby inhibiting the maturation and secretion of IL-1 ⁇ and IL-18, thereby inhibiting the development of related diseases caused by the activation of NLRP3 inflammatory bodies. .
  • the NLRP3 inflammatory body inhibitor is used to treat, alleviate or prevent an inflammatory disease caused by an NLRP3 inflammatory body.
  • the above inflammation preferably includes inflammation of the central system and/or inflammation of the peripheral system.
  • the above inflammatory diseases preferably include:
  • Type II diabetes atherosclerosis, fatty liver, metabolic syndrome, acute and chronic tissue damage caused by infection, gout, arthritis, enteritis, hepatitis, peritonitis, silicosis, UV-induced skin sunburn, contact hypersensitivity
  • gout arthritis
  • enteritis hepatitis
  • peritonitis hepatitis
  • silicosis UV-induced skin sunburn
  • contact hypersensitivity One or more of the reaction, sepsis, tumor, neurodegenerative disease, multiple sclerosis, and Mu-Wey's syndrome.
  • the above peritonitis and gout are preferably inflammatory diseases caused by urate crystal accumulation.
  • the above sepsis is preferably acute peripheral inflammation caused by intraperitoneal injection of bacterial lipopolysaccharide (LPS).
  • LPS bacterial lipopolysaccharide
  • the above Mu-Wei's syndrome is preferably a clinical hereditary disease Mu-Wey's syndrome caused by mutation of the NLRP3 protein resulting in sustained activation of the NLRP3 inflammatory body.
  • the above-described administration method of the NLRP3 inflammatory body inhibitor for peritonitis, gout, sepsis and Mu-Web syndrome is preferably intraperitoneal injection.
  • the dose for intraperitoneal injection is preferably from 2.5 mg/kg to 20 mg/kg, preferably 20 mg/kg.
  • the administration of the above-described NLRP3 inflammatory body inhibitor for metabolic syndrome is preferably oral administration or intraperitoneal injection.
  • the dose for oral administration is preferably from 20 mg/kg to 40 mg/kg, preferably 40 mg/kg.
  • the dose for intraperitoneal injection is preferably from 2.5 mg/kg to 20 mg/kg, preferably 2.5 mg/kg.
  • the type II diabetes, fatty liver, and atherosclerosis are induced by high-fat food (HFD, 60% Fat), with obvious insulin resistance accompanied by impaired glucose tolerance and metabolism of hepatic steatosis and arterial plaque formation.
  • HFD high-fat food
  • compositions of the present invention when applied to the preparation of NLRP3 inflammatory body inhibitors, may be used alone or in combination with other drugs.
  • the present invention also provides a thiazolinone heterocyclic compound having the structure of Formula I or Formula II or an isomer, prodrug, pharmaceutically acceptable solvate or salt thereof:
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from:
  • R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from:
  • X 1 is O, X 2 is S, or X 1 is S, and when X 2 is O, n is an arbitrary integer from 1 to 6;
  • n is an arbitrary integer from 0 to 6;
  • Q is C or N
  • Q 1 , Q 2 , Q 3 , Q 4 are independently selected from -CR 1 , -CR 2 , -CR 3 , -CR 4 , -CR 5 , N, -NR 1 , S or O;
  • Ar 2 is a substituted or unsubstituted aryl, heteroaryl or fused ring group
  • m 0 or 1.
  • thiazolinone heterocyclic compound does not include the following compounds:
  • the above substituted or unsubstituted amino group is preferably an amino group, a C1-C10 alkyl-substituted amino group, a C1-C10 dialkyl-substituted amino group, a C3-C7 cycloalkyl-substituted amino group or an acyl-substituted amino group.
  • the substituent of the above substituted C1-C6 oxyalkyl group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the substituent of the above substituted C1-C6 alkoxy group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the substituent of the above substituted C1-C6 alkyl group is preferably any one or more of a halogen, a nitro group and a cyano group.
  • the substituent of the above substituted C3-C7 cycloalkyl group is preferably any one or more of a halogen, a nitro group and a cyano group.
  • said R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from:
  • the C1-C6 alkyl group is preferably a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, an isopentyl group or a neopentyl group.
  • the above C1-C6 alkyl group may be substituted with any one or more of a halogen, a nitro group and a cyano group.
  • the C1-C6 fluoroalkyl group is preferably substituted with any one or more of halogen, nitro, amino and cyano groups, and in certain embodiments of the invention, it is a trifluoromethyl group.
  • the C1-C6 oxyalkyl group is preferably a methoxyethyl group or a methoxyethoxymethyl group.
  • the above C1-C6 oxyalkyl group may be substituted by any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the C1-C6 alkoxy group is preferably a methoxy group or an ethoxy group.
  • the above C1-C6 alkoxy group may be substituted by any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • the C1-C6 fluoroalkoxy group is preferably substituted by any one or more of a halogen, a nitro group, an amino group and a cyano group, and in some embodiments of the invention, it is a trifluoromethoxy group.
  • the cycloalkyl group is preferably a cyclopropyl group, a cyclobutyl group, a cyclopentyl group or a cyclohexyl group.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently selected from:
  • any three or four of R 1 , R 2 , R 3 , R 4 and R 5 are simultaneously H.
  • the substituted or unsubstituted formyl group is preferably a C1-C6 acyl group.
  • R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from:
  • the substituent of the substituted C1-C6 alkyl group is preferably any one or more of a halogen, a nitro group, a cyano group, an ester group and a carboxyl group.
  • the ester group is preferably a methyl formate group.
  • the above substituted C1-C6 alkyl group has the structure:
  • R 11 is preferably a C1-C6 alkyl group.
  • R 11 is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl.
  • the substituent of the substituted C1-C6 oxyalkyl group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group. .
  • the substituent of the substituted C1-C6 alkoxy group is preferably any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • said R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from:
  • the C1-C10 alkylsulfonyl group is methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl or isobutylsulfonyl.
  • the C1-C10 alkylaminosulfonyl group is N-methylaminosulfonyl, N-ethylaminosulfonyl, N-propylaminosulfonyl, N-isopropylamino Sulfonyl, N-butylaminosulfonyl, N-isobutylaminosulfonyl, N,N-dimethylaminosulfonyl, N,N-diethylaminosulfonyl or N,N-dipropylamino Sulfonyl.
  • the C3 to C10 cycloalkylaminosulfonyl group is N-cyclopropylaminosulfonyl, N-cyclobutylaminosulfonyl, N-cyclopentylaminosulfonyl or N - cyclohexylaminosulfonyl group.
  • the C6-C12 arylaminosulfonyl group is an N-phenylaminosulfonyl group.
  • the C5-C12 heteroarylaminosulfonyl group is a five-membered heteroarylaminosulfonyl group or a six-membered heteroarylaminosulfonyl group, such as N-pyridylaminosulfonyl, N -Thienylaminosulfonyl, N-furylaminosulfonyl or N-pyrrolylaminosulfonyl.
  • the C1-C10 sulfonylamino group is methanesulfonylamino, ethanesulfonylamino, propylsulfonylamino, isopropylsulfonylamino, butylsulfonylamino, isobutylsulfonylamino , sulfonic acid group, aminosulfonyl group.
  • the above C1 to C10 alkylsulfonyl group, C1 to C10 alkylaminosulfonyl group, C3 to C10 cycloalkylaminosulfonyl group, C6 to C12 arylaminosulfonyl group, C5 to C12 heteroarylaminosulfonyl group, Or a C1 to C10 sulfonylamino group may be bonded to any one or more of a halogen, a nitro group, an amino group and a cyano group.
  • any three or four of R 6 , R 7 , R 8 , R 9 , and R 10 are simultaneously H.
  • X 1 is O, X 2 is S, or X 1 is S, and when X 2 is O, n is an arbitrary integer from 1 to 6;
  • n is an arbitrary integer of 0 to 6; preferably, X 1 is O or S, and X 2 is O.
  • X 1 is S
  • X 2 is O
  • n is an arbitrary integer from 1 to 6, and specifically, n is 1, 2, 3, 4, 5, or 6
  • n is 1, 2 or 3.
  • X 1 and X 2 are simultaneously O, and n is any integer from 0 to 6, in particular, n is 0, 1, 2, 3, 4, 5 or 6; , n, is 0, 1, 2 or 3.
  • n 1, 2 or 3
  • the compound has a selective inhibitory effect on inflammatory bodies.
  • Q is C or N.
  • R 1 , R 2 , R 3 , R 4 and R 5 are not H at the same time.
  • R 8 is not a methoxyformyl group.
  • n 2 or 3
  • Q is C
  • any one of R 6 , R 7 , R 8 , R 9 and R 10 is a carboxyl group, R 1 , R 2 , R 3 , R Both 4 and R 5 are not H.
  • X 1 and X 2 are both O
  • R 6 , R 7 , R 8 , R 9 and R 10 are a carboxyl group
  • R 1 , R 2 , R 3 , R 4 and R 5 are not trifluoromethyl.
  • X 1 and X 2 are both O
  • R 6 , R 7 , R 8 , R 9 and R 10 are a carboxyl group
  • R 1 , R 2 , R 3 , R 4 and R 5 are not methoxyformyl groups.
  • the Formula II has the structure of Formula II-a or Formula II-b:
  • Ar 2 is a substituted or unsubstituted aryl group, a heteroaryl group or a fused ring group.
  • Ar 2 is a substituted or unsubstituted phenyl, five- or six-membered heteroaryl, or a substituted or unsubstituted phenyl with a substituted or unsubstituted aryl, heteroaryl, hetero A fused ring group formed by the condensation of a ring group.
  • the aryl group is preferably a C6-C10 aryl group, and more preferably a phenyl group.
  • the heteroaryl group is preferably a five- or six-membered heteroaryl group, preferably one or more hetero atoms in N, O, and S. Further preferred is a pyridyl group, a pyrimidinyl group, a pyridazinyl group, a pyrazinyl group or a pyranyl group.
  • the heterocyclic group is preferably a C2-C10 heterocyclic group.
  • the Ar 2 is a substituted or unsubstituted phenyl, pyrrolyl, thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, triazolyl, tetra An azolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyranyl group, or a group:
  • the substituent is independently hydrogen, halogen, nitro, hydroxy, cyano, substituted or unsubstituted amino, methylsulfonyl, p-toluenesulfonyl or substituted or unsubstituted C1-C6 oxygen.
  • the substituted or unsubstituted C6-C10 aryl group is a substituted phenyl group.
  • the substituent of the phenyl group is a halogen, a nitro group, an amino group, a hydroxyl group, a cyano group or a C1-C6 alkyl group.
  • the thiazolinone heterocyclic compound has any of the following structures or cis-trans isomers thereof:
  • C1-C6 oxyalkyl group refers to a group in which a C1-C6 alkyl skeleton is substituted by one or more C1-C6 alkoxy groups, for example, a methoxyethyl group, a methoxy group B. Oxymethyl group and the like.
  • the C1-C6 alkoxy group means that the C1-C6 alkyl group is bonded to the mother nucleus via an oxygen atom, such as a methoxy group, an ethoxy group or the like.
  • C 1 -C 6 alkyl refers to any straight or branched chain group containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, tert-butyl, sec-butyl, n-pentyl, tert-amyl, n-hexyl and the like.
  • C 3 -C 7 cycloalkyl refers to a 3- to 7-membered all-carbon monocyclic ring which may contain one or more double bonds but does not have a fully conjugated ⁇ - electronic system.
  • cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutane, cyclopentyl, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptyl, ring Heptenyl, cycloheptadienyl.
  • each of the above substituents may be further substituted with one or more of the above-exemplified groups.
  • halogen atom means a fluorine, chlorine, bromine or iodine atom.
  • cyano refers to the -CN residue.
  • nitro refers to a -NO 2 group.
  • alkoxy refers to any of the above C 1 -C 6 alkyl, C 3 -C 7 naphthenes A aryl, aryl or heterocyclic group attached to the remainder of the molecule through an oxygen atom (-O-).
  • sulfonyl refers to a -S(O) 2- R residue.
  • R is a substituent group defined by the present invention, such as a generally substituted or unsubstituted amino group or an alkyl group.
  • penta-heteroaryl refers to a heteroaryl group having a carbon atom and a total of five hetero atoms.
  • heteroaryl refers to a heteroaryl group having a carbon atom and a total of six hetero atoms.
  • arylamino refers to an amino group substituted with an aryl group, wherein the aryl group is as defined above.
  • alkylthio, alkylamino, dialkylamino, alkoxycarbonyl, alkoxycarbonylamino, heterocyclylcarbonyl, heterocyclylcarbonylamino, cycloalkyloxycarbonyl, etc. are as defined above, wherein alkyl, The alkoxy, aryl, C 3 -C 7 cycloalkyl and heterocyclic groups are as defined above.
  • prodrug refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the invention. Prodrugs undergo this reaction to become active compounds only under biological conditions or are active in their unreacted form. Prodrugs can generally be prepared using well-known methods, such as those described in 1 Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, ed. 5th edition).
  • the invention also provides a preparation method of the above thiazolinone heterocyclic compound, comprising the following steps:
  • the above carbon dichloride is also referred to as thiophosgene.
  • the compound represented by the formula b and the ethyl carbazate are subjected to a base-catalyzed cyclization reaction under the action of a basic compound to obtain a compound of the formula c.
  • the basic compound may be triethylamine or the like.
  • the addition reaction of the step C) is preferably carried out under the conditions of a basic compound such as ammonium acetate or sodium acetate.
  • the phosgene is known as trichloromethyl carbonate.
  • the compound represented by the formula b' and the ethyl carbazate are subjected to a base-catalyzed cyclization reaction under the action of a basic compound to obtain a compound of the formula c'.
  • the basic compound may be triethylamine or the like.
  • the addition reaction of the step C) is preferably carried out under the conditions of a basic compound such as ammonium acetate or sodium acetate.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 in the above structure are the same as those in the above, and will not be described herein.
  • the preparation method of the above compound provided by the present invention is not limited to the above production method, and other preparation methods well known to those skilled in the art are also within the scope of the present invention.
  • Thin layer chromatography was carried out on a silica gel GF254 precoated plate (Qingdao Marine Chemical Plant). Column chromatography was carried out by silica gel (300-400 mesh, Yantai Zhihuang Silica Gel Development Reagent Factory) under medium pressure or by column chromatography using a pre-packed silica gel cartridge (ISCO or Welch) using an ISCO Combiflash Rf200 rapid purification system. The ingredients were developed by UV light ( ⁇ : 254 nm) and by iodine vapor.
  • the compound was prepared by preparative HPLC on a Waters Symmetry C18 (19 x 50 mm, 5 ⁇ m) column or via a Waters X Terra RP 18 (30 x 150 mm, 5 ⁇ m) column using a Waters preparative HPLC 600 equipped with a 996 Waters PDA detector. And Micromass mod.ZMD single quadrupole mass spectrometry (electrospray ionization, cationic mode).
  • Method 1 Phase A: 0.1% TFA / MeOH 95/5 ; phase B: MeOH / H 2 O 95/5 . Gradient: 10 to 90% B for 8 min, 90% B 2 min; flow rate 20 mL/min.
  • Method 2 Phase A: 0.05% NH 4 OH / MeOH 95/5; phase B: MeOH / H 2 O 95/5 . Gradient: 10 to 100% B for 8 min, maintaining 100% B 2 min. The flow rate was 20 mL/min.
  • Electrospray (ESI) mass spectra were obtained on a Finnigan LCQ ion trap.
  • HPLC-UV-MS analysis for evaluating compound purity was performed by combining an ion trap MS apparatus with an HPLC system SSP4000 (Thermo Separation Products) equipped with an autosampler LC Pal (CTC Analytics) and a UV6000LP diode array Detector (UV detection 215-400 nm). Device control, data acquisition and processing with Xcalibur 1.2 software (Finnigan). HPLC chromatography was carried out at room temperature and a flow rate of 1 mL/min using a Waters X Terra RP 18 column (4.6 x 50 mm; 3.5 [mu]m).
  • Mobile phase A was ammonium acetate 5 mM buffer (pH 5.5 with acetic acid): acetonitrile 90:10
  • mobile phase B ammonium acetate 5 mM buffer (pH 5.5 with acetic acid): acetonitrile 10:90; gradient 0 to 100% B Perform for 7 minutes and then maintain 100% B for 2 minutes before rebalancing.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above thiazolinone heterocyclic compound or a salt thereof or a thiazolinone heterocyclic compound prepared by the above preparation method or a salt thereof, and a pharmaceutically acceptable carrier, an excipient, Diluent, adjuvant, vehicle or combination thereof.
  • the carrier, the excipient, the diluent, the adjuvant, and the vehicle are not particularly limited, and may be a carrier, an excipient, a diluent, an adjuvant suitable for a pharmaceutical composition well known to those skilled in the art. vehicle.
  • the present inventors have found a series of compounds having inhibitory activity against inflammatory bodies, providing compounds having the structure of formula I, tautomers, prodrugs, pharmaceutically acceptable solvents thereof.
  • the compound or salt is used as an inhibitor of NLRP3 inflammatory body which has excellent specific inhibitory activity against NLRP3 inflammatory bodies.
  • Figure 1 CY-09 inhibits Nigericin and MSU-induced NLRP3 inflammatory body activation and IL-1 ⁇ secretion electropherogram and histogram;
  • Figure 2 Activation histogram of CY-09 specific inhibition of classical and non-canonical NLRP3 inflammatory bodies
  • Figure 3 CY-09 inhibits the formation of ASC dimers and the assembled electrophoresis pattern of NLRP3 inflammatory body complexes
  • FIG. 4 Electrophoresis pattern of CY-09 specific interaction with NLRP3 protein
  • ATP binding site in the NACHT functional segment of NLRP3 protein is an electrophoresis map of the direct binding site of CY-09;
  • Figure 6 Bar graph of CY-09 specific inhibition of ATPase activity of NLRP3 protein
  • Figure 7 Cytogram of CY-09 inhibiting MSU-induced peritonitis
  • Figure 8 CY-09 inhibits MSU-induced gout curve and histogram
  • Figure 9 Column diagram of CY-09 inhibiting LPS-induced sepsis
  • Figure 11 Bar graph and graph of obesity and insulin resistance induced by intraperitoneal injection of CY-09 in high-fat diets
  • Figure 12 Bar graph and graph of obesity and insulin resistance induced by oral administration of CY-09 in the treatment of high fat diets.
  • Compound IA-1 was prepared according to the following route:
  • Table 1 The compounds shown in Table 1 were prepared according to the above procedure, and Table 1 shows the structure and structural characterization data of the compounds.
  • Table 2 The compounds shown in Table 2 were prepared according to the procedure described in Example 1, and Table 2 is the structure and structural characterization data of the compounds.
  • Table 3 The compounds shown in Table 3 were prepared according to the procedure described in Example 1, and Table 3 is the structure and structural characterization data of the compounds.
  • the compound of formula II-1 was prepared according to the following route:
  • the crude product compound 2 (187 mg, 1.0 mmol) and 50 mg of sodium salt were dissolved in anhydrous toluene (1.0 mL), and methyl thioglycolate (0.13 mL, 1.0 mmol) was quickly taken into the tube at 120 ° C for 2 h. After completion of the reaction, it was naturally cooled to room temperature and directly concentrated to give the crude compound 3 as a white solid.
  • Table 4 The compounds shown in Table 4 were prepared according to the above procedure, and Table 4 is the structure and structural characterization data of the compounds.
  • test materials used in the following examples were purchased from conventional biochemical reagent stores, as shown in the table below.
  • Example 5 CY-09 (IA-1) and analogues inhibit the activation of NLRP3 inflammatory bodies in macrophages in vitro
  • CFTR(inh)-172(IC-1) inhibits NLRP3 inflammatory body activation
  • BMDM cells cultured in vitro to day 4 were divided into 24-well plates, adhered to the wall overnight, the medium was removed, and cultured in Opti-MEM medium containing 1% FBS and LPS for 3 hours.
  • the wells were added with CFTR(inh)-172 and various CY-09 analogs at a dose of 10 ⁇ M, and DMSO control wells were set at the same time.
  • the culture was continued for 30 minutes, and an equal amount of the classical NLRP3 inflammatory body activator Nigericin was added to each well. Stimulate for 20 minutes.
  • the IA group, the IB group compound, and the compound II have a specific inhibitory effect on the NLRP3 inflammatory body.
  • BMDM bone marrow-derived macrophages
  • BMDM cells were divided into 12-well plates at 3-6*10 5 cells per well.
  • the first group a blank control group.
  • the second group the negative control group, only the same dose of ultra-LPS was added.
  • Group 3 An equal volume of DMSO was added to each well for half an hour, followed by stimulation with Nigericin at a final concentration of 5 ⁇ M for 30 minutes or 3.5 ⁇ L of MSU (concentration at 200 ⁇ g/ml) for 4.5 hours.
  • each well was pretreated with CY-09 at a final concentration of 1 ⁇ M for 30 minutes, and then added with a final concentration of 5 ⁇ M of Nigericin for half an hour or 3.5 ⁇ L of MSU (concentration of 200 ⁇ g/ml) for 4.5 hours.
  • Group 5 Each well was pretreated with CY-09 at a final concentration of 5 ⁇ M for 30 minutes, and then added with a final concentration of 5 ⁇ M of Nigericin for half an hour or 3.5 ⁇ L of MSU (concentration of 200 ⁇ g/ml) for 4.5 hours.
  • step 4 After the cells treated in step 3, the cell culture supernatant (SN) and the cell lysate (Input) were collected, and a part of the supernatant was taken, and IL-1 ⁇ was detected by ELISA, and the protein in the supernatant was extracted by a conventional method. WB analysis was performed with an anti-mouse IL-1 ⁇ antibody and an anti-mouse p20 antibody.
  • NLRP3 inflammatory bodies were activated by stimulation with Nigericin/MSU, and WB and ELISA assays showed that CY-09 concentration-dependently inhibited the maturation and secretion of p20 and IL-1 ⁇ .
  • CY-09 specifically inhibits the activation of classical and non-canonical NLRP3 inflammatory bodies, but has no inhibitory effect on Poly(dA:dT)-activated AIM2 inflammatory bodies and Salmonella-activated IPAF inflammatory bodies.
  • BMDM bone marrow-derived macrophages
  • BMDM was divided into 24-well plates at 1.5-3*10 5 cells per well.
  • CY-09 inhibits the activation of ATP-stimulated classical NLRP3 inflammatory bodies.
  • the final concentration of ATP was 5 mM and the stimulation time was 20 minutes.
  • CY-09 inhibits the activation of non-canonical NLRP3 inflammatory bodies induced by intracellular transfection of LPS.
  • Ultra-LPS was transfected into BMDM cells at a final concentration of 2 ⁇ g/ml by Lipofectamine 2000, and cultured for 16 hours overnight. The cell culture supernatant was collected, and the secretion level of IL-1 ⁇ in the supernatant was detected by ELISA.
  • CY-09 did not inhibit the activation of AIM2 inflammatory bodies induced by Poly(dA:dT).
  • Poly(dA:dT) was transfected into BMDM cells at a final concentration of 1 ⁇ g/ml using Lipofectamine 2000. The cells were cultured for 3-4 hours, and the cell culture supernatant was collected. The secretion level of IL-1 ⁇ in the supernatant was determined by ELISA.
  • CY-09 does not inhibit the activation of IPAF inflammatory bodies induced by Salmonella.
  • the Salmonella solution was cultured overnight by centrifugation, the supernatant was discarded, and resuspended in 1 ml Opti-MEM, and 0.5 ⁇ L of the suspension was added to a culture supernatant of a 24-well plate containing 250 ⁇ L of the medium at a dilution ratio of 1:500. After 4 hours, the cell culture supernatant was collected, and the secretion level of IL-1 ⁇ in the supernatant was measured by ELISA.
  • CY-09 inhibits the activation of NLRP3 inflammatory bodies induced by the classical NLRP3 inflammatory agonist ATP, as well as the activation of non-canonical NLRP3 inflammatory bodies induced by intracellular transfection of LPS. Concentration-dependent inhibition effect. However, there is no inhibitory effect on AIM2 inflammatory bodies activated by Poly(dA:dT) and Salmonella-activated IPAF inflammatory bodies.
  • V. CY-09 inhibits the formation of ASC dimer and assembly of NLRP3 inflammatory body complex
  • BMDM bone marrow-derived macrophages
  • CY-09 inhibited the formation of ASC dimers. Divided into BMDM to a six-well plate with 1*10 6 cells per well. LPS was stimulated for 3 hours, and the corresponding concentration of CY-09 or DMSO was added to each well for 30 minutes, followed by stimulation with Nigericin at a final concentration of 10 ⁇ M for 30 minutes. The culture supernatant of each well was collected, and the protein was precipitated. WB was used to detect the inhibitory effect of CY-09 on the activation of NLRP3 inflammatory bodies.
  • CY-09 inhibits the interaction of NLRP3 with NLRP3.
  • the 293T cells in the six-well plate were fused to about 80%, and the NLRP3 plasmid carrying the FLAG and mCherry tags was transferred separately or simultaneously. After transfection for 2 hours, CY-09 was added, and the culture was continued for 20 hours. The NP-40 was lysed, the supernatant was centrifuged, and the FLAG-coated beads were added and incubated at 4 ° C for 2.5 hours with WB detection. Reference to exogenous co-ip methods and procedures (Shi et al., 2015, Nature Immunology).
  • CY-09 inhibits the formation of NLRP3 multimers and interaction with ASC.
  • WB method methods and procedures, see Shi et al., 2015, Nature Immunology
  • CY-09 can inhibit the formation of ASC dimer and the interaction between NLRP3 and ASC and NLRP3 and NLRP3 in a concentration-dependent manner, and the results of molecular sieve experiments further confirm that CY-09 can inhibit NLRP3. Formation of the polymer and interaction of NLRP3 with ASC.
  • BMDM bone marrow-derived macrophages
  • Biotinylated CY-09 binds to NLRP3 protein in a concentration-dependent manner.
  • the BMDM cells stimulated by LPS for 3 hours were lysed on NP-40, centrifuged to precipitate, and the supernatants were separately separated into EP tubes of different treatment groups.
  • Each group was added with CY-09 without biotin (free CY-09).
  • different concentrations of biotinylated CY-09 Biotin-CY-09
  • Biotin-CY-09 different concentrations of biotinylated CY-09 (Biotin-CY-09)
  • Incubated at 4 °C for 1.5 hours Thereafter, equal amounts of avidin-coated beads were added to each group and incubation was continued for 2 hours with rotation.
  • the beads were washed 4 times with PBS containing 1% tween-20 and PBS containing 0.1% NP-40, respectively, and detected by WB.
  • each group was added with the same molar concentration of biotin-labeled CY-09 (1 ⁇ M), and incubated at 4 ° C for 2 hours with rotation, and the beads were washed 4 times with PBS containing 1% tween-20 and PBS containing 0.1% NP-40. , WB detection.
  • the THP-1 cell line used in the experiment was purchased from ATCC, USA.
  • CY-09 specifically interacts with the NLRP3 protein and does not interact with NOD1/2, AIM2 and NLRC4 proteins.
  • the 293T cells in the six-well plate were fused to about 80%, and transferred to the FLAG-tagged empty plasmid (EV), NLRP3 plasmid, NOD1 plasmid, NOD2 plasmid, AIM2 plasmid and NLRC4 plasmid, and culture was continued for 24 hours.
  • the cells of each well were lysed on NP-40 ice, and the subsequent Pulldown system was subjected to reference experiment 1 and WB detection.
  • CY-09 binds directly to the purified GST-NLRP3 protein.
  • the purified GST-NLRP3 recombinant protein purchased from Abnova was diluted into PBS to a final concentration of 0.4 pg/ ⁇ L, and different concentrations of Biotin-CY-09 were added, respectively, and incubated at 4 ° C for 1.5 hours. Afterwards, equal amounts of avidin-coated beads were added to each tube and incubation was continued for 2 hours with rotation. The beads were washed 4 times with PBS containing 1% tween-20 and PBS containing 0.1% NP-40, respectively, and detected by WB.
  • the pulldown system combined with the competitive inhibition protocol, WB detection found that the direct binding of recombinant GST-NLRP3 protein to Biotin-CY-09 can be inhibited in a concentration-dependent manner by free CY-09.
  • Biotin-CY-09 is a concentration-dependent pulldown NLRP3 protein, and this pulldown of NLRP3 protein can be competitively inhibited by free CY-09.
  • the ATP binding site in the NACHT functional segment of the NLRP3 protein is the direct binding site of CY-09.
  • 1.293T cells were subcultured and transfected as before.
  • CY-09 binds to the NLRP3 protein by interacting with the NACHT functional segment of the NLRP3 protein.
  • the 293T cells in the six-well plate were fused to about 80%, and transferred into the FLAG-tagged full-length NLRP3 plasmid, NACHT plasmid, LRR plasmid and PYD plasmid, and cultured for 24 hours.
  • the cells of each well were lysed on NP-40 ice, and the subsequent Pulldown system was carried out in the first experiment of Reference 4, and WB was detected.
  • CY-09 directly binds to the ATP binding site in the NACHT functional segment of the NLRP3 protein.
  • the ATPase active region of the NLRP3 protein is located in the NACHT functional segment, including the ATP binding site (walker A) and the catalytic site of the ATPase (walker B).
  • NLRP3 plasmids with FLAG tags and containing walker A and walker B point mutations were constructed separately. Transfer to a 293T cell line that is fused to about 80% and continue to culture for 24 hours. The cells of each well were lysed on NP-40 ice, and the subsequent Pulldown system was carried out in the first experiment of Reference 4, and WB was detected.
  • Biotin-CY-09 is only combined with the NACHT functional segment of NLRP3 and is not capable of combining with the LRR and PYD functional segments.
  • the mutated plasmids (walker A and walker B) of the NLRP3 protein ATPase active site were constructed to further confirm that Biotin-CY-09 binds to the NACHT functional segment by interacting with its ATP binding site.
  • CY-09 specifically inhibits ATPase activity of NLRP3 protein
  • 1.293T cells were subcultured and transfected as before.
  • Experiment 1 CY-09 dose-dependently inhibits the ATPase activity of the NLRP3 protein.
  • the FLAG-tagged NLRP3 protein, NLRP1 protein and NLRC4 protein were peptdowned from 293T cell lysate according to exogenous co-ip method, followed by coating the target protein from FLAG antibody with a final concentration of 5 mg/ml of peptide solution. The beads are eluted. This step is to purify the protein of interest. The above operation can be repeated once to increase the purity of the target protein.
  • Experiment 2 The ATPase activity was detected after purification of the target protein. 1 5 uL of further purified flag-NLRP3 protein or flag-NLRP1 protein or flag-NLRC4 protein was added to a white 96-well plate containing 25 uL of kinase reaction buffer and various concentrations of CY-09, and incubated at 37 ° C for 15 minutes. 2 Add ATP at a final concentration of 250 uM and continue to incubate at 37 ° C for 40-60 minutes. 3 Add 25 uL of ADP-Glo Reagent to stop the kinase reaction and remove residual ATP, incubate for 40 minutes at room temperature. 4 After the incubation was completed, 50 uL of Kinase Detection Reagent was added and incubation was continued for 30-60 minutes at room temperature. 5 Detect the absorbance of luminescence on a multi-function microplate reader.
  • CY-09 dose-dependently inhibits the ATPase activity of the NLRP3 protein, but does not significantly inhibit the ATPase activity of the NLRP1 protein and the NLRC4 protein.
  • Example 6 CY-09 inhibits acute inflammation induced by urate crystals
  • CY-09 inhibits urate crystal (MSU)-induced peritonitis
  • mice of 10 weeks old were divided into 3 groups of 6 rats each.
  • peritoneal lavage supernatant obtained in step 3 was assayed for cytokines such as IL-1 ⁇ by ELISA; the precipitated cells were labeled with anti-CD11b and anti-Ly6G antibodies, and the peritoneal neutrophils were detected by flow cytometry. Infiltration situation.
  • neutrophils were recruited after intraperitoneal injection of MSU, and neutrophil infiltration was significantly inhibited after injection of CY-09. Simultaneous injection of CY-09 also significantly inhibited the secretion of IL-1 ⁇ . This indicates that CY-09 can effectively inhibit MSU-induced peritonitis.
  • CY-09 inhibits gout induced by urate crystallisation (MSU) accumulation
  • mice of 10 weeks old were divided into two groups of 6 rats each.
  • the initial width of the knee joint on each side of each rat was measured and recorded with a vernier caliper.
  • a mixture of DMSO and PBS was injected into the knee joint cavity of the left leg, and CY-09 and MSU were simultaneously injected into the knee joint cavity of the right leg.
  • Example 7 CY-09 inhibits LPS-induced acute peripheral inflammation
  • the first group of negative controls were injected intraperitoneally with PBS.
  • the second group of positive controls was injected intraperitoneally with LPS solution in PBS at a dose of 20 mg/kg.
  • the treatment group was intraperitoneally injected with LPS and CY-09, and the doses of LPS and CY-09 were both 20 mg/kg.
  • the eyeballs were bled, left at room temperature for half an hour, and then centrifuged at low speed for half an hour to collect serum.
  • ELISA detection step 3 secretion level of IL-1 ⁇ in serum.
  • IL-1 ⁇ level was significantly increased under the action of LPS compared with the control group, and intraperitoneal injection of CY-09 was effective in inhibiting IL-1 ⁇ secretion. This indicates that CY-09 can effectively inhibit acute peripheral inflammation induced by LPS.
  • NLRP3 inflammatory body After the 351th amino acid of NLRP3 protein is mutated to phenylalanine by arginine, the NLRP3 inflammatory body will continue to activate, which will produce a large amount of mature IL-1 ⁇ and IL-18.
  • the peripheral high inflammatory environment can cause the development of mice to stagnate. Death occurs shortly after birth (generally 4-12 days).
  • mice with 8-week old C57BL/6J background and Lyz-Cre label were mated with Nlrp3A350VneoR female (male) mice, also from CjBL3J background, purchased from Jackson Lab.
  • control group was intraperitoneally injected with the same volume of DMSO, once every two days, during which weight and survival were recorded.
  • the body weight and the survival period of the CY-09-administered group were significantly improved as compared with the DMSO-injected control group. After stopping the administration on the 21st day, the mice can continue to survive for 27 days. This indicates that CY-09 has a significant therapeutic effect on the hereditary disease Mu-Wey's syndrome caused by NLRP3 mutation leading to the continuous activation of NLRP3 inflammatory bodies.
  • Example 9 CY-09 prevents and treats obesity-related metabolic syndrome
  • Group 1 Wild control mice fed a normal diet were injected intraperitoneally with a comparable volume of DMSO/PBS mixture (10%/90%) per day.
  • the second group wild rats induced by high-fat diet for 12 weeks, continued high-fat feed every day, and intraperitoneally injected a considerable volume of DMSO/PBS mixture.
  • the third group wild rats induced by high-fat diet for 12 weeks, continued high-fat diet feeding, and a considerable volume of DMSO/PBS suspension containing CY-09 was intraperitoneally injected at a dose of 2.5 mg/kg per day.
  • Group 4 NLRP3-/- control mice fed a normal diet were injected intraperitoneally with a comparable volume of DMSO/PBS mixture (10%/90%) per day.
  • Group 5 NLRP3-/- mice induced by high-fat diet for 12 weeks, continued high-fat diet feeding, and intraperitoneally injected a considerable volume of DMSO/PBS mixture per day.
  • Group 6 NLRP3-/- mice induced by high-fat diet for 12 weeks, continued high-fat diet feeding, daily injection of a considerable volume of DMSO containing CY-09 according to the injection dose of 2.5 mg/kg.BW-1. /PBS suspension.
  • Oral administration of CY-09 can treat HFD-induced obesity and insulin resistance
  • the CY-09 powder was emulsified by using a 0.5% sodium carboxymethylcellulose solution to uniformly emulsify the CY-09 powder. According to the administration of 20 mg/kg. BW-1 or 40 mg/kg. BW-1, it is administered once a day by gavage. The HFD used induced mice as before.

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Abstract

本发明提供了一种噻唑啉酮杂环化合物、其制备方法、药用组合物及应用，其中，本发明提供了噻唑啉酮杂环化合物在制备 NLRP3 炎症小体抑制剂中的应用，所述噻唑啉酮杂环化合物具有式Ⅰ或式Ⅱ所示结构或其异构体、前药、药学上可接受的溶剂化物或盐：(I), (II)

Description

噻唑啉酮杂环化合物、其制备方法、药用组合物及应用
本申请要求于2017年06月26日提交中国专利局、申请号为201710494473.5、发明名称为“噻唑啉酮杂环化合物、其制备方法、药用组合物及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物化学技术领域，尤其涉及一种噻唑啉酮杂环化合物、其制备方法、药用组合物及应用。
背景技术
NLRP3(NACHT,LRR and PYD domains-containing protein 3)是存在于胞内的模式识别受体NOD样受体(NLRs)中的重要一员，在受到外源的病原相关分子模式(如细菌，真菌)或危险相关分子模式(如尿酸盐结晶，饱和脂肪酸)刺激时，NLRP3招募接头蛋白ASC(apoptosis-associated speck-like protein containing a CARD)和caspase-1前体形成一个多聚复合物，称之为NLRP3炎症小体。NLRP3炎症小体组装后，发生自剪切，形成具有剪切活性的caspase-1，进而诱导IL-1β和IL-18的成熟和分泌，介导免疫应答及炎症反应，维持机体的免疫稳态。但是，当NLRP3炎症小体的活化失调时，与多种疾病的发生发展密切相关，如肠炎、肝炎、硅肺、紫外线所诱导的皮肤晒伤和接触性超敏反应、脓毒血症、肿瘤、神经退行性疾病，甚至是2型糖尿病和动脉粥样硬化。这表明NLRP3炎症小体是治疗这些疾病的潜在靶标。
目前临床治疗NLRP3炎症小体相关疾病的药物主要集中在靶标IL-1上，包括IL-1受体激动剂阿那白滞素(anakinra)，IL-1单克隆抗体康纳单抗(canakinumab)以及IL-1受体诱导剂利纳西普(rilonacept)。这些靶标IL-1的药物虽然对一些疾病有一定疗效，但是IL-1并不是NLRP3炎症小体活化后产生的唯一效应分子，也不是NLRP3炎症小体诱导相关疾病发生的唯一途径。因此，靶标IL-1的药物并不能有效治疗所有NLRP3炎症小体相关疾病。此外，IL-1不仅仅来源于NLRP3炎症小体，其他炎症小体(如NLRP1，NLRC4)的活化也能引起IL-1的成熟及分泌。因此，与靶标IL-1及其受体的干预手段相比，靶向NLRP3炎症小体活化的干预手段则能从根本上抑制IL-1、IL-18和HMGB1等介导的疾病，取得更好更特异的治疗效果，因此将NLRP3作为一个潜在靶标更有价值，靶向NLRP3炎症小体治疗相关炎症性疾病也更有意义。
尽管目前已经报道多个小分子化合物可以有效抑制NLRP3炎症小体的活化，如MCC950,sulforaphane,isoliquiritigenin,β-hydroxybutyrate,flufenamic acid,mefenamic acid,3,4-methylenedioxy-β-nitrostyrene(MNS),parthenolide,Bay 11-7082等，但是目前还没有明确的证据表明上述化合物是NLRP3直接和特异性的抑制剂。实际上，只有直接靶向NLRP3蛋白本身才有可能特异性抑制NLRP3炎症小体活化。靶向NLRP3炎症小体其他蛋白组分，比如ASC或者caspase-1，会抑制AIM2和NLRC4等炎症小体从而抑制机体的抗感染能力；靶向NLRP3炎症小体相关的信号通路或者信号分子，比如NEK7、线粒体损伤、钾离子外流等必然会有不可避免的非特异效果，因为这些信号分子或者信号事件也会影响其他的生命活动过程。目前已经报道的小分子都没有证据表明他们直接靶向NLRP3本身。Sulforaphane,isoliquiritigenin,β-hydroxybutyrate,parthenolide和Bay 11-7082已经被报道可以抑制NF-kB信号通路的活化。3,4-methylenedioxy-β-nitrostyrene(MNS)则是一种激酶抑制剂，能够抑制Src和Syk等激酶的活性。Flufenamic acid和mefenamic acid已经被报道是通过抑制氯离子外流从而抑制NLRP3炎症小体活化。尽管MCC950目前没有报道非特异性的效果，但是该分子并不直接抑制炎症小体的组装，比如NLRP3多聚和NLRP3-ASC相互作用，表明其可能靶向NLRP3炎症小体的上游信号通路。总之，到目前为止还没有发现能够靶向NLRP3本身的特异性抑制剂。找到一种能够特异性抑制NLRP3炎症小体活化，对NLRP3炎症小体相关疾病的治疗足够安全且高特异性的小分子化学药物意义重大且势在必行。
发明内容
有鉴于此，本发明要解决的技术问题在于提供一种噻唑啉酮杂环化合物、其制备方法、药用组合物及应用，发现了一系列对于NLRP3炎症小体具有特异性抑制活性的化合物。
本发明提供了噻唑啉酮杂环化合物在制备NLRP3炎症小体抑制剂中的应用，所述噻唑啉酮杂环化 合物具有式Ⅰ或式Ⅱ所示结构或其异构体、前药、药学上可接受的溶剂化物或盐：
Figure PCTCN2018092824-appb-000001
其中，
R 1，R 2，R 3，R 4和R 5各自独立地选自：
氢，卤素，硝基，羟基，氰基，苯基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基；
R 6，R 7，R 8，R 9和R 10各自独立地选自：
氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1-C6含氧烷基、C1-C6烷氧基、C3-C7环烷基；取代或非取代的甲酰基、磺酰基、磺酰基氨基；
X 1和X 2各自独立地选自O或S；
n为0～6的任意整数；
Q为C或N；
Q 1、Q 2、Q 3、Q 4独立的选自-C-R 1，-C-R 2，-C-R 3，-C-R 4、-C-R 5、N，-N-R 1，S或O；
Ar 2为取代或非取代芳基、杂芳基或稠环基；
m为0或1。
上述式Ⅱ中，虚线表示连接键可以为单键或双键。
上述取代或非取代的氨基优选为氨基，C1～C10烷基取代的氨基，C1～C10二烷基取代的氨基，C3-C7环烷基取代的氨基或酰基取代的氨基。
上述取代的C1-C6含氧烷基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
上述取代的C1-C6烷氧基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
上述取代的C1-C6烷基的取代基优选为卤素、硝基、氰基中的任意一种或多种。
上述取代的C3-C7环烷基的取代基优选为卤素、硝基和氰基中的任意一种或多种。
本发明优选的，所述R 1，R 2，R 3，R 4和R 5各自独立地选自：
氢，卤素，硝基，羟基，氰基，苯基；氨基，甲基氨基，N,N-二甲基氨基，乙基氨基，N,N-二乙基氨基，异丙基氨基，N,N-二异丙基氨基，环丙基氨基，环丁基氨基，环戊基氨基，环己基氨基，乙酰基氨基，N,N-二乙酰基氨基，2-N,N-二甲基氨基乙基氨基，2-羟基乙基氨基，甲磺酰基氨基，N,N-二甲磺酰基氨基，甲磺酰基、对甲苯磺酰基；C1-C6烷基，C1-C6含氟烷基，C1-C6含氧烷基，C1-C6烷氧基，C1～C6含氟烷氧基或C3-C7环烷基。
所述C1-C6烷基优选为甲基、乙基、丙基、异丙基、丁基、异丁基、戊基、异戊基、新戊基。
上述C1-C6烷基可被卤素、硝基和氰基中的任意一种或多种取代。
所述C1-C6含氟烷基优选被卤素、硝基、氨基和氰基中的任意一种或多种取代，在本发明的某些具体实施例中，其为三氟甲基。
所述C1-C6含氧烷基优选为甲氧基乙基或甲氧基乙氧基甲基。
上述C1-C6含氧烷基可被卤素、硝基、氨基和氰基中的任意一种或多种取代。
所述C1-C6烷氧基优选为甲氧基或乙氧基。
上述C1-C6烷氧基可被卤素、硝基、氨基和氰基中的任意一种或多种取代。
所述C1～C6含氟烷氧基优选被卤素、硝基、氨基和氰基中的任意一种或多种取代，在本发明的某些具体实施例中，其为三氟甲氧基。
所述C3-C7环烷基优选为环丙基、环丁基、环戊基或环己基。
更优选的，R 1，R 2，R 3，R 4和R 5各自独立地选自：
氢、氟、氯、溴、硝基、苯基、羟基、甲基、甲氧基，三氟甲基，三氟甲氧基，氨基，乙酰基氨基，甲磺酰基、N,N-二甲磺酰基氨基、N，N-二甲基氨基或AcNH-。
本发明优选的，R 1，R 2，R 3，R 4和R 5中的任意三个或四个同时为H。
本发明中，R 6，R 7，R 8，R 9和R 10各自独立地选自：
氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1-C6含氧烷基、C1-C6烷氧基、C3-C7环烷基；取代或非取代的甲酰基、磺酰基、磺酰基氨基。
上述R 6，R 7，R 8，R 9或R 10中，所述取代的C1-C6烷基的取代基优选为卤素、硝基、氰基、酯基、羧基中的任意一种或多种。
所述酯基优选为甲酸甲酯基。
在本发明的某些具体实施例中，上述取代的C1-C6烷基具有以下结构：
Figure PCTCN2018092824-appb-000002
其中，R 11优选为C1～C6烷基。
在本发明的一些具体实施例中，所述R 11为氢、甲基、乙基、丙基、异丙基、丁基、异丁基或戊基。
上述R 6，R 7，R 8，R 9或R 10中，所述取代的C1-C6含氧烷基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
上述R 6，R 7，R 8，R 9或R 10中，所述取代的C1-C6烷氧基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
优选的，所述R 6，R 7，R 8，R 9和R 10各自独立地选自：
氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1～C6烷氧基或C1-C6含氧烷基；氨基甲酰基，甲胺基甲酰基，乙胺基甲酰基，丙胺基甲酰基，异丙胺基甲酰基，N,O-二甲羟胺基甲酰基，环丙基胺基甲酰基，环丁基胺基甲酰基，环戊基胺基甲酰基，哌啶基-1-甲酰基，4-羟基哌啶基-1-甲酰基，4-N,N-二甲基哌啶基-1-甲酰基，4-N,N-二乙基哌啶基-1-甲酰基，四氢吡咯基-1-甲酰基，3-N,N-二甲基四氢吡咯基-1-甲酰基，3-N,N-二乙基四氢吡咯基-1-甲酰基，哌嗪基-1-甲酰基，N-甲基哌嗪基-1-甲酰基，N-乙基哌嗪基-1-甲酰基，N-乙酰基哌嗪基-1-甲酰基，N-叔丁氧甲酰基哌嗪基-1-甲酰基，N-(2-羟基乙基)哌嗪基-1-甲酰基，N-(2-氰基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二甲基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二乙基乙基)哌嗪基-1-甲酰基，N-(3-羟基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二甲基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二乙基丙基)哌嗪基-1-甲酰基，吗啡啉基-1-甲酰基，3,5-二甲基吗啡啉基-1-甲酰基，4-(N-甲基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙酰基-1-哌嗪基)哌啶基-1-甲酰基，N-(N-甲基-4-哌啶基)哌嗪基-1-甲酰基，2-羟基乙胺基甲酰基，2-乙酰基羟基乙胺基甲酰基，苯胺基甲酰基，苄基甲酰基，苄基氨基甲酰基，3-甲氧基苄基甲酰基，3-甲氧基苄基氨基甲酰基；羟基甲酰基，甲氧基甲酰基，乙氧基甲酰基，丙氧基甲酰基，异丙氧基甲酰基，正丁氧基甲酰基，异丁氧基甲酰基，叔丁氧基甲酰基，氨基磺酰基、C1～C10烷基磺酰基，C1～C10烷氨基磺酰基，C3～C10环烷基氨基磺酰基，C6～C12芳基氨基磺酰基，C5～C12杂芳基氨基磺酰基，或C1～C10磺酰基氨基。
在本发明的一些具体实施例中，所述C1～C10烷基磺酰基为甲磺酰基，乙磺酰基，丙磺酰基，异丙磺酰基，丁磺酰基或异丁磺酰基。
在本发明的一些具体实施例中，所述C1～C10烷氨基磺酰基为N-甲基氨基磺酰基，N-乙基氨基磺酰基，N-丙基氨基磺酰基，N-异丙基氨基磺酰基，N-丁基氨基磺酰基，N-异丁基氨基磺酰基，N,N-二甲基氨基磺酰基、N,N-二乙基氨基磺酰基或N,N-二丙基氨基磺酰基。
在本发明的一些具体实施例中，所述C3～C10环烷基氨基磺酰基为N-环丙基氨基磺酰基，N-环丁基氨基磺酰基，N-环戊基氨基磺酰基或N-环己基氨基磺酰基。
在本发明的一些具体实施例中，所述C6～C12芳基氨基磺酰基为N-苯基氨基磺酰基。
在本发明的一些具体实施例中，所述C5～C12杂芳基氨基磺酰基为五元杂芳基氨基磺酰基或六元杂芳基氨基磺酰基，如N-吡啶基氨基磺酰基，N-噻吩基氨基磺酰基，N-呋喃基氨基磺酰基或N-吡咯基氨基磺酰基。
在本发明的一些具体实施例中，所述C1～C10磺酰基氨基为甲磺酰氨基，乙磺酰氨基，丙磺酰氨基，异丙磺酰氨基，丁磺酰氨基，异丁磺酰氨基，磺酸基，氨基磺酰基。
本发明中，上述C1～C10烷基磺酰基，C1～C10烷氨基磺酰基，C3～C10环烷基氨基磺酰基，C6～C12芳基氨基磺酰基，C5～C12杂芳基氨基磺酰基，或C1～C10磺酰基氨基可以连接有卤素、硝基、氨基和氰基中的任意一种或多种取代基。
优选的，R 6，R 7，R 8，R 9，R 10中的任意三个或四个同时为H。
X 1和X 2各自独立地选自O或S。
优选的，X 1为O或S，X 2为O。
即，在本发明的某些具体实施例中，X 1为S，X 2为O。
在本发明另外一些具体实施例中，X 1和X 2同时为O。
n为0，1，2、3、4、5或6；优选的，n为0，1，2或3。
Q为C或N。
本发明中，当X 1为S，X 2为O时，n优选为0，1，2或3。
当X 1和X 2同时为O时，n优选为0或1。
在本发明的一些具体实施例中，所述式Ⅱ具有以下式Ⅱ-a或式Ⅱ-b结构：
Figure PCTCN2018092824-appb-000003
本发明式Ⅱ结构中，或式Ⅱ-a或式Ⅱ-b结构中，Ar 2为取代或非取代芳基、杂芳基或稠环基。
在本发明的一些具体实施例中，Ar 2为取代或非取代苯基、五元或六元杂芳基、或者为取代或非取代苯基与取代或非取代芳基、杂芳基、杂环基稠合形成的稠环基团。
在本发明的一些具体实施例中，上述稠环基团中，所述芳基优选为C6～C10芳基，进一步优选为苯基。
在本发明的一些具体实施例中，上述稠环基团中，所述杂芳基优选为五元或六元杂芳基，优选含N、O、S中的一个或多个杂原子。进一步优选为吡啶基、嘧啶基、哒嗪基、吡嗪基或吡喃基。
在本发明的一些具体实施例中，上述稠环基团中，所述杂环基优选为C2～C10的杂环基。
在本发明的一些具体实施例中，所述Ar 2为取代或非取代苯基、吡咯基、噻吩基、呋喃基、咪唑基、吡唑基、噻唑基、噁唑基、三唑基、四唑基、吡啶基、嘧啶基、哒嗪基、吡嗪基、吡喃基，或为以下基团：
Figure PCTCN2018092824-appb-000004
所述Ar 2中，取代基独立的优选为氢，卤素，硝基，羟基，氰基，取代或非取代的氨基，甲磺酰 基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基和C6～C10芳基中的一种或多种。
在本发明的一些具体实施例中，上述取代或非取代的C6～C10芳基为取代的苯基。
在本发明的一些具体实施例中，所述苯基的取代基为卤素、硝基、氨基、羟基、氰基或C1～C6的烷基。
本发明经研究发现，上述化合物通过特异性抑制NLRP3炎症小体的活化，进而抑制IL-1β和IL-18的成熟及分泌，来抑制由NLRP3炎症小体的活化而引起的相关疾病的发生发展。
优选的，所述NLRP3炎症小体抑制剂用于治疗、减轻或预防NLRP3炎症小体引发的炎症性疾病。
上述炎症优选包括中枢系统炎症和/或外周系统炎症。
上述炎症性疾病优选包括：
II型糖尿病、动脉粥样硬化、脂肪肝、代谢综合征、感染引起的急、慢性组织损伤、痛风、关节炎、肠炎、肝炎、腹膜炎、硅肺、紫外线诱导的皮肤晒伤、接触性超敏反应、脓毒血症、肿瘤、神经退行性疾病、多发性硬化症和穆-韦氏综合征中的一种或多种。
上述腹膜炎和痛风优选为尿酸盐结晶堆积引起的炎症性疾病。
上述脓毒血症优选为细菌脂多糖(LPS)腹腔注射引起的外周急性炎症。
上述穆-韦氏综合征优选为由NLRP3蛋白突变导致NLRP3炎症小体持续活化引起的临床遗传性疾病穆-韦氏综合征。
上述用于腹膜炎、痛风、脓毒血症和穆-韦氏综合征的NLRP3炎症小体抑制剂的给药方式优选为腹腔注射。
所述腹腔注射的剂量优选为2.5mg/kg～20mg/kg，优选20mg/kg。
上述用于代谢综合征的NLRP3炎症小体抑制剂的给药方式优选为口服给药或腹腔注射。
所述口服给药的剂量优选为20mg/kg～40mg/kg，优选40mg/kg。
所述腹腔注射的剂量优选为2.5mg/kg～20mg/kg，优选2.5mg/kg。
所述II型糖尿病、脂肪肝以及动脉粥样硬化为高脂食物(HFD，60％Fat)诱导而成，有明显胰岛素抵抗伴随糖耐量受损发生以及肝脂肪变性和动脉血管斑块形成等代谢紊乱症状。
本发明上述化合物或组合物在应用于制备NLRP3炎症小体抑制剂时，可以单独使用，或与其他药物联合使用。
本发明还提供了一种噻唑啉酮杂环化合物，具有式Ⅰ或式Ⅱ所示结构或其异构体、前药、药学上可接受的溶剂化物或盐：
Figure PCTCN2018092824-appb-000005
其中，
R 1，R 2，R 3，R 4和R 5各自独立地选自：
氢，卤素，硝基，羟基，氰基，苯基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基；
R 6，R 7，R 8，R 9和R 10各自独立地选自：
氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1-C6含氧烷基、C1-C6烷氧基、C3-C7环烷基；取代或非取代的甲酰基、磺酰基、磺酰基氨基；
X 1为O，X 2为S，或X 1为S，X 2为O时，n为1～6的任意整数；
X 1和X 2同时为O或S时，n为0～6的任意整数；
Q为C或N；
Q 1、Q 2、Q 3、Q 4独立的选自-C-R 1，-C-R 2，-C-R 3，-C-R 4、-C-R 5、N，-N-R 1，S或O；
Ar 2为取代或非取代芳基、杂芳基或稠环基；
m为0或1。
其中，所述噻唑啉酮杂环化合物不包括以下化合物：
Figure PCTCN2018092824-appb-000006
上述取代或非取代的氨基优选为氨基，C1～C10烷基取代的氨基，C1～C10二烷基取代的氨基，C3-C7环烷基取代的氨基或酰基取代的氨基。
上述取代的C1-C6含氧烷基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
上述取代的C1-C6烷氧基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
上述取代的C1-C6烷基的取代基优选为卤素、硝基和氰基中的任意一种或多种。
上述取代的C3-C7环烷基的取代基优选为卤素、硝基和氰基中的任意一种或多种。
优选的，所述R 1，R 2，R 3，R 4和R 5各自独立地选自：
氢，卤素，硝基，羟基，氰基，苯基；氨基，甲基氨基，N,N-二甲基氨基，乙基氨基，N,N-二乙基氨基，异丙基氨基，N,N-二异丙基氨基，环丙基氨基，环丁基氨基，环戊基氨基，环己基氨基，乙酰基氨基，N,N-二乙酰基氨基，2-N,N-二甲基氨基乙基氨基，2-羟基乙基氨基，甲磺酰基氨基，N,N-二甲磺酰基氨基，甲磺酰基、对甲苯磺酰基；C1-C6烷基，C1-C6含氟烷基，C1-C6含氧烷基，C1-C6烷氧基，C1～C6含氟烷氧基或C3-C7环烷基。
所述C1-C6烷基优选为甲基、乙基、丙基、异丙基、丁基、异丁基、戊基、异戊基、新戊基。
上述C1-C6烷基可被卤素、硝基和氰基中的任意一种或多种取代。
所述C1-C6含氟烷基优选被卤素、硝基、氨基和氰基中的任意一种或多种取代，在本发明的某些具体实施例中，其为三氟甲基。
所述C1-C6含氧烷基优选为甲氧基乙基或甲氧基乙氧基甲基。
上述C1-C6含氧烷基可被卤素、硝基、氨基和氰基中的任意一种或多种取代。
所述C1～C6烷氧基优选为甲氧基或乙氧基。
上述C1-C6烷氧基可被卤素、硝基、氨基和氰基中的任意一种或多种取代。
所述C1～C6含氟烷氧基优选被卤素、硝基、氨基和氰基中的任意一种或多种取代，在本发明的某 些具体实施例中，其为三氟甲氧基。
所述环烷基优选为环丙基、环丁基、环戊基或环己基。
更优选的，R 1，R 2，R 3，R 4和R 5各自独立地选自：
氢、氟、氯、溴、硝基、苯基、羟基、甲基、甲氧基，三氟甲基，三氟甲氧基，氨基，乙酰基氨基，甲磺酰基、N,N-二甲磺酰基氨基、N，N-二甲基氨基或AcNH-。
本发明优选的，R 1，R 2，R 3，R 4和R 5中的任意三个或四个同时为H。
所述取代或非取代的甲酰基优选为C1～C6的酰基。
本发明中，R 6，R 7，R 8，R 9和R 10各自独立地选自：
氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1-C6含氧烷基、C1-C6烷氧基、C3-C7环烷基；取代或非取代的甲酰基、磺酰基、磺酰基氨基。
上述R 6，R 7，R 8，R 9或R 10中，所述取代的C1-C6烷基的取代基优选为卤素、硝基、氰基、酯基、羧基中的任意一种或多种。
所述酯基优选为甲酸甲酯基。
在本发明的某些具体实施例中，上述取代的C1-C6烷基具有以下结构：
Figure PCTCN2018092824-appb-000007
其中，R 11优选为C1～C6烷基。
在本发明的一些具体实施例中，所述R 11为氢、甲基、乙基、丙基、异丙基、丁基、异丁基或戊基。
上述R 6，R 7，R 8，R 9或R 10中，所述取代的C1-C6含氧烷基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
上述R 6，R 7，R 8，R 9或R 10中，所述取代的C1-C6烷氧基的取代基优选为卤素、硝基、氨基和氰基中的任意一种或多种。
优选的，所述R 6，R 7，R 8，R 9和R 10各自独立地选自：
氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基或C1-C6含氧烷基；氨基甲酰基，甲胺基甲酰基，乙胺基甲酰基，丙胺基甲酰基，异丙胺基甲酰基，N,O-二甲羟胺基甲酰基，环丙基胺基甲酰基，环丁基胺基甲酰基，环戊基胺基甲酰基，哌啶基-1-甲酰基，4-羟基哌啶基-1-甲酰基，4-N,N-二甲基哌啶基-1-甲酰基，4-N,N-二乙基哌啶基-1-甲酰基，四氢吡咯基-1-甲酰基，3-N,N-二甲基四氢吡咯基-1-甲酰基，3-N,N-二乙基四氢吡咯基-1-甲酰基，哌嗪基-1-甲酰基，N-甲基哌嗪基-1-甲酰基，N-乙基哌嗪基-1-甲酰基，N-乙酰基哌嗪基-1-甲酰基，N-叔丁氧甲酰基哌嗪基-1-甲酰基，N-(2-羟基乙基)哌嗪基-1-甲酰基，N-(2-氰基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二甲基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二乙基乙基)哌嗪基-1-甲酰基，N-(3-羟基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二甲基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二乙基丙基)哌嗪基-1-甲酰基，吗啡啉基-1-甲酰基，3,5-二甲基吗啡啉基-1-甲酰基，4-(N-甲基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙酰基-1-哌嗪基)哌啶基-1-甲酰基，N-(N-甲基-4-哌啶基)哌嗪基-1-甲酰基，2-羟基乙胺基甲酰基，2-乙酰基羟基乙胺基甲酰基，苯胺基甲酰基，苄基甲酰基，苄基氨基甲酰基，3-甲氧基苄基甲酰基，3-甲氧基苄基氨基甲酰基；羟基甲酰基，甲氧基甲酰基，乙氧基甲酰基，丙氧基甲酰基，异丙氧基甲酰基，正丁氧基甲酰基，异丁氧基甲酰基，叔丁氧基甲酰基，氨基磺酰基、C1～C10烷基磺酰基，C1～C10烷氨基磺酰基，C3～C10环烷基氨基磺酰基，C6～C12芳基氨基磺酰基，C5～C12杂芳基氨基磺酰基，或C1～C10磺酰基氨基。
在本发明的一些具体实施例中，所述C1～C10烷基磺酰基为甲磺酰基，乙磺酰基，丙磺酰基，异丙磺酰基，丁磺酰基或异丁磺酰基。
在本发明的一些具体实施例中，所述C1～C10烷氨基磺酰基为N-甲基氨基磺酰基，N-乙基氨基磺酰基，N-丙基氨基磺酰基，N-异丙基氨基磺酰基，N-丁基氨基磺酰基，N-异丁基氨基磺酰基，N,N-二甲 基氨基磺酰基、N,N-二乙基氨基磺酰基或N,N-二丙基氨基磺酰基。
在本发明的一些具体实施例中，所述C3～C10环烷基氨基磺酰基为N-环丙基氨基磺酰基，N-环丁基氨基磺酰基，N-环戊基氨基磺酰基或N-环己基氨基磺酰基。
在本发明的一些具体实施例中，所述C6～C12芳基氨基磺酰基为N-苯基氨基磺酰基。
在本发明的一些具体实施例中，所述C5～C12杂芳基氨基磺酰基为五元杂芳基氨基磺酰基或六元杂芳基氨基磺酰基，如N-吡啶基氨基磺酰基，N-噻吩基氨基磺酰基，N-呋喃基氨基磺酰基或N-吡咯基氨基磺酰基。
在本发明的一些具体实施例中，所述C1～C10磺酰基氨基为甲磺酰氨基，乙磺酰氨基，丙磺酰氨基，异丙磺酰氨基，丁磺酰氨基，异丁磺酰氨基，磺酸基，氨基磺酰基。
本发明中，上述C1～C10烷基磺酰基，C1～C10烷氨基磺酰基，C3～C10环烷基氨基磺酰基，C6～C12芳基氨基磺酰基，C5～C12杂芳基氨基磺酰基，或C1～C10磺酰基氨基可以连接有卤素、硝基、氨基和氰基中的任意一种或多种取代基。
优选的，R 6，R 7，R 8，R 9，R 10中的任意三个或四个同时为H。
X 1为O，X 2为S，或X 1为S，X 2为O时，n为1～6的任意整数；
X 1和X 2同时为O或S时，n为0～6的任意整数；优选的，X 1为O或S，X 2为O。
即，在本发明的某些具体实施例中，X 1为S，X 2为O，此时n为1～6的任意整数，具体的，n为1，2、3、4、5或6；优选的，n为1，2或3。
在本发明另外一些具体实施例中，X 1和X 2同时为O，此时n为0～6的任意整数，具体的，n为0，1，2、3、4、5或6；优选的，n,为0，1，2或3。
其中，当n为1，2或3时，所述化合物对炎症小体具有选择抑制作用。
Q为C或N。
本发明优选的，当n＝1，Q为C时，R 1，R 2，R 3，R 4和R 5不同时为H。
本发明优选的，当Q为N时，R 8不为甲氧基甲酰基。
本发明优选的，当n＝2或3，Q为C时，当R 6，R 7，R 8，R 9和R 10中的任意一个为羧基时，R 1，R 2，R 3，R 4和R 5均不为H。
本发明优选的，当Q为C，X 1和X 2同时为O时，当R 6，R 7，R 8，R 9和R 10中的任意一个为羧基时，R 1，R 2，R 3，R 4和R 5均不为三氟甲基。
本发明优选的，当Q为C，X 1和X 2同时为O时，当R 6，R 7，R 8，R 9和R 10中的任意一个为羧基时，R 1，R 2，R 3，R 4和R 5均不为甲氧基甲酰基。
在本发明的一些具体实施例中，所述式Ⅱ具有以下式Ⅱ-a或式Ⅱ-b结构：
Figure PCTCN2018092824-appb-000008
本发明式Ⅱ结构中，或式Ⅱ-a或式Ⅱ-b结构中，Ar 2为取代或非取代芳基、杂芳基或稠环基。
在本发明的一些具体实施例中，Ar 2为取代或非取代苯基、五元或六元杂芳基、或者为取代或非取代苯基与取代或非取代芳基、杂芳基、杂环基稠合形成的稠环基团。
在本发明的一些具体实施例中，上述稠环基团中，所述芳基优选为C6～C10芳基，进一步优选为苯基。
在本发明的一些具体实施例中，上述稠环基团中，所述杂芳基优选为五元或六元杂芳基，优选含N、O、S中的一个或多个杂原子。进一步优选为吡啶基、嘧啶基、哒嗪基、吡嗪基或吡喃基。
在本发明的一些具体实施例中，上述稠环基团中，所述杂环基优选为C2～C10的杂环基。
在本发明的一些具体实施例中，所述Ar 2为取代或非取代苯基、吡咯基、噻吩基、呋喃基、咪唑基、吡唑基、噻唑基、噁唑基、三唑基、四唑基、吡啶基、嘧啶基、哒嗪基、吡嗪基、吡喃基，或为以下基团：
Figure PCTCN2018092824-appb-000009
所述Ar 2中，取代基独立的优选为氢，卤素，硝基，羟基，氰基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基和C6～C10芳基中的一种或多种。
在本发明的一些具体实施例中，上述取代或非取代的C6～C10芳基为取代的苯基。
在本发明的一些具体实施例中，所述苯基的取代基为卤素、硝基、氨基、羟基、氰基或C1～C6的烷基。
在本发明的某些具体实施例中，所述的噻唑啉酮杂环化合物具有以下任一结构或其顺反异构体：
Figure PCTCN2018092824-appb-000010
Figure PCTCN2018092824-appb-000011
Figure PCTCN2018092824-appb-000012
Figure PCTCN2018092824-appb-000013
Figure PCTCN2018092824-appb-000014
Figure PCTCN2018092824-appb-000015
Figure PCTCN2018092824-appb-000016
Figure PCTCN2018092824-appb-000017
Figure PCTCN2018092824-appb-000018
Figure PCTCN2018092824-appb-000019
本发明中，除非特殊说明，上述基团和取代基具有药物化学领域的普通含义。
需要说明的是，C1-C6含氧烷基是指C1-C6烷基骨架被一个或多个C1-C6烷氧基取代所成的基团，例如，甲氧基乙基，甲氧基乙氧基甲基等。
C1～C6烷氧基是指C1～C6的烷基通过氧原子与母核相连，如甲氧基、乙氧基等。
术语“C 1-C 6烷基”指的是任意的含有1－6个碳原子的直链或支链基团，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、叔戊基、正己基等。
除非另有提供，术语“C 3-C 7环烷基”指的是3-至7-元全-碳单环，其可以包含一个或多个双键，但不具有完全共轭的π-电子系统。环烷基的实例包括但不限于环丙烷基、环丁烷基、环戊烷基、环戊烯基、环己烷基、环己烯基、环己二烯基、环庚烷基、环庚烯基、环庚二烯基。
术语“C 1-C 6酰基”指的是-C(＝O)-H或-C(＝O)-C 1-C 5烷基，例如甲酰基、乙酰基、丙酰基、丁酰基等。
根据本发明和除非另有提供，任意上述基团可以任选地在其任意自由位置上被一个或多个基团取代，例如被1-6个基团取代，该基团独立地选自:卤素原子、硝基、氧代(＝O)、氰基、C 1-C 6烷基、多氟化烷基、多氟化烷氧基、烯基、炔基、羟基烷基、羟基烷基氨基、羟基杂环基、芳基、芳基-烷基、杂芳基、杂芳基-烷基、杂环基、杂环基-烷基、C 3-C 7环烷基、环烷基-烷基、烷基-芳基、烷基-杂芳基、烷基-杂环基、烷基-环烷基、烷基-芳基-烷基、烷基-杂芳基-烷基、烷基-杂环基-烷基、烷基-环烷基-烷基、烷基-杂环基-杂环基、杂环基-杂环基、杂环基-烷基-杂环基、杂环基-烷基氨基、烷基-杂环基-烷基-氨基、羟基、烷氧基、芳氧基、杂环基氧基、烷基-杂环基氧基、亚甲二氧基、烷基羰基氧基、芳基羰基氧基、环烯基氧基、杂环基羰基氧基、亚烷基氨基氧基、羧基、烷氧基羰基、芳氧基羰基、环烷基氧基羰基、杂环基氧基羰基、氨基、脲基、烷基氨基、氨基-烷基氨基、二烷基氨基、二烷基氨基-杂环基、二烷基氨基-烷基氨基、芳基氨基、芳基烷基氨基、二芳基氨基、杂环基氨基、烷基-杂环基氨基、烷基-杂环基羰基、甲酰基氨基、烷基羰基氨基、芳基羰基氨基、杂环基羰基氨基、烷基-杂环基羰基氨基、氨基羰基、烷基氨基羰基、二烷基氨基羰基、芳基氨基羰基、杂环基氨基羰基、烷氧基羰基氨基、烷氧基羰基氨基-烷基氨基、烷氧基羰基杂环基-烷基氨基、烷氧基-芳基-烷基、羟基氨基-羰基、烷氧基亚氨基、烷基磺酰基氨基、芳基磺酰基氨基、杂环基磺酰基氨基、甲酰基、烷基羰基、芳基羰基、环烷基羰基、杂环基羰基、烷基磺酰基、芳基磺酰基、氨基磺酰基、烷基氨基磺酰基、二烷基氨基磺酰基、芳基氨基磺酰基、杂环基氨基磺酰基、芳硫基、烷硫基、膦酸酯基和烷基膦酸酯基。
进一步的，上述取代基各自可以进一步被一个或多个上述举出的基团取代。
其中，术语“卤原子”指氟、氯、溴或碘原子。
术语“氰基”指-CN残基。
术语“硝基”指-NO 2基团。
术语“烷氧基”、“环基氧基”、“芳基氧基”，“杂环基氧基”及其衍生物指任意上述C 1-C 6烷基、C 3-C 7环烷基、芳基或杂环基，其通过氧原子(-O-)连接到分子的其余部分。
术语“磺酰基”指-S(O) 2-R残基。其中，R为一般取代或非取代氨基、烷基等本发明限定的取代基团。
术语“五元杂芳基”指碳原子和杂原子总数为五的杂芳基。
术语“六元杂芳基”指碳原子和杂原子总数为六的杂芳基。
从所有上述描述中，对本领域技术人员显而易见的是，其名称是相应基团的复合名称，例如“芳基氨基”，指被芳基取代的氨基，其中芳基如上文所定义。
术语烷硫基、烷基氨基、二烷基氨基、烷氧基羰基、烷氧基羰基氨基、杂环基羰基、杂环基羰基氨基、环烷基氧基羰基等定义同上，其中烷基、烷氧基、芳基、C 3-C 7环烷基和杂环基范围如上文所定义。
化学缩写符号Ms指甲磺酰基。
如本文所使用，除非另外说明，术语“前药”是指可以在生物学条件(体外或体内)下水解、氧化或进行其他反应以提供本发明的化合物的衍生物。前药仅在生物学条件下经过该反应成为活性化合物，或者在它们不反应的形式中具有活性。通常可以使用公知的方法制备前药，例如1Burger's Medicinal Chemistry and Drug Discovery(1995)172-178,949-982(Manfred E.Wolff编,第5版)中描述的方法。
本发明还提供了上述噻唑啉酮杂环化合物的制备方法，包括以下步骤：
A)式a所示的芳香胺类化合物与二氯硫化碳，在碱性条件下进行反应，得到式b所示化合物；
B)式b所示化合物进行碱催化的环化反应，得到式c所示化合物；
C)式c所示化合物与式d所示化合物进行加成反应，得到式Ⅰ-1所示化合物；
Figure PCTCN2018092824-appb-000020
上述二氯硫化碳也称为硫代光气。
所述步骤B)具体为，式b所示化合物与巯基甲酸乙酯，在碱性化合物的作用下进行碱催化的环化反应，得到式c所示化合物。所述碱性化合物可以为三乙胺等。
所述步骤C)的加成反应，优选在醋酸铵、醋酸钠等碱性化合物的条件下进行。
上述反应的反应路线如下所示：
Figure PCTCN2018092824-appb-000021
或者包括以下步骤：
A＇)式a所示的芳香胺类化合物与三光气，在碱性条件下进行羰基化反应，得到式b＇所示化合物；
B＇)式b＇所示化合物进行碱催化的环化反应，得到式c＇所示化合物；
C＇)式c＇所示化合物与式d所示化合物进行加成反应，得到式Ⅱ所示化合物；
Figure PCTCN2018092824-appb-000022
所述三光气学名碳酸三氯甲基酯。
所述步骤B)具体为，式b＇所示化合物与巯基甲酸乙酯，在碱性化合物的作用下进行碱催化的环化反应，得到式c＇所示化合物。所述碱性化合物可以为三乙胺等。
所述步骤C)的加成反应，优选在醋酸铵、醋酸钠等碱性化合物的条件下进行。
上述反应的反应路线如下所示：
Figure PCTCN2018092824-appb-000023
上述结构中的R 1，R 2，R 3，R 4，R 5，R 6，R 7，R 8，R 9和R 10同上，在此不再赘述。
本发明提供的上述化合物的制备方法不限定于上述制备方法，本领域技术人员熟知的其他制备方法也在本发明的保护范围之内。
本发明在制备上述化合物的过程中，所采用的纯化和分析方法均为本领域技术人员公知的方法， 其中通用纯化和分析方法如下：
在硅胶GF254预涂覆板(青岛海洋化工厂)上进行薄层色谱。在中压下经硅胶(300-400目，烟台芝黄务硅胶开发试剂厂)进行柱色谱分离或通过使用ISCO Combiflash Rf200快速纯化系统用预装的硅胶筒(ISCO或Welch)进行柱色谱分离。成分通过UV光(λ:254nm)和通过碘蒸气显影。当必要时，将化合物通过制备型HPLC制备经Waters Symmetry C18(19x 50mm,5μm)柱或经Waters X Terra RP 18(30x150mm,5μm)柱纯化，使用装配有996Waters PDA检测器的Waters制备型HPLC 600和Micromass mod.ZMD单四级质谱(电喷雾离子化，阳离子模式)。
方法1：相A:0.1％TFA/MeOH 95/5；相B:MeOH/H 2O 95/5。梯度：10至90％B进行8min，保持90％B 2min；流速20mL/min。
方法2：相A:0.05％NH 4OH/MeOH 95/5；相B:MeOH/H 2O 95/5。梯度：10至100％B进行8min，保持100％B 2min。流速20mL/min。
将 1H-NMR谱在DMSO-d 6或CDCl 3中经在600MHz操作的BrukerAvance 600谱仪(对于 1H而言)进行记录。将残留溶剂信号用作参比(δ＝2.50或7.27ppm)。化学位移(δ)以百万分率(ppm)进行报道且偶合常数(J)以Hz计。以下缩写用于峰裂分:s＝单；br.s.＝宽信号；d＝双；t＝三；m＝多重；dd＝双双。
电喷雾(ESI)质谱经Finnigan LCQ离子阱获得。
除非另外说明，所有最终化合物均是均质的(纯度不低于95％)，如高效液相色谱(HPLC)所确定。用于评价化合物纯度的HPLC-UV-MS分析通过组合离子阱MS设备与HPLC系统SSP4000(Thermo Separation Products)来进行，所述HPLC系统装配有自动进样器LC Pal(CTC Analytics)和UV6000LP二极管阵列检测器(UV检测215-400nm)。用Xcalibur 1.2软件(Finnigan)进行设备控制、数据采集和处理。HPLC色谱法在室温和1mL/min流速下进行，其使用Waters X Terra RP 18柱(4.6x 50mm；3.5μm)。流动相A是乙酸铵5mM缓冲液(采用乙酸得到pH 5.5):乙腈90:10，流动相B乙酸铵5mM缓冲液(采用乙酸得到pH 5.5):乙腈10:90；梯度为0至100％B进行7分钟，然后在再平衡前保持100％B达2分钟。
试剂纯化参考Purification of Laboratory Chemicals(Perrin,D.D.,Armarego,W.L.F.and PerrinsEds,D.R.；Pergamon Press:Oxford,1980)一书进行。石油醚是60-90℃馏分、乙酸乙酯、甲醇、二氯甲烷均为分析纯。
本发明还提供了一种药物组合物，包括上述噻唑啉酮杂环化合物或其盐或上述制备方法制备的噻唑啉酮杂环化合物或其盐，以及药学上可接受的载体，赋形剂，稀释剂，辅剂，媒介物或它们的组合。
本发明对所述载体，赋形剂，稀释剂，辅剂，媒介物并无特殊限定，可以为本领域技术人员熟知的适用于药物组合物的载体，赋形剂，稀释剂，辅剂，媒介物。
与现有技术相比，本发明发现了一系列对炎症小体具有抑制活性的化合物，提供了具有式Ⅰ所示结构的化合物，其互变异构体、前药、药学上可接受的溶剂化物或盐，用作NLRP3炎症小体抑制剂，其对于NLRP3炎症小体具有优异的特异性抑制活性。
附图说明
图1：CY-09抑制Nigericin和MSU诱导的NLRP3炎症小体活化及IL-1β的分泌电泳图和柱形图；
图2：CY-09特异性抑制经典及非经典NLRP3炎症小体的活化柱形图；
图3：CY-09抑制ASC二聚体的形成及NLRP3炎症小体复合物的组装电泳图；
图4：CY-09特异性的与NLRP3蛋白发生相互作用电泳图；
图5:NLRP3蛋白NACHT功能区段中的ATP结合位点是CY-09的直接结合部位电泳图；
图6：CY-09特异性的抑制NLRP3蛋白的ATP酶活性柱形图；
图7：CY-09抑制MSU诱导的腹膜炎柱形图；
图8：CY-09抑制MSU诱导的痛风曲线图及柱形图；
图9：CY-09抑制LPS诱导的脓毒血症柱形图；
图10：CY-09显著提升穆-韦氏综合征模型鼠的生存率柱形图；
图11：腹腔注射CY-09治疗高脂食物诱导的肥胖及胰岛素抵抗柱形图及曲线图；
图12：口服给药CY-09治疗高脂食物诱导的肥胖及胰岛素抵抗柱形图及曲线图。
具体实施方式
为了进一步说明本发明，下面结合实施例对本发明提供的噻唑啉酮杂环化合物、其制备方法、药用组合物及其应用进行详细描述。
在如下实施例和本申请的上下文中，下列缩写具有如下含义。如果不定义，则术语具有其普遍可接受的含义。
Figure PCTCN2018092824-appb-000024
实施例1
按照以下路线制备化合物ⅠA-1：
Figure PCTCN2018092824-appb-000025
1)化合物2的制备
Figure PCTCN2018092824-appb-000026
将化合物1(3.23g，20mmol)溶于40mL氯仿中，加入由8g NaHCO 3和80mL水配成的混合溶液，0℃下缓慢加入硫代光气(1.84mL，24mmol)溶于40mL氯仿所得的溶液，然后继续反应1-2h。静置，分液，水相用氯仿萃取，合并有机相用饱和食盐水洗涤，无水Na 2SO 4干燥。过滤，浓缩，快速柱层析(CH 2Cl 2)，得到化合物2(3.43g，84.2％)，淡黄色油状液体。
MS(ESI)m/z:217[M+H] +。
2)化合物3的制备
Figure PCTCN2018092824-appb-000027
将化合物2(3.43g，16.88mmol)和巯基乙酸甲酯(1.83g，1.53mL，16.88mmol)溶于15.0mL无水二氯甲烷中，冰浴中冷却至0℃，逐滴缓慢加入TEA(0.5mL)，冰浴下继续反应15min后，撤去冰浴，室温反应3-5h，反应体系呈酒红色。150mL CH 2Cl 2稀释体系，70mL水萃取一遍，70mL饱和食盐水洗一遍，静置分层，有机相用无水Na 2SO 4干燥，过滤，浓缩，油泵抽1-3h，得酒红色固体3(3.97g，84.8％)。
MS(ESI)m/z:291[M+H] +。
3)化合物IA-1的制备
Figure PCTCN2018092824-appb-000028
称取化合物3(1.11g，4.0mmol)，对甲醛苯甲酸(600mg，4.0mmol)和无水醋酸钠(656mg，8.0mmol)加入150mL旋塞瓶中，加入无水醋酸32mL，油浴130℃反应6-8h，有亮黄色固体析出。待体系冷却至室温，转移到50mL离心管内，用甲醇反复离心(3000r/min)3-5遍，转移得到亮黄色固体产物IA-1(1.33g，81.2％)。
MS(ESI)m/z:423[M+H] +。
按照以上方法，制备表1所示化合物，表1是化合物结构以及结构表征数据。
表1.化合物IA-1—IA-81的结构及表征
Figure PCTCN2018092824-appb-000029
Figure PCTCN2018092824-appb-000030
Figure PCTCN2018092824-appb-000031
Figure PCTCN2018092824-appb-000032
Figure PCTCN2018092824-appb-000033
Figure PCTCN2018092824-appb-000034
Figure PCTCN2018092824-appb-000035
Figure PCTCN2018092824-appb-000036
Figure PCTCN2018092824-appb-000037
Figure PCTCN2018092824-appb-000038
Figure PCTCN2018092824-appb-000039
Figure PCTCN2018092824-appb-000040
Figure PCTCN2018092824-appb-000041
Figure PCTCN2018092824-appb-000042
Figure PCTCN2018092824-appb-000043
Figure PCTCN2018092824-appb-000044
Figure PCTCN2018092824-appb-000045
Figure PCTCN2018092824-appb-000046
Figure PCTCN2018092824-appb-000047
Figure PCTCN2018092824-appb-000048
Figure PCTCN2018092824-appb-000049
Figure PCTCN2018092824-appb-000050
Figure PCTCN2018092824-appb-000051
Figure PCTCN2018092824-appb-000052
Figure PCTCN2018092824-appb-000053
实施例2
按照实施例1所述方法，制备表2所示化合物，表2是化合物结构以及结构表征数据。
表2.化合物IB-1-IB-7的结构及表征
Figure PCTCN2018092824-appb-000054
Figure PCTCN2018092824-appb-000055
实施例3
按照实施例1所述方法，制备表3所示化合物，表3是化合物结构以及结构表征数据。
表3.化合物IC-1—IC-64的结构及表征
Figure PCTCN2018092824-appb-000056
Figure PCTCN2018092824-appb-000057
Figure PCTCN2018092824-appb-000058
Figure PCTCN2018092824-appb-000059
Figure PCTCN2018092824-appb-000060
Figure PCTCN2018092824-appb-000061
Figure PCTCN2018092824-appb-000062
Figure PCTCN2018092824-appb-000063
Figure PCTCN2018092824-appb-000064
Figure PCTCN2018092824-appb-000065
实施例4
按照以下路线制备式II-1所示化合物：
Figure PCTCN2018092824-appb-000066
1)化合物2的制备
Figure PCTCN2018092824-appb-000067
称取化合物1(161mg，1.0mmol)溶于3.0mL无水1,2-二氯乙烷，0℃加入三光气(BTC，445mg，1.5mmol)，70℃反应0.5h，83℃反应4h，反应体系由最初的白色浑浊状变成暗紫色澄清，待反应结束后自然冷却至室温，浓缩，油泵抽2h得粗产物化合物2，浅紫色固体，粗品直接用于下一步反应。
MS(ESI)m/z:188[M+H] +.
2)化合物3的制备
Figure PCTCN2018092824-appb-000068
将粗产物化合物2(187mg，1.0mmol)和钠块50mg溶解在无水甲苯(1.0mL)内，迅速取巯基乙酸甲酯(0.13mL，1.0mmol)加入管内，120℃，微波反应2h。待反应结束后自然冷却至室温，直接浓缩得粗产物化合物3，白色固体，粗品3直接用于下一步反应。
MS(ESI)m/z:262[M+H] +.
3)化合物II-1的制备
Figure PCTCN2018092824-appb-000069
将化合物3(147mg，0.56mmol)，对醛基苯甲酸(85mg，0.56mmol)和无水醋酸铵(130mg，1.7mmol)加入20mL微波管，再加入5mL干燥处理过的醋酸，使用微波反应器，180℃，反应50min，体系由无色透明变成黄色透明。自然冷却至室温，直接浓缩，甲醇洗3-5遍得化合物II-1(8.5mg，3.8％)，淡黄色固体。
按照以上方法制备表4所示化合物，表4是化合物结构以及结构表征数据。
表4.化合物II-1—II-5的结构及表征
Figure PCTCN2018092824-appb-000070
Figure PCTCN2018092824-appb-000071
生物实验
下述实施案例中的实验方法，如无特殊说明，均为常规实验方法。下述实施案例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到，见下表。
表一：材料及实验动物
Figure PCTCN2018092824-appb-000072
Figure PCTCN2018092824-appb-000073
实施例5 CY-09(ⅠA-1)及类似物在体外抑制巨噬细胞NLRP3炎症小体的活化
一、CFTR(inh)-172(ⅠC-1)抑制NLRP3炎症小体活化
1.取体外分化培养至第4天的BMDM细胞，分至12孔板中，过夜贴壁，去除培养基，换用含1％FBS及LPS的Opti-MEM培养基培养3个小时，加入不同浓度的CFTR(inh)-172(0.5，1,5，10和20μM)，同时加入DMSO作为对照,继续培养30分钟，每孔加入等量的经典NLRP3炎症小体活化剂Nigericin，刺激20分钟。
2.收取上清，WB和ELISA分别检测上清中IL-1β和p20的分泌量。
二、CFTR(inh)-172及CY-09类似物对NLRP3炎症小体和CFTR活性的抑制分析
1.CFTR(inh)-172及CY-09类似物对NLRP3炎症小体的抑制分析。
(1)取体外分化培养至第4天的BMDM细胞，分至24孔板中，过夜贴壁，去除培养基，换用含1％FBS及LPS的Opti-MEM培养基培养3个小时，每孔按照10μM的给药剂量，分别加入CFTR(inh)-172及各种CY-09类似物，同时设置DMSO对照孔，继续培养30分钟，每孔加入等量的经典NLRP3炎症小体活化剂Nigericin，刺激20分钟。
(2)收取上清，ELISA分别检测各孔上清中IL-1β的分泌量。
(3)计算各化合物相对于DMSO组IL-1β分泌的抑制效率(活性数据见表5～表8)
2.CFTR(inh)-172及CY-09类似物对CFTR活性的抑制分析
(1)利用CFTR高表达的肠道上皮细胞系HT29，结合Forsklin和IBMX刺激，检测CFTR(inh)-172及各种CY-09类似物对CFTR活性的影响。
(2)取处于对数生长期的HT29，消化并分至12孔板中，过夜贴壁，待细胞融合80％左右，去除上清，换用无血清的Opti-MEM培养基，继续培养12小时，每孔按照20μM的给药剂量，分别加入CFTR(inh)-172及各种CY-09类似物，同时设置DMSO对照孔，继续培养30分钟，之后每孔加入终浓度分别为10μM和20μM的Forsklin/MIBMX cocktail，继续孵育35分钟。
(3)去除上清，每孔加入300μL的MilliQ级无菌水，反复冻融数次，使孔中HT29彻底裂解，离心取上清，按照1:20的稀释比例，将浓度为1mM的氯离子染料MAQE加入到离心上清中，避光染色30分钟，上机比色并读取MAQE的荧光度值，扣除背景荧光后，计算各孔裂解液中氯离子的含量。
(4)将计算得到的各孔氯离子含量值分别除以DMSO对照组的氯离子含量值即为该小分子化合物对CFTR活性的抑制(活性数据见表5～表8)。
表5-1 化合物IA活性数据
Figure PCTCN2018092824-appb-000074
Figure PCTCN2018092824-appb-000075
*抑制率低于10％，标为Inactive。
表5-2 化合物IA活性数据
Figure PCTCN2018092824-appb-000076
Figure PCTCN2018092824-appb-000077
*“--”表示未检测。
表6 化合物IB活性数据
Figure PCTCN2018092824-appb-000078
*抑制率低于10％，标为Inactive。
表7 化合物IC活性数据：
Figure PCTCN2018092824-appb-000079
Figure PCTCN2018092824-appb-000080
表8 化合物II活性数据：
Figure PCTCN2018092824-appb-000081
由表5～表8可知，IA组、IB组化合物，和化合物II，对于NLRP3炎症小体具有特异性抑制作用。
三.CY-09抑制IL-1β的分泌
1.BMDM(骨髓来源的巨噬细胞)：制备方法见文献(Yan et al.,2013，Immunity)。
2.第五天，将BMDM细胞分到十二孔板上，每孔3-6*10 5个细胞。
3.第六天，去上清，每孔加入终浓度为50ng/ml的ultra-LPS，含1％胎牛血清的opti-MEM，预处理3个小时。之后分别加入终浓度为1μM,5μM,10μM的CY-09继续孵育30分钟，分成6组，每组6个复孔。
第一组:空白对照组。
第二组：阴性对照组，仅加入等剂量的ultra-LPS。
第三组:每孔加入等体积的DMSO预处理半小时，再加入终浓度为5μM的Nigericin刺激30分钟或加入3.5μL MSU(浓度为200μg/ml)刺激4.5个小时。
第四组：每孔加入终浓度为1μM的CY-09预处理30分钟，再加入终浓度为5μM的Nigericin半小时或加入3.5μL MSU(浓度为200μg/ml)刺激4.5个小时。
第五组：每孔加入终浓度为5μM的CY-09预处理30分钟，再加入终浓度为5μM的Nigericin半小时或加入3.5μL MSU(浓度为200μg/ml)刺激4.5个小时。
第六组：每孔加入终浓度为10μM的CY-09预处理30分钟，再加入终浓度为5μM的Nigericin半小时或加入3.5μL MSU(浓度为200μg/ml)刺激4.5个小时。
4.经步骤3处理后的各组细胞，收集细胞培养上清(SN)和细胞裂解液(Input)，留取一部分上清，ELISA检测IL-1β，其余按常规方法提取上清中的蛋白，用抗鼠IL-1β抗体和抗鼠p20抗体进行WB分析。
如图1所示，在Nigericin/MSU的刺激下，NLRP3炎症小体被活化，WB和ELISA检测显示CY-09浓度依赖性的抑制p20及IL-1β的成熟和分泌。
四.CY-09特异性抑制经典及非经典NLRP3炎症小体的活化，而对于Poly(dA:dT)激活的AIM2炎症小体及沙门氏菌激活的IPAF炎症小体无抑制效果
1.BMDM(骨髓来源的巨噬细胞)：制备方法见文献(Yan et al.,2013，Immunity)。
2.第五天，将BMDM分到24孔板上，每孔1.5-3*10 5个细胞。
3.第六天，去上清，每孔加入终浓度为50ng/ml的ultra-LPS或终浓度为100ng/ml的Pam3CSK4，含1％胎牛血清的opti-MEM，预处理3个小时。后分别加入终浓度为1μM,5μM,10μM的CY-09继续孵育30分钟。
实验一：CY-09抑制ATP刺激的经典NLRP3炎症小体的活化。ATP加入终浓度为5mM，刺激时间为20分钟。
实验二：CY-09抑制胞内转染LPS诱导的非经典NLRP3炎症小体的活化。利用Lipofectamine2000转染终浓度为2μg/ml的ultra-LPS至BMDM细胞中，过夜培养16小时，收集细胞培养上清，ELISA检测上清中IL-1β的分泌水平。
实验三：CY-09对由Poly(dA:dT)诱导的AIM2炎症小体的活化不具有抑制作用。利用Lipofectamine2000转染终浓度为1μg/ml的Poly(dA:dT)至BMDM细胞中，继续培养3-4个小时，收集细胞培养上清，ELISA检测上清中IL-1β的分泌水平。
实验四：CY-09对由沙门氏菌诱导的IPAF炎症小体的活化不具有抑制作用。离心沉淀培养过夜的沙门氏菌液，弃上清，加入1ml Opti-MEM重悬，按照1:500的稀释比例，取0.5μL重悬液加入到含250μL培养基的24孔板的培养上清中，4小时后，收集细胞培养上清，ELISA检测上清中IL-1β的分泌水平。
4.如图2所示，CY-09抑制由经典NLRP3炎症小体激动剂ATP诱导的NLRP3炎症小体的活化，同时对于由胞内转染LPS诱导的非经典NLRP3炎症小体的活化亦具有浓度依赖性的抑制效果。然而对于由Poly(dA:dT)激活的AIM2炎症小体及沙门氏菌激活的IPAF炎症小体不具有抑制作用。
五.CY-09抑制ASC二聚体的形成及NLRP3炎症小体复合物的组装
1.BMDM(骨髓来源的巨噬细胞)：制备方法见文献(Yan et al.,2013，Immunity)。
实验一：CY-09抑制ASC二聚体的形成。分BMDM至六孔板中，每孔1*10 6个细胞。LPS刺激3小时，每孔加入相应浓度的CY-09或DMSO继续培养30分钟，接着加入终浓度为10μM的Nigericin刺激30分钟。收集每孔培养上清，沉淀蛋白，WB检测CY-09对NLRP3炎症小体活化的抑制效果。每孔加入等体积的含蛋白酶抑制剂的NP-40，冰上裂解30分钟，收集裂解液，离心沉淀，可见一小团白色沉淀物，吸去上清，加入终浓度为2mM的新鲜交联剂DSS，室温交联30分钟，冰冷PBS洗涤交联沉淀物两次，离心沉淀交联物，加入相应体积的蛋白裂解缓冲液，100℃，5分钟，WB检测。
实验二：CY-09抑制ASC与NLRP3相互作用。BMDM处理方法及NLRP3炎症小体刺激体系同前，内源co-ip方法和步骤参考(Shi et al.,2015，Nature Immunology)。
实验三：CY-09抑制NLRP3与NLRP3相互作用。待六孔板中293T细胞融合80％左右，分别或同时转入带FLAG和mCherry标签的NLRP3质粒，转染2小时后加入CY-09，继续培养20小时。NP-40裂解，离心取上清，加入FLAG抗体包被的珠子，4℃旋转孵育2.5小时，WB检测。外源co-ip方法和步骤参考(Shi et al.,2015，Nature Immunology)。
实验四：CY-09抑制NLRP3多聚体的形成及与ASC的相互作用。利用分子筛实验体系结合WB方法(方法和步骤参考Shi et al.,2015，Nature Immunology)，进一步确定CY-09不但可以抑制NLRP3多 聚体的形成，同时还可抑制NLRP3与ASC的相互作用。
2.如图3所示，CY-09可以浓度依赖性的抑制ASC二聚体的形成及NLRP3与ASC和NLRP3与NLRP3之间的相互作用，而分子筛实验结果进一步证实CY-09可以抑制NLRP3多聚体的形成及NLRP3与ASC的相互作用。
六、CY-09特异性的与NLRP3蛋白发生相互作用
1.BMDM(骨髓来源的巨噬细胞)：制备方法见文献(Yan et al.,2013，Immunity)。
实验一：生物素标记的CY-09可以浓度依赖性的结合NLRP3蛋白。NP-40冰上裂解经LPS刺激3小时的BMDM细胞，离心去沉淀，均分上清至不同处理组别的EP管中，每组分别加入不连生物素的CY-09(free CY-09)或不同浓度的生物素标记的CY-09(Biotin-CY-09)，4℃旋转孵育1.5小时。之后，每组加入等量亲和素包被的珠子，继续旋转孵育2小时。用含1％tween-20的PBS和含0.1％NP-40的PBS先后分别洗涤珠子4次，WB检测。
实验二：free CY-09可以竞争性抑制Biotin-CY-09与NLRP3蛋白的相互作用。NP-40冰上裂解经LPS刺激3小时的BMDM细胞，离心去沉淀，均分上清至不同处理组别的EP管中，每组分别加入不同浓度的free CY-09(0μM,10Μm，20μM，40μM，80μM)，4℃旋转孵育过夜。之后，每组加入相同摩尔浓度生物素标记的CY-09(1μM)，4℃旋转孵育2小时，用含1％tween-20的PBS和含0.1％NP-40的PBS先后分别洗涤珠子4次，WB检测。实验中所用THP-1细胞系购自美国ATCC。
实验三：CY-09特异性的与NLRP3蛋白发生相互作用，而不能与NOD1/2，AIM2和NLRC4蛋白发生相互作用。待六孔板中293T细胞融合80％左右，分别转入带FLAG标签的空载质粒(EV)，NLRP3质粒，NOD1质粒，NOD2质粒，AIM2质粒以及NLRC4质粒，继续培养24小时。NP-40冰上裂解各孔细胞，后续Pulldown体系参考实验一进行，WB检测。
实验四：CY-09直接与纯化的GST-NLRP3蛋白发生结合。取购自Abnova公司的纯化GST-NLRP3重组蛋白稀释至PBS中，使其终浓度为0.4pg/μL,分别加入不同浓度的Biotin-CY-09,4℃旋转孵育1.5小时。之后每管加入等量亲和素包被的珠子，继续旋转孵育2小时。用含1％tween-20的PBS和含0.1％NP-40的PBS先后分别洗涤珠子4次，WB检测。Pulldown体系结合竞争性抑制方案,WB检测发现重组GST-NLRP3蛋白与Biotin-CY-09的直接结合可以被free CY-09浓度依赖性的抑制。
2.如图4所示，Biotin-CY-09可以浓度依赖性的pulldown NLRP3蛋白，且NLRP3蛋白的这种pulldown可以被free CY-09竞争性抑制。利用293T细胞系过表达带有FLAG标签的空载质粒(EV)，NLRP3质粒，NOD1质粒，NOD2质粒，AIM2质粒以及NLRC4质粒，结合pulldown和WB实验体系，进一步证实Biotin-CY-09特异性的与NLRP3蛋白发生相互作用。
七.NLRP3蛋白NACHT功能区段中的ATP结合位点是CY-09的直接结合部位
1.293T细胞传代培养及转染方法同前。
实验一：CY-09通过与NLRP3蛋白的NACHT功能区段发生相互作用而与NLRP3蛋白相结合。待六孔板中293T细胞融合80％左右，分别转入带FLAG标签的全长NLRP3质粒，NACHT质粒，LRR质粒以及PYD质粒，继续培养24小时。NP-40冰上裂解各孔细胞，后续Pulldown体系参考四中的实验一进行，WB检测。
实验二：CY-09与NLRP3蛋白NACHT功能区段中的ATP结合位点发生直接结合。NLRP3蛋白的ATP酶活性区域位于NACHT功能区段，包括ATP结合位点(walker A)和ATP酶的催化位点(walker B)。分别构建带有FLAG标签并且包含walker A和walker B点突变的NLRP3质粒。转入融合80％左右的293T细胞系中，继续培养24小时。NP-40冰上裂解各孔细胞，后续Pulldown体系参考四中的实验一进行，WB检测。
2.如图5所示，Biotin-CY-09仅与NLRP3的NACHT功能区段相结合，而不能够与LRR和PYD功能区段相结合。构建NLRP3蛋白ATP酶活性位点的突变质粒(walker A和walker B)，进一步证实Biotin-CY-09与NACHT功能区段相结合是通过与其ATP结合位点发生相互作用。
八.CY-09特异性抑制NLRP3蛋白的ATP酶活性
1.293T细胞传代培养及转染方法同前。
实验一：CY-09剂量依赖性的抑制NLRP3蛋白的ATP酶活性。按照外源co-ip方法将带有FLAG标签的NLRP3蛋白，NLRP1蛋白以及NLRC4蛋白自293T细胞裂解液中pulldown下来，接着用终浓度为5mg/ml的flag peptide溶液将目的蛋白从FLAG抗体包被的珠子上洗脱下来，此步为纯化目的蛋白，为提高目的蛋白纯度可重复上述操作一次。
实验二：目的蛋白纯化出来之后进行ATP酶活性检测。①取5uL经进一步纯化的flag-NLRP3蛋白或flag-NLRP1蛋白或flag-NLRC4蛋白加入到含有25uL kinase reaction buffer以及不同浓度CY-09的白色96孔板中，于37℃孵育15分钟。②加入终浓度为250uM的ATP，继续于37℃孵育40-60分钟。③加入25uL ADP-Glo Reagent以终止激酶反应并去除残留的ATP，室温孵育40分钟。④结束孵育后，再加入50uL的Kinase Detection Reagent，室温继续孵育30-60分钟。⑤于多功能酶标仪上，检测luminescence的吸光度值。
实验三：取购自Abnova公司的纯化GST-NLRP3重组蛋白，按照实验二方案进行体外ATP ase活性检测，结果显示GST-NLRP3重组蛋白的ATP ase活性可以被CY-09浓度依赖性的抑制。
2.如图6所示，CY-09剂量依赖性的抑制NLRP3蛋白的ATP酶活性，而对于NLRP1蛋白和NLRC4蛋白的ATP酶活性不具有明显的抑制作用。
实施例6 CY-09抑制尿酸盐结晶诱导的急性炎症
一.CY-09抑制尿酸盐结晶(MSU)诱导的腹膜炎
1.取10周龄雄性C57BL/6J鼠，分成3组，每组6只。
2.第一组，腹腔注射一定体积DMSO，半小时后注射适当体积的PBS。
第二组，腹腔注射一定体积DMSO，半小时后注射MSU，剂量为每只鼠0.5mg。
第三组，腹腔注射20mg/kg的CY-09，半小时后注射MSU，剂量为每只鼠0.5mg。
3.六小时后，向鼠的腹腔中注入10ml预冷PBS，取腹腔灌洗液离心。
4.步骤3得到的腹腔灌洗液上清用ELISA的方法进行IL-1β等细胞因子的测定；沉淀的细胞用抗CD11b和抗Ly6G抗体进行标记，细胞流式分析仪检测腹腔中性粒细胞的浸润情况。
如图7所示，腹腔注射MSU后，中性粒细胞明显被招募，而注射CY-09后可显著抑制中性粒细胞浸润。同时注射CY-09后也可显著抑制IL-1β的分泌。说明CY-09可有效抑制MSU诱导的腹腔炎。
二.CY-09抑制由尿酸盐结晶(MSU)堆积诱导的痛风
1.取10周龄雄性C57BL/6J鼠，分成两组，每组6只。用游标卡尺分别测量并记录每只鼠两侧膝关节的起始宽度。
2.第一组，向左腿膝关节腔中注入DMSO和PBS混合液，向右腿膝关节腔中同时注入DMSO和MSU。
第二组，向左腿膝关节腔中注入DMSO和PBS混合液，向右腿膝关节腔中同时注入CY-09和MSU。
3.分别在注射后12小时和24小时测量并记录小鼠两腿膝关节的宽度。
4.24小时后，摘眼球取血，剪取小鼠膝盖骨置于24孔板，每孔加入200μl opti-MEM(含1％双抗)培养基培养1小时，收集培养上清，ELISA检测培养上清中IL-1β的分泌水平。
如图8所示，关节腔内注射MSU后，可使小鼠膝关节明显发生肿胀，加入CY-09后可有效缓解这种肿胀。与对照组相比，小鼠膝盖骨培养上清中IL-1β的分泌水平明显升高，而注射CY-09后可有效抑制IL-1β的分泌，这表明，CY-09可有效抑制MSU诱导的关节炎。
实施例7 CY-09抑制LPS诱导的急性外周炎症
1.取8周龄雄性C57BL/6J鼠，分成3组，每组6只。
2.第一组阴性对照，腹腔注射PBS。
第二组阳性对照，腹腔注射溶于PBS的LPS溶液，注射剂量为20mg/kg。
第三组给药治疗组，同时腹腔注射LPS和CY-09，LPS和CY-09的注射剂量均为20mg/kg。
3.4小时后，摘眼球放血，室温放置半小时，接着低速离心半小时，收集血清。
4.ELISA检测步骤3血清中IL-1β的分泌水平。
结果如图9所示，与对照组相比，在LPS的作用下IL-1β水平明显升高，而腹腔注射CY-09可有效抑制IL-1β的分泌。说明CY-09可以有效抑制LPS诱导的急性外周炎症。
实施例8 CY-09显著提升穆-韦氏综合征模型鼠的生存率
NLRP3蛋白第351位氨基酸由精氨酸突变成苯丙氨酸后，NLRP3炎症小体会持续活化，继而产生大量成熟IL-1β和IL-18，外周高炎性环境可致小鼠发育停滞，出生不久(一般在4-12天)即发生死亡。
1.分别取8周龄C57BL/6J背景且带有Lyz-Cre标签的雄(雌)鼠与同样是C57BL/6J背景，购自Jackson Lab公司的Nlrp3A350VneoR雌(雄)鼠进行交配。
2.在确定雌鼠怀孕后，雌雄分开饲养至雌鼠产下突变小鼠，母乳喂养4天后，腹腔注射CY-09，注射剂量为20mg/kg，每两天注射一次，期间记录体重及存活情况。
3.对照组腹腔注射同样体积的DMSO，每两天注射一次，期间记录体重及存活情况。
4.给药10次后，停止给药，继续精心饲养，记录小鼠的最终存活期限。
结果如图10所示，与注射DMSO的对照组相比，CY-09给药组小鼠的体重以及存活期限都明显得到了提升。从第21天停止给药后，小鼠依然可以继续存活27天。说明CY-09对由NLRP3突变导致NLRP3炎症小体持续活化的遗传性疾病穆-韦氏综合征具有显著的治疗效果。
实施例9 CY-09预防并治疗肥胖相关代谢紊乱综合征
一.腹腔注射CY-09可治疗HFD诱导的肥胖和胰岛素抵抗
1.取体重和随机血糖接近的6周龄雄性C57BL/6J鼠和NLRP3-/-鼠若干，一部分用正常食物进行喂养，另一部分用HFD进行喂养。结束12周的HFD喂养之后，将随机血糖值＞11.1mM的野生小鼠按照体重分成2组，每组6只。根据体重和随机血糖值将同样被HFD喂养的NLRP3-/-鼠分成2组，每组6只。
2.每组处理方式如下：
第一组：使用正常饲料喂养的野生对照组小鼠，每天腹腔注射相当体积的DMSO/PBS混合液(10％/90％)。
第二组：高脂饲料诱导12周后的野生鼠，每天继续高脂饲料喂养，腹腔注射相当体积的DMSO/PBS混合液。
第三组：高脂饲料诱导12周后的野生鼠，继续高脂饲料喂养，每天按照2.5mg/kg的注射剂量，腹腔注射相当体积的含有CY-09的DMSO/PBS混悬液。
第四组：使用正常饲料喂养的NLRP3-/-对照小鼠，每天腹腔注射相当体积的DMSO/PBS混合液(10％/90％)。
第五组：高脂饲料诱导12周后的NLRP3-/-小鼠，继续高脂饲料喂养，每天腹腔注射相当体积的DMSO/PBS混合液。
第六组：高脂饲料诱导12周后的NLRP3-/-小鼠，继续高脂饲料喂养，每天按照2.5mg/kg.BW-1的注射剂量，腹腔注射相当体积的含有CY-09的DMSO/PBS混悬液。
3.在完成9周的给药试验后，分别检测各组小鼠的体重、随机及禁食血糖，并完成葡萄糖耐量试验(GTT)和胰岛素敏感性试验(ITT)。
4.摘眼球取血并分离血清，称取部分肝脏和附睾脂肪组织进行组织培养，收集组织培养上清。ELISA检测血清中和组织培养上清中IL-1β的分泌水平。WB检测附睾脂肪组织中成熟caspase-1的表达。
5.结果如图11所示，经9周CY-09治疗后，与对照组小鼠相比，CY-09可显著改善由高脂饲料诱导的糖耐量受损和胰岛素抵抗，进而抑制高血糖。对外周以及肝脏和附睾脂肪组织中IL-1β的分泌水平、p20的表达水平均具有明显的抑制效果，这说明CY-09通过平衡机体的炎症状态，增强了机体对胰岛素和葡萄糖的敏感性，进而抑制高血糖并改善代谢紊乱。
二.口服给药CY-09可治疗HFD诱导的肥胖和胰岛素抵抗
1.利用0.5％的羧甲基纤维素钠盐溶液乳化CY-09粉末，使之均匀乳化其中。按照20mg/kg.BW-1 或40mg/kg.BW-1的给药计量，采用灌胃法每天给药一次。所用HFD诱导小鼠同前。
2.结果如图12所示，经7周CY-09口服治疗后，与对照组小鼠相比，CY-09抑制HFD诱导的体重增加并显著改善由高脂饲料诱导的糖耐量受损和胰岛素抵抗，进而抑制血糖升高。对外周以及肝脏和附睾脂肪组织中IL-1β的分泌水平、p20的表达水平均具有明显的抑制效果，这说明口服给药CY-09同样可以达到类似腹腔注射给药的治疗效果。对机体炎症状态的平衡，增强了机体对胰岛素和葡萄糖的敏感性，进而抑制血糖升高并改善代谢紊乱。
由上述实施例可知，本发明提供的化合物，对NLRP3炎症小体及其诱导的一系列炎症性疾病具有特异性抑制作用，其中n＝1,2或3的化合物，具有特异性抑制活性，尤其具有优良的药物应用前景。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (22)

  1. 噻唑啉酮杂环化合物在制备NLRP3炎症小体抑制剂中的应用，所述噻唑啉酮杂环化合物具有式Ⅰ或式Ⅱ所示结构或其异构体、前药、药学上可接受的溶剂化物或盐：
    Figure PCTCN2018092824-appb-100001
    其中，
    R 1，R 2，R 3，R 4和R 5各自独立地选自：
    氢，卤素，硝基，羟基，氰基，苯基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基；
    R 6，R 7，R 8，R 9和R 10各自独立地选自：
    氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1-C6含氧烷基、C1-C6烷氧基、C3-C7环烷基；取代或非取代的甲酰基、磺酰基、磺酰基氨基；
    X 1和X 2各自独立地选自O或S；
    n为0～6的任意整数；
    Q为C或N；
    Q 1、Q 2、Q 3、Q 4独立的选自-C-R 1，-C-R 2，-C-R 3，-C-R 4、-C-R 5、N，-N-R 1，S或O；
    Ar 2为取代或非取代芳基、杂芳基或稠环基；
    m为0或1。
  2. 根据权利要求1所述的应用，其特征在于，所述R 1，R 2，R 3，R 4和R 5各自独立地选自：
    氢，卤素，硝基，羟基，氰基，苯基；氨基，甲基氨基，N,N-二甲基氨基，乙基氨基，N,N-二乙基氨基，异丙基氨基，N,N-二异丙基氨基，环丙基氨基，环丁基氨基，环戊基氨基，环己基氨基，乙酰基氨基，N,N-二乙酰基氨基，2-N,N-二甲基氨基乙基氨基，2-羟基乙基氨基，甲磺酰基氨基，N,N-二甲磺酰基氨基，甲磺酰基，对甲苯磺酰基；C1-C6烷基，C1-C6含氟烷基，C1-C6含氧烷基，C1-C6烷氧基、C1～C6含氟烷氧基或C3-C7环烷基。
  3. 根据权利要求1所述的应用，其特征在于，所述R 6，R 7，R 8，R 9和R 10各自独立地选自：
    氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基或C1-C6含氧烷基；氨基甲酰基，甲胺基甲酰基，乙胺基甲酰基，丙胺基甲酰基，异丙胺基甲酰基，N,O-二甲羟胺基甲酰基，环丙基胺基甲酰基，环丁基胺基甲酰基，环戊基胺基甲酰基，哌啶基-1-甲酰基，4-羟基哌啶基-1-甲酰基，4-N,N-二甲基哌啶基-1-甲酰基，4-N,N-二乙基哌啶基-1-甲酰基，四氢吡咯基-1-甲酰基，3-N,N-二甲基四氢吡咯基-1-甲酰基，3-N,N-二乙基四氢吡咯基-1-甲酰基，哌嗪基-1-甲酰基，N-甲基哌嗪基-1-甲酰基，N-乙基哌嗪基-1-甲酰基，N-乙酰基哌嗪基-1-甲酰基，N-叔丁氧甲酰基哌嗪基-1-甲酰基，N-(2-羟基乙基)哌嗪基-1-甲酰基，N-(2-氰基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二甲基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二乙基乙基)哌嗪基-1-甲酰基，N-(3-羟基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二甲基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二乙基丙基)哌嗪基-1-甲酰基，吗啡啉基-1-甲酰基，3,5-二甲基吗啡啉基-1-甲酰基，4-(N-甲基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙酰基-1-哌嗪基)哌啶基-1-甲酰基，N-(N-甲基-4-哌啶基)哌嗪基-1-甲酰基，2-羟基乙胺基甲酰基，2-乙酰基羟基乙胺基甲酰基，苯胺基甲酰基，苄基甲酰基，苄基氨基甲酰基，3-甲氧基苄基甲酰基，3-甲氧基苄基氨基甲酰基；羟基甲酰基，甲氧基甲酰基，乙氧基甲酰基，丙氧基甲酰基，异丙氧基甲酰基，正丁氧基甲酰基，异丁 氧基甲酰基，叔丁氧基甲酰基，氨基磺酰基、C1～C10烷基磺酰基，C1～C10烷氨基磺酰基，C3～C10环烷基氨基磺酰基，C6～C12芳基氨基磺酰基，C5～C12杂芳基氨基磺酰基，或C1～C10磺酰基氨基。
  4. 根据权利要求3所述的应用，其特征在于，所述C1～C10烷基磺酰基为甲磺酰基，乙磺酰基，丙磺酰基，异丙磺酰基，丁磺酰基或异丁磺酰基；
    所述C1～C10烷氨基磺酰基为N-甲基氨基磺酰基，N-乙基氨基磺酰基，N-丙基氨基磺酰基，N-异丙基氨基磺酰基，N-丁基氨基磺酰基，N-异丁基氨基磺酰基，N,N-二甲基氨基磺酰基或N,N-二乙基氨基磺酰基；
    所述C3～C10环烷基氨基磺酰基为N-环丙基氨基磺酰基，N-环丁基氨基磺酰基，N-环戊基氨基磺酰基或N-环己基氨基磺酰基；
    所述C6～C12芳基氨基磺酰基为N-苯基氨基磺酰基；
    所述C5～C12杂芳基氨基磺酰基为N-吡啶基氨基磺酰基，N-噻吩基氨基磺酰基，N-呋喃基氨基磺酰基或N-吡咯基氨基磺酰基；
    所述C1～C10磺酰基氨基为甲磺酰氨基，乙磺酰氨基，丙磺酰氨基，异丙磺酰氨基，丁磺酰氨基，异丁磺酰氨基，磺酸基，氨基磺酰基。
  5. 根据权利要求1所述的应用，其特征在于，所述式Ⅱ具有以下式Ⅱ-a或式Ⅱ-b结构：
    Figure PCTCN2018092824-appb-100002
  6. 根据权利要求1所述的应用，其特征在于，所述Ar 2为取代或非取代苯基、五元或六元杂芳基、或者为取代或非取代苯基与取代或非取代芳基、杂芳基、杂环基稠合形成的稠环基团。
  7. 根据权利要求6所述的应用，其特征在于，所述Ar 2为取代或非取代苯基、吡咯基、噻吩基、呋喃基、咪唑基、吡唑基、噻唑基、噁唑基、三唑基、四唑基、吡啶基、嘧啶基、哒嗪基、吡嗪基、吡喃基，或为以下基团：
    Figure PCTCN2018092824-appb-100003
  8. 根据权利要求1、6或7所述的应用，其特征在于，所述Ar 2中，取代基独立的选自氢，卤素，硝基，羟基，氰基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基和C6～C10芳基中的一种或多种。
  9. 根据权利要求1所述的应用，其特征在于，所述NLRP3炎症小体抑制剂用于治疗、减轻或预防NLRP3炎症小体引发的炎症性疾病。
  10. 根据权利要求9所述的应用，其特征在于，所述炎症性疾病包括：
    II型糖尿病、动脉粥样硬化、脂肪肝、代谢综合征、感染引起的急、慢性组织损伤、痛风、关节炎、肠炎、肝炎、腹膜炎、硅肺、紫外线诱导的皮肤晒伤、接触性超敏反应、脓毒血症、肿瘤、神经退行性疾病、多发性硬化症和穆-韦氏综合征中的一种或多种。
  11. 一种噻唑啉酮杂环化合物，其特征在于，具有式Ⅰ或式Ⅱ所示结构或其异构体、前药、药学上可接受的溶剂化物或盐：
    Figure PCTCN2018092824-appb-100004
    其中，
    R 1，R 2，R 3，R 4和R 5各自独立地选自：
    氢，卤素，硝基，羟基，氰基，苯基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基；
    R 6，R 7，R 8，R 9和R 10各自独立地选自：
    氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基、C1-C6含氧烷基、C1-C6烷氧基、C3-C7环烷基；取代或非取代的甲酰基、磺酰基、磺酰基氨基；
    X 1为O，X 2为S，或X 1为S，X 2为O时，n为1～6的任意整数；
    X 1和X 2同时为O或S时，n为0～6的任意整数；
    Q为C或N；
    Q 1、Q 2、Q 3、Q 4独立的选自-C-R 1，-C-R 2，-C-R 3，-C-R 4、-C-R 5、N，-N-R 1，S或O；
    Ar 2为取代或非取代芳基、杂芳基或稠环基；
    m为0或1；
    其中，所述噻唑啉酮杂环化合物不包括以下化合物：
    Figure PCTCN2018092824-appb-100005
  12. 根据权利要求11所述的噻唑啉酮杂环化合物，其特征在于，所述R 1，R 2，R 3，R 4和R 5各自独立地选自：
    氢，卤素，硝基，羟基，氰基，苯基；氨基，甲基氨基，N,N-二甲基氨基，乙基氨基，N,N-二乙基氨基，异丙基氨基，N,N-二异丙基氨基，环丙基氨基，环丁基氨基，环戊基氨基，环己基氨基，乙酰基氨基，N,N-二乙酰基氨基，2-N,N-二甲基氨基乙基氨基，2-羟基乙基氨基，甲磺酰基氨基，N,N-二甲磺酰基氨基，甲磺酰基，对甲苯磺酰基；C1-C6烷基，C1-C6含氟烷基，C1-C6含氧烷基，C1-C6烷氧基，C1～C6含氟烷氧基或C3-C7环烷基。
  13. 根据权利要求11所述的噻唑啉酮杂环化合物，其特征在于，所述R 6，R 7，R 8，R 9和R 10各自独立地选自：
    氢，卤素，硝基，羟基，巯基，氨基，氰基，四唑基；取代或非取代的C1-C6烷基或C1-C6含氧烷基；氨基甲酰基，甲胺基甲酰基，乙胺基甲酰基，丙胺基甲酰基，异丙胺基甲酰基，N,O-二甲羟胺基甲酰基，环丙基胺基甲酰基，环丁基胺基甲酰基，环戊基胺基甲酰基，哌啶基-1-甲酰基，4-羟基哌啶基-1-甲酰基，4-N,N-二甲基哌啶基-1-甲酰基，4-N,N-二乙基哌啶基-1-甲酰基，四氢吡咯基-1-甲酰基，3-N,N-二甲基四氢吡咯基-1-甲酰基，3-N,N-二乙基四氢吡咯基-1-甲酰基，哌嗪基-1-甲酰基，N-甲基哌嗪基-1-甲酰基，N-乙基哌嗪基-1-甲酰基，N-乙酰基哌嗪基-1-甲酰基，N-叔丁氧甲酰基哌嗪基-1-甲酰基，N-(2-羟基乙基)哌嗪基-1-甲酰基，N-(2-氰基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二甲基乙基)哌嗪基-1-甲酰基，N-(2-N,N-二乙基乙基)哌嗪基-1-甲酰基，N-(3-羟基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二甲基丙基)哌嗪基-1-甲酰基，N-(3-N,N-二乙基丙基)哌嗪基-1-甲酰基，吗啡啉基-1-甲酰基，3,5-二甲基吗啡啉基-1-甲酰基，4-(N-甲基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙基-1-哌嗪基)哌啶基-1-甲酰基，4-(N-乙酰基-1-哌嗪基)哌啶基-1-甲酰基，N-(N-甲基-4-哌啶基)哌嗪基-1-甲酰基，2-羟基乙胺基甲酰基，2-乙酰基羟基乙胺基甲酰基，苯胺基甲酰基，苄基甲酰基，苄基氨基甲酰基，3-甲氧基苄基甲酰基，3-甲氧基苄基氨基甲酰基；羟基甲酰基，甲氧基甲酰基，乙氧基甲酰基，丙氧基甲酰基，异丙氧基甲酰基，正丁氧基甲酰基，异丁氧基甲酰基，叔丁氧基甲酰基，氨基磺酰基、C1～C10烷基磺酰基，C1～C10烷氨基磺酰基，C3～C10环烷基氨基磺酰基，C6～C12芳基氨基磺酰基，C5～C12杂芳基氨基磺酰基，或C1～C10磺酰基氨基。
  14. 根据权利要求13所述的噻唑啉酮杂环化合物，其特征在于，所述C1～C10烷基磺酰基为甲磺酰基，乙磺酰基，丙磺酰基，异丙磺酰基，丁磺酰基或异丁磺酰基；
    所述C1～C10烷氨基磺酰基为N-甲基氨基磺酰基，N-乙基氨基磺酰基，N-丙基氨基磺酰基，N-异丙基氨基磺酰基，N-丁基氨基磺酰基，N-异丁基氨基磺酰基，N,N-二甲基氨基磺酰基或N,N-二乙基氨基磺酰基；
    所述C3～C10环烷基氨基磺酰基为N-环丙基氨基磺酰基，N-环丁基氨基磺酰基，N-环戊基氨基磺酰基或N-环己基氨基磺酰基；
    所述C6～C12芳基氨基磺酰基为N-苯基氨基磺酰基；
    所述C5～C12杂芳基氨基磺酰基为N-吡啶基氨基磺酰基，N-噻吩基氨基磺酰基，N-呋喃基氨基磺酰基或N-吡咯基氨基磺酰基；
    所述C1～C10磺酰基氨基为甲磺酰氨基，乙磺酰氨基，丙磺酰氨基，异丙磺酰氨基，丁磺酰氨基，异丁磺酰氨基，磺酸基，氨基磺酰基。
  15. 根据权利要求11所述的噻唑啉酮杂环化合物，其特征在于，所述式Ⅱ具有以下式Ⅱ-a或式Ⅱ-b结构：
    Figure PCTCN2018092824-appb-100006
  16. 根据权利要求11所述的噻唑啉酮杂环化合物，其特征在于，所述Ar 2为取代或非取代苯基、五元或六元杂芳基、或者为取代或非取代苯基与取代或非取代芳基、杂芳基、杂环基稠合形成的稠环基团。
  17. 根据权利要求16所述的噻唑啉酮杂环化合物，其特征在于，所述Ar 2为取代或非取代苯基、吡咯基、噻吩基、呋喃基、咪唑基、吡唑基、噻唑基、噁唑基、三唑基、四唑基、吡啶基、嘧啶基、哒嗪基、吡嗪基、吡喃基，或为以下基团：
    Figure PCTCN2018092824-appb-100007
  18. 根据权利要求11、16或17所述的噻唑啉酮杂环化合物，其特征在于，所述Ar 2中，取代基独立的选自氢，卤素，硝基，羟基，氰基，取代或非取代的氨基，甲磺酰基，对甲苯磺酰基或者取代或非取代的C1-C6含氧烷基、C1-C6烷氧基、C1-C6烷基、C3-C7环烷基和C6～C10芳基中的一种或多种。
  19. 根据权利要求11所述的噻唑啉酮杂环化合物，其特征在于，当n＝1，Q为C时，R 1，R 2，R 3，R 4和R 5不同时为H。
  20. 根据权利要求1所述的应用或权利要求11所述的噻唑啉酮杂环化合物，其特征在于，所述噻唑啉酮杂环化合物具有以下任一结构或其顺反异构体：
    Figure PCTCN2018092824-appb-100008
    Figure PCTCN2018092824-appb-100009
    Figure PCTCN2018092824-appb-100010
    Figure PCTCN2018092824-appb-100011
    Figure PCTCN2018092824-appb-100012
    Figure PCTCN2018092824-appb-100013
    Figure PCTCN2018092824-appb-100014
    Figure PCTCN2018092824-appb-100015
    Figure PCTCN2018092824-appb-100016
    Figure PCTCN2018092824-appb-100017
  21. 权利要求11～20任一项所述的噻唑啉酮杂环化合物的制备方法，其特征在于，包括以下步骤：
    A)式a所示的芳香胺类化合物与二氯硫化碳，在碱性条件下进行反应，得到式b所示化合物；
    B)式b所示化合物进行碱催化的环化反应，得到式c所示化合物；
    C)式c所示化合物与式d所示化合物进行加成反应，得到式Ⅰ-1所示化合物；
    Figure PCTCN2018092824-appb-100018
    或者包括以下步骤：
    A＇)式a所示的芳香胺类化合物与三光气，在碱性条件下进行羰基化反应，得到式b＇所示化合物；
    B＇)式b＇所示化合物进行碱催化的环化反应，得到式c＇所示化合物；
    C＇)式c＇所示化合物与式d所示化合物进行加成反应，得到式Ⅱ所示化合物；
    Figure PCTCN2018092824-appb-100019
  22. 一种药物组合物，包括权利要求11～20任意一项所述的噻唑啉酮杂环化合物或其盐或权利要求21所述的制备方法制备的噻唑啉酮杂环化合物或其盐，以及药学上可接受的载体，赋形剂，稀释剂，辅剂，媒介物或它们的组合。
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