WO2023137885A1 - 一种母核为吡啶并咪唑的化合物及其制备方法和应用 - Google Patents

一种母核为吡啶并咪唑的化合物及其制备方法和应用 Download PDF

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WO2023137885A1
WO2023137885A1 PCT/CN2022/086781 CN2022086781W WO2023137885A1 WO 2023137885 A1 WO2023137885 A1 WO 2023137885A1 CN 2022086781 W CN2022086781 W CN 2022086781W WO 2023137885 A1 WO2023137885 A1 WO 2023137885A1
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compound
alkyl
trifluoromethyl
nitro
halogen
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徐莉莉
狄斌
李冰艳
江程
李沛
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中国药科大学
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    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
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Definitions

  • the invention belongs to the field of medicine, and in particular relates to a compound whose parent nucleus is pyridoimidazole, a preparation method and application thereof.
  • the inflammasome is a complex of proteins that is activated when cells are infected or stimulated.
  • the innate immune system recognizes pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP) through pattern recognition receptors (PRR).
  • PRR pattern recognition receptors
  • a variety of PRRs have been discovered, including the nucleotide-binding oligomerization domain (NOD)-like receptor (NOD-like receptor, NLR).
  • NOD nucleotide-binding oligomerization domain
  • NLR nuclear factor receptor
  • the NLR family includes many members, among which NOD like receptor containing pyrin domain 3 (NLRP3) is the most thoroughly studied.
  • NLRP3 contains three domains: pyrin domain (PYD), nucleotide binding domain (nucleoside triphosphate domain, NACHT), and leucine-rich repeat domain (LRR).
  • PYD pyrin domain
  • NACHT nucleoside triphosphate domain
  • LRR leucine-rich repeat domain
  • NLRP3 inflammasome The activation of NLRP3 inflammasome can be divided into two stages: initiation and activation.
  • microbial components or endogenous cytokines stimulate Toll-like receptors (Toll-like receptors, TLRs), activate the transcription factor NF- ⁇ B, and NF- ⁇ B up-regulates the expression of NLRP3 and pro-IL-1 ⁇ .
  • TLRs Toll-like receptors
  • NF- ⁇ B transcription factor- ⁇ B
  • NF- ⁇ B up-regulates the expression of NLRP3 and pro-IL-1 ⁇ .
  • stimuli such as ATP, K + ionophores, heme, pathogen-associated RNA, bacteria, and mycotoxins activate NLRP3, causing the assembly of the NLRP3 inflammasome.
  • NLRP3 and its inflammasome are potential targets for the treatment of a variety of diseases, including rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, inflammatory bowel disease, gout, atherosclerosis, nonalcoholic fatty liver disease, cryopyrin-related periodic syndrome, chronic kidney disease, diabetes, Parkinson's disease, multiple sclerosis, Alzheimer's disease, Huntington's disease, systemic lupus erythematosus, etc.
  • the abnormal increase in the level of NLRP3 inflammasome may lead to the above diseases. Therefore, the development of NLRP3 inhibitors is expected to bring breakthroughs in solving the above diseases.
  • NLRP3 inflammasome For diseases related to NLRP3 inflammasome, some drugs have been used clinically, such as anakinra, canakinumab, and rilonacept. But these drugs all target IL-1 or the IL-1 receptor downstream in the pathway, which creates two problems. On the one hand, there is more than one inflammatory factor that causes these diseases, and the disease cannot be fundamentally eliminated; on the other hand, there is more than one pathway that produces this inflammatory factor, so it will cause immunosuppression. Therefore, inhibitors specifically targeting the NLRP3 signaling pathway are expected to avoid the above problems. At present, many small molecules targeting the NLRP3 signaling pathway have been discovered.
  • the object of the present invention is to provide a compound whose parent nucleus is pyridimidazole and its preparation method and application.
  • the present invention relates to the discovery of a new class of NLRP3 inflammasome inhibitors with pyridimidazole as the mother nucleus, and the application of such compounds in inhibiting the secretion of IL-1 ⁇ and treating related diseases caused by abnormal activation of NLRP3 inflammasomes; the present invention also includes the preparation process of such inhibitors, the pharmacophore composition of the novel inhibitors, and their application in the field of anti-inflammation.
  • the present invention is based on computer-aided drug design.
  • a series of derivatives with pyridimidazole as the mother nucleus were designed and synthesized in the previous research. Through further pharmacological activity screening, it was found that the derivatives with pyridimidazole as the mother nucleus have good activity in inhibiting the release of IL-1 ⁇ , and can be used as NLRP3 inflammasome inhibitors.
  • R 1 is C 1 -C 30 alkyl, aryl, heteroaryl, C 1 -C 30 alkylaryl, C 1 -C 30 alkyl heteroaryl or C 1 -C 12 heterocycle; the C 1 -C 12 heterocycle is substituted by one or more of the following substituents which are the same or different: C 1 -C 30 alkyl, alkoxy, alkylamino, amido, nitro, halogen, benzyl, nitrile group, amino, carboxyl, carbonyl, trifluoromethyl;
  • R 2 is hydrogen, C 1 -C 30 alkyl, aryl, heteroaryl, C 1 -C 30 alkylaryl, C 1 -C 30 alkyl heteroaryl or C 1 -C 12 heterocycle; the C 1 -C 12 heterocycle is substituted by one or more of the same or different substituents below: C 1 -C 30 alkyl, alkoxy, alkylamino, amido, nitro, halogen, benzyl, nitrile, Amino, carboxyl, carbonyl, trifluoromethyl.
  • R 1 is Wherein R 3 -R 7 are independently H, alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, amino, nitro, hydroxyl, ester, trifluoromethyl;
  • R 3 -R 7 are alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, amino, nitro, hydroxyl, ester, trifluoromethyl, and the rest are H;
  • R 3 -R 7 are alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, amino, nitro, hydroxyl, ester, trifluoromethyl, and the rest are H;
  • R1 is Wherein Y 1 -Y 5 are independently CH, N, R 8 is H, alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, amino, nitro, hydroxyl, ester, trifluoromethyl;
  • one or two of Y 1 -Y 3 are N, the rest are CH, R 8 is H, alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, amino, nitro, hydroxyl, ester, trifluoromethyl;
  • one of Y 1 -Y 3 is N, the rest are CH, R 8 is H, alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, amino, nitro, hydroxyl, ester, trifluoromethyl;
  • one of Y 1 -Y 3 is N, the rest are CH, R 8 is H, F;
  • R 1 is C 1 -C 30 alkyl, haloalkyl, cycloalkyl, halocycloalkyl, heterocycloalkyl;
  • R 1 is C 1 -C 10 alkyl, haloalkyl, cycloalkyl, halocycloalkyl, heterocycloalkyl;
  • R 1 is C 3 -C 6 alkyl, cycloalkyl
  • R 1 is C 3 -C 6 cycloalkyl
  • the R2 is wherein Z 1 -Z 2 are independently CH, N, R 9 -R 10 have and only one is alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, imino, nitro, hydroxyl, ester, trifluoromethyl, and the other is H;
  • one and only one of Z 1 -Z 2 is N, the other is CH, one and only one of R 9 -R 10 is alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, imino, nitro, hydroxyl, ester, trifluoromethyl, and the other is H;
  • Z 1 -Z 2 is CH, R 9 is H, R 10 is alkyl, alkoxy, halogen, cyano, carbonyl, carboxyl, acyl, sulfonyl, imino, nitro, hydroxyl, ester, trifluoromethyl;
  • Z 1 -Z 2 is CH
  • R 9 is H
  • R 10 is acyl group, imino group.
  • a kind of preparation method that parent nucleus is the compound of pyridoimidazole comprises the steps:
  • Step 1 Compound II forms a ring to obtain Compound III;
  • Step 2 subjecting the compound III to a nucleophilic substitution reaction under alkaline conditions to obtain compound IV;
  • Step 3 subjecting the compound IV to a nucleophilic substitution reaction under basic conditions to obtain compound I;
  • R 1 is C 1 -C 30 alkyl, aryl, heteroaryl, C 1 -C 30 alkylaryl, C 1 -C 30 alkyl heteroaryl or C 1 -C 12 heterocycle; the C 1 -C 12 heterocycle is substituted by one or more of the following substituents which are the same or different: C 1 -C 30 alkyl, alkoxy, alkylamino, amido, nitro, halogen, benzyl, nitrile group, amino, carboxyl, carbonyl, trifluoromethyl;
  • R 2 is hydrogen, C 1 -C 30 alkyl, aryl, heteroaryl, C 1 -C 30 alkylaryl, C 1 -C 30 alkyl heteroaryl or C 1 -C 12 heterocycle; the C 1 -C 12 heterocycle is substituted by one or more of the same or different substituents below: C 1 -C 30 alkyl, alkoxy, alkylamino, amido, nitro, halogen, benzyl, nitrile, Amino, carboxyl, carbonyl, trifluoromethyl.
  • the step 1 is: i. adding compound II and carbon disulfide to ethanol, and heating to reflux for 4 hours to obtain compound III;
  • the step 2 is: ii. adding compound III and anhydrous sodium carbonate to N,N-dimethylformamide, heating and reacting at 40°C for 1h; iii. slowly adding The reaction was continued for 4h to obtain compound IV.
  • the step 3 is, iv. dissolving compound IV in N,N-dimethylformamide, slowly adding sodium hydride while stirring, and reacting at room temperature for 0.5h; v. slowly adding The reaction was continued for 4h to obtain compound I.
  • a pharmaceutical composition comprising: a therapeutically effective amount of the compound whose core is pyridimidazole according to claim 1, a pharmaceutically acceptable salt, hydrate or solvate, and a pharmaceutically acceptable carrier.
  • the present invention also protects the above-mentioned compound whose core is pyridimidazole or its pharmaceutically acceptable salt, or the application of the above-mentioned pharmaceutical composition in the preparation of NLRP3 inflammasome inhibitors and the preparation of medicines for treating inflammation-related diseases.
  • the inflammation-related diseases are selected from rheumatoid arthritis, rheumatoid arthritis, inflammatory bowel disease, gout, atherosclerosis, non-alcoholic fatty liver disease, cryopyrin-related periodic syndrome, chronic kidney disease, diabetes and neurodegenerative diseases.
  • the neurodegenerative disease is selected from Parkinson's disease, multiple sclerosis, Alzheimer's disease, and Huntington's disease.
  • the present invention also provides the application of the pyridimidazole compound in inhibiting the release of pro-inflammatory factor IL-1 ⁇ .
  • C 1 -C 30 alkyl refers to straight or branched hydrocarbon chains containing one to twelve carbon atoms, optionally substituted by C 1 -C 30 alkyl;
  • Substitution refers to the replacement of a hydrogen atom in a molecule with a different atom or molecule.
  • the minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix ( Ca - Cb )alkyl indicates any alkyl group containing "a" to "b” carbon atoms.
  • C 1 -C 30 alkyl refers to an alkyl group containing 1 to 30 carbon atoms.
  • Aryl means an aryl carbocyclic group with a single ring, multiple rings or multiple condensed rings, at least one of which is aromatic, C 1 ⁇ C 30 alkyl, alkoxy, alkylamino, amido, nitro, halogen, benzyl, nitrile, amino, carboxyl, carbonyl, trifluoromethyl, etc., mono-, di-, tri-, tetra-, penta-substituted.
  • Heteroaryl represents one or more 5-, 6- or 7-membered aromatic ring systems containing at least one heteroatom selected from nitrogen, oxygen or sulfur, which may be mono-, di-, tri-, tetra-, penta-substituted by halogen, nitro, trifluoromethyl, nitrile, C 1 -C 12 alkyl, etc.
  • C 1 -C 30 alkylaryl means that an alkyl group containing C 1 -C 30 is connected to an aryl group, and is optionally substituted by a C 1 -C 30 alkyl group.
  • C 1 -C 30 alkylheteroaryl means that an alkyl group containing C 1 -C 30 is connected to a heteroaryl group, optionally substituted by C 1 -C 30 .
  • Heterocycles refer to stable monocyclic, bicyclic or tricyclic rings containing heteroatoms or heteroatom groups, which may be saturated, partially unsaturated or unsaturated.
  • Alkoxy refers to the group formed by the loss of one hydrogen atom from the corresponding alcohol.
  • Halogen refers to fluorine, chlorine, bromine and iodine.
  • the compound of the present invention refers to the compound represented by formula I.
  • the term also includes various crystalline forms of the compound of formula I, pharmaceutically acceptable salts, hydrates or solvates and pharmaceutically acceptable carriers.
  • a pharmaceutically acceptable carrier refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic or side effects on the host or patient.
  • the compound of the present invention can be prepared into common pharmaceutical preparations by adding pharmaceutically acceptable carriers, such as tablets, capsules, powders, syrups, suspensions, injections, and common pharmaceutical excipients such as spices, sweeteners, liquid or solid fillers or diluents can be added.
  • pharmaceutically acceptable carriers such as tablets, capsules, powders, syrups, suspensions, injections, and common pharmaceutical excipients such as spices, sweeteners, liquid or solid fillers or diluents can be added.
  • the clinical administration of the compound of the present invention can be oral administration, injection and the like.
  • “Therapeutically effective amount” or “effective amount” refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect.
  • the invention provides a compound with pyridimidazole as the core, as shown in formula I.
  • This type of compound has a novel structure and a simple preparation method. By specifically inhibiting the activation of NLRP3 inflammasome, it has significant inhibitory activity on the release of interleukin IL-1 ⁇ , thereby reducing inflammatory damage, improving the inflammatory microenvironment, and has potential anti-inflammatory activity. At the same time, it has no obvious toxicity to THP-1 cells.
  • It can be used for the preparation of anti-inflammatory drugs and for inflammatory damage in many inflammation-related diseases, including cryopyrin-related periodic syndrome, inflammatory bowel disease, chronic obstructive pulmonary disease, diabetes, rheumatoid arthritis, rheumatoid arthritis, gout, non-alcoholic fatty liver disease, chronic kidney disease, atherosclerosis, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis.
  • inflammation-related diseases including cryopyrin-related periodic syndrome, inflammatory bowel disease, chronic obstructive pulmonary disease, diabetes, rheumatoid arthritis, rheumatoid arthritis, gout, non-alcoholic fatty liver disease, chronic kidney disease, atherosclerosis, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis.
  • Fig. 1 is the Western Blot test result of compound I-20 obtained in Example 45.
  • reaction solution was cooled to room temperature, poured into a separatory funnel, diluted with a large amount of water, extracted with n-butanol, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, separated and purified by dichloromethane/methanol column chromatography to obtain 519.6 mg of a white solid, yield: 65.11%.
  • Embodiment 7 synthetic compound IV-6
  • Embodiment 21 synthetic compound IV-20
  • reaction solution was cooled to room temperature, poured into a separatory funnel, diluted with a large amount of water, extracted with n-butanol, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. It was separated and purified by dichloromethane/methanol column chromatography to obtain 418.7 mg of white solid with a yield of 56.56%.
  • the synthesis method is the same as in Example 3, with compound IV-8 (400mg, 1.47mmol), sodium hydride (61.91mg, 1.55mmol), 4-(bromomethyl)-N-(3-methoxypropyl base) benzamide (421.86mg, 1.47mmol) as raw material to obtain 405.6mg of light yellow solid with a yield of 57.73%.
  • the synthesis method is the same as in Example 3, with compound IV-9 (400mg, 1.29mmol), sodium hydride (54.31mg, 1.36mmol), 4-(bromomethyl)-N-(3- Methoxypropyl)benzamide (370.07mg, 1.29mmol) was used as starting material to obtain 379.4mg of white solid with a yield of 57.02%.
  • the synthesis method is the same as in Example 3, with compound IV-10 (400mg, 1.57mmol), sodium hydride (65.79mg, 1.64mmol), 4-(bromomethyl)-N-(3-methyl Oxypropyl)benzamide (448.30mg, 1.57mmol) was used as starting material to obtain 388.3mg of white solid with a yield of 53.82%.
  • the synthesis method is the same as in Example 3, with compound IV-12 (400mg, 1.57mmol), sodium hydride (65.79mg, 1.64mmol), 4-(bromomethyl)-N-(3-methyl Oxypropyl)benzamide (448.3mg, 1.57mmol) was used as starting material to obtain 401.8mg of white solid with a yield of 55.69%.
  • the synthesis method is the same as in Example 3, with compound IV-13 (400mg, 1.40mmol), sodium hydride (58.67mg, 1.47mmol), 4-(bromomethyl)-N-(3-methyl Oxypropyl) benzamide (399.80 mg, 1.40 mmol) was used as starting material to obtain 397.1 mg of light yellow solid with a yield of 58.72%.
  • the synthesis method is the same as in Example 3, with compound IV-17 (400mg, 1.65mmol), sodium hydride (69.33mg, 1.73mmol), 4-(bromomethyl)-N-(3- Methoxypropyl)benzamide (472.42mg, 1.65mmol) was used as starting material to obtain 297.4mg of yellow solid with a yield of 40.25%.
  • the synthesis method is the same as in Example 3, with compound IV-18 (400mg, 1.65mmol), sodium hydride (69.33mg, 1.73mmol), 4-(bromomethyl)-N-(3- Methoxypropyl)benzamide (472.42mg, 1.65mmol) was used as starting material to obtain 278.4mg of light yellow solid with a yield of 37.68%.
  • N-(3-methoxypropyl)-4-((2-((pyridin-2-ylmethyl)thio)-3-hydrogen-imidazol[4,5-c]pyridin-3-yl)methyl)benzamide (I-19).
  • the synthesis method is the same as in Example 3, with compound IV-19 (400mg, 1.65mmol), sodium hydride (69.33mg, 1.73mmol), 4-(bromomethyl)-N-(3- Methoxypropyl)benzamide (472.42mg, 1.65mmol) was used as starting material to obtain 296.7mg of yellow solid with a yield of 40.16%.
  • N-(3-methoxypropyl)-4-((2-((3-methyl-2-en-1-yl)thio)-3-hydrogen-imidazo[4,5-c]pyridin-3-yl)methyl)benzamide (I-24).
  • the synthesis method is the same as in Example 3, using compound IV-24 (400mg, 1.82mmol), sodium hydride (76.60mg, 1.92mmol), 4-(bromomethyl) -N-(3-Methoxypropyl)benzamide (521.95 mg, 1.82 mmol) was used as starting material to obtain 517.6 mg of white solid with a yield of 66.84%.
  • the secretion of IL-1 ⁇ in the cell culture supernatant was detected by double antibody sandwich ELISA. After the freshly collected serum samples were allowed to stand at room temperature for 20 minutes, they were centrifuged at 3000g for 10 minutes at 4°C, and the supernatant was ready for detection and analysis. The samples and standards were added to the antibody-coated 96-well plate, and the biotinylated antibody was added, incubated on a shaker at 37°C for 1 hour, and the plate was washed 5 times. Add enzyme-binding working solution, incubate at 37°C in the dark for 30 min, and wash the plate 5 times. Add a chromogenic substrate and incubate at 37°C for 15 minutes in the dark.
  • the inhibitory rate of the compound to interleukin IL-1 ⁇ is calculated as follows:
  • Inhibition rate (%) 1-(drug well OD value-blank well OD value)/(drug well OD value-blank well OD value), the cell well without drug and inducing factor is used as blank control, and the cell well with no drug added and LPS and ATP added is a negative control.
  • MCC950 is used as a positive control
  • DMSO group is used as a negative control
  • cell lysate is used as a blank background
  • 3 duplicate wells are set.
  • the results of the compound's inhibitory rate on IL-1 ⁇ are expressed as the average value ⁇ SEM of the data of each group.
  • Table 1 shows the inhibition rate of some compounds of the present invention on interleukin IL-1 ⁇ .
  • the compounds of the present invention have inhibitory activity on interleukin IL-1 ⁇ , which provides a basis for the development of NLRP3 inhibitors with high efficiency, safety, novel structure and strong specificity, and drugs for the treatment of NLRP3-mediated inflammation-related diseases such as rheumatoid arthritis, rheumatoid arthritis, inflammatory bowel disease, gout, atherosclerosis, non-alcoholic fatty liver disease, cryopyrin-related periodic syndrome, chronic kidney disease, diabetes and neurodegenerative diseases.
  • NLRP3-mediated inflammation-related diseases such as rheumatoid arthritis, rheumatoid arthritis, inflammatory bowel disease, gout, atherosclerosis, non-alcoholic fatty liver disease, cryopyrin-related periodic syndrome, chronic kidney disease, diabetes and neurodegenerative diseases.
  • MTT phosphate-buffered saline
  • PBS phosphate-buffered saline
  • DMSO phosphate-buffered saline
  • Inhibition rate (%) 1-(OD value of drug well-OD value of blank well)/(OD value of drug well-OD value of blank well), and the cell well without drug and inducing factor was used as blank control.
  • the cytotoxicity evaluation of some compounds of the present invention is shown in Table 2.
  • Cell protein preparation Aspirate the cell supernatant from the six-well plate, rinse twice with ice-cold PBS buffer, and then use a cell scraper to scrape the cells from the six-well plate and collect them. Centrifuge at 1000g for 5min, add RIPA protein lysate at a ratio of 1:5 (volume of somatic cells: volume of lysate), lyse on ice for 60min, centrifuge at 16000g for 10min, absorb the supernatant to obtain the whole cell protein, take 4 ⁇ L to measure the protein concentration by BCA method, add the remaining protein supernatant to 5 ⁇ loading buffer by volume, denature in a boiling water bath for 10min, and store at -20°C after aliquoting.
  • the western blot experiment shows that the compound I-20 of the present invention can significantly inhibit the expression of NLRP3 protein in a concentration-dependent manner.

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Abstract

本发明公开了一种母核为吡啶并咪唑的化合物及其制备方法和应用。本发明提供的母核为吡啶并咪唑的化合物结构新颖，制备方法简单，可通过特异性抑制NLRP3炎症小体的活化，减少白介素IL-1β的成熟分泌，从而减轻炎性损伤，改善炎症微环境，具有显著的抗炎活性，同时对THP-1细胞无明显毒性。本发明化合物可制备成抗炎药物并用于炎症相关疾病的治疗，包括风湿性关节炎，类风湿性关节炎，炎性肠道疾病，痛风，动脉粥样硬化，非酒精性脂肪肝病，冷吡啉相关周期性综合征，慢性肾脏疾病，糖尿病和神经退行性疾病等。

Description

一种母核为吡啶并咪唑的化合物及其制备方法和应用 技术领域
本发明属于医药领域，具体涉及一种母核为吡啶并咪唑的化合物及其制备方法和应用。
背景技术
炎症小体是一种由多种蛋白组成的复合物，当细胞受到感染或刺激时可被激活。先天免疫系统通过模式识别受体(pattern recognition receptor，PRR)来识别病原体相关分子模式(pathogen associated molecular patterns，PAMP)或损伤相关的分子模式(damage associated molecular patterns，DAMP)。人们已经发现多种PRR，其中就包括核苷酸结合寡聚域(nucleotide-binding oligomerization domain，NOD)样受体(NOD-like receptor，NLR)。NLR家族又包括众多成员，其中，包含吡啶结构域3的NOD样受体(NOD like receptor containing pyrin domain 3，NLRP3)的研究最为透彻。
NLRP3包含三个结构域：热蛋白结构域(pyrin domain,PYD)，核苷酸结合结构域(nucleoside triphosphatase domain,NACHT)，以及亮氨酸富集结构域(leucine-rich repeat,LRR)。当NLRP3被激活后，随即发生寡聚，通过PYD的同型相互作用引起ASC的寡聚，再通过ASC的CARD结构域引起procaspase-1的聚集，并将其水解为成熟的caspase-1，随后caspase-1再将pro-IL-1β和pro-IL-18水解为成熟的IL-1β和IL-18，引发炎症风暴，导致细胞焦亡。NLRP3炎症小体的活化可以分启动和激活两个阶段。在启动阶段，微生物成分或内源性细胞因子刺激Toll样受体(Toll-like receptor，TLR)，激活转录因子NF-κB，NF-κB上调NLRP3和pro-IL-1β的表达。在激活阶段，ATP、K +离子载体、血红素、病原体相关RNA、细菌和真菌毒素等刺激物激活NLRP3，引起NLRP3炎症小体的组装。
NLRP3及其炎症小体是治疗多种疾病的潜在靶点，包括风湿性关节炎，类风湿性关节炎，通风性关节炎，炎性肠道疾病，痛风，动脉粥样硬化，非酒精性脂肪肝病，冷吡啉相关周期性综合征，慢性肾脏疾病，糖尿病，帕金森症，多发性硬化症，阿尔兹海默症，亨廷顿病，系统性红斑狼疮等。NLRP3炎症小体水平的异常升高，有可能导致以上疾病，因此，开发出NLRP3抑制剂有望为解决以上疾病带来突破。
针对NLRP3炎症小体相关疾病，目前已经有一些药物用于临床，如阿那白滞素(anakinra)，卡那津单抗(canakinumab)，列洛西普(rilonacept)。但这些药物都针对通路下游的IL-1或IL-1受体，这会产生两个问题。一方面，导致这些疾病的炎症因子不止这一种，无法从根本上消除疾病；另一方面，产生这种炎症因子的通路不止这一条，因此会造成免疫抑制。所以，特异性针对NLRP3信 号通路的抑制剂有望避免上述问题。目前，人们已经发现多个针对NLRP3信号通路的小分子。16673-34-0通过改变NLRP3蛋白的构象或抑制NLRP3与ASC的结合来抑制NLRP3炎症小体的形成；Bay 11-7082可抑制NLRP3的ATPase活性和ASC寡聚活性，此外还可抑制IKκB激酶从而抑制NF-κB通路的激活；β-羟基丁酸(BHB)通过抑制K +外排从而减少ASC的寡聚，抑制NLRP3炎症小体的形成，最终降低IL-1β和IL-18的产生；CY-09可与NLRP3作用，阻止其与ATP的结合，从而抑制炎症小体的形成；FC11A-2可抑制casepase-1的成熟，从而抑制IL-1β的生成；格列本脲可抑制ATP敏感的K +通道，同时抑制ASC的聚集；异甘草素通过抑制TLR4/MD-2复合物阻断NF-κB通路；JC124可降低NLRP3、ASC、caspase-1、pro-IL-1β的表达，抑制炎症小体的生成；MCC950是目前最高效的NLRP3抑制剂，可直接与NLRP3的NACHT结构域作用，阻断ATP水解而激活NLRP3，达到抑制炎症小体生成的目的；MNS可直接与NLRP3结合，抑制其ATP酶活性；小白菊内酯(Parthenolide)可直接作用于NLRP3的ATP酶结构域，同时抑制casepase-1的激活。尽管已有不少化合物能抑制NLRP3通路，但有些是针对通路中下游的信号分子，不止影响这一条通路，有些虽针对NLRP3本身，但同时有其它生物活性，副作用巨大，有些针对NLRP3本身且选择性很好，比如MCC950，但因为肝毒性而无法成为药物。因此，发现新颖的靶向NLRP3炎症小体的特异性抑制剂，对于治疗NLRP3炎症小体相关的疾病更有研究意义。
发明内容
针对现有问题的不足，本发明的目的是提供一种母核为吡啶并咪唑的化合物及其制备方法和应用。本发明涉及以吡啶并咪唑为母核的一类新型NLRP3炎症小体抑制剂的发现，此类化合物在抑制IL-1β的分泌和治疗由NLRP3炎症小体异常激活引起的相关疾病的应用；本发明还包括此类抑制剂的制备过程和新型抑制剂的药效团组成以及在抗炎领域的应用。
本发明基于计算机辅助药物设计，在前期研究中设计并合成了一系列吡啶并咪唑为母核的衍生物，通过进一步药理活性筛选，发现吡啶并咪唑为母核的衍生物具有很好的抑制IL-1β释放的活性，可作为NLRP3炎症小体抑制剂。
本发明解决其技术问题采用的技术方案是：
一种母核为吡啶并咪唑的化合物，结构如式Ⅰ所示：
Figure PCTCN2022086781-appb-000001
式中，R 1为C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取 代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基；
R 2为氢、C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基。
在一些技术方案中：所述的R 1为
Figure PCTCN2022086781-appb-000002
其中R 3-R 7分别独立地为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
优选：R 3-R 7中有一个或两个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基，且其余为H；
进一步优选：R 3-R 7中有一个或两个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基，且其余为H；
或：所述的R 1为
Figure PCTCN2022086781-appb-000003
其中Y 1-Y 5分别独立地为CH、N，R 8为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
优选：Y 1-Y 3中有一个或两个为N，其余为CH，R 8为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
进一步优选：Y 1-Y 3中有一个为N，其余为CH，R 8为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
最优选：Y 1-Y 3中有一个为N，其余为CH，R 8为H、F；
或：所述的R 1为C 1-C 30烷基、卤代烷基、环烷基、卤代环烷基、杂环烷基；
优选：R 1为C 1-C 10烷基、卤代烷基、环烷基、卤代环烷基、杂环烷基；
进一步优选：R 1为C 3-C 6烷基、环烷基；
最优选：R 1为C 3-C 6环烷基、
Figure PCTCN2022086781-appb-000004
在一些技术方案中：所述的R 2为
Figure PCTCN2022086781-appb-000005
其中Z 1-Z 2分别独立地为CH、N，R 9-R 10有且仅有一个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、亚胺基、硝基、羟基、酯基、三氟甲基，且另一个为H；
优选：Z 1-Z 2有且仅有一个为N，另一个为CH，R 9-R 10有且仅有一个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、亚胺基、硝基、羟基、酯基、三氟甲基，且另一个为H；
进一步优选：Z 1-Z 2为CH，R 9为H，R 10为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、亚胺基、硝基、羟基、酯基、三氟甲基；
最优选：Z 1-Z 2为CH，R 9为H，R 10为酰基、亚胺基。
在一些具体的技术方案中，所述的化合物结构式如下：
Figure PCTCN2022086781-appb-000006
Figure PCTCN2022086781-appb-000007
Figure PCTCN2022086781-appb-000008
一种母核为吡啶并咪唑的化合物的制备方法，包括如下步骤：
步骤1，化合物Ⅱ成环，得到化合物Ⅲ；
步骤2，将所述化合物Ⅲ在碱性条件下发生亲核取代反应，得到化合物Ⅳ；
步骤3，将所述化合物Ⅳ在碱性条件下发生亲核取代反应，得到化合物I；
Figure PCTCN2022086781-appb-000009
式中，R 1为C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基；
R 2为氢、C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基。
作为本申请的优选技术方案，所述步骤1为，i.将化合物II与二硫化碳加入乙醇中，加热回流4h，得到化合物III；
作为本申请的优选技术方案，所述步骤2为，ii.将化合物III与无水碳酸钠加入N,N-二甲基甲酰胺中，40℃加热反应1h；iii.向反应液中缓慢加入
Figure PCTCN2022086781-appb-000010
继续反应4h得到化合物IV。
作为本申请的优选技术方案，所述步骤3为，iv.将化合物IV溶解于N,N-二甲基甲酰胺中，边搅边缓慢加入氢化钠，室温反应0.5h；v.向反应液中缓慢加入
Figure PCTCN2022086781-appb-000011
继续反应4h，得到化合物I。
一种药物组合物，包括：含有治疗有效量的权利要求1所述的母核为吡啶并咪唑的化合物、药学上可接受的盐、水合物或溶剂合物及药学上可接受的载体。
本发明还保护上述母核为吡啶并咪唑的化合物或者其药学上可接受的盐，或者上述的药物组合物在制备NLRP3炎症小体抑制剂以及在制备治疗炎症相关疾病的药物中的应用。
作为本申请的优选技术方案，所述炎症相关疾病选自风湿性关节炎，类风湿性关节炎，炎性肠道疾病，痛风，动脉粥样硬化，非酒精性脂肪肝病，冷吡啉相关周期性综合征，慢性肾脏疾病，糖尿病和神经退行性疾病。
作为本申请的优选技术方案，所述神经退行性疾病选自帕金森症，多发性硬化症，阿尔兹海默症，亨廷顿病。
本发明还提供了以吡啶并咪唑的化合物在抑制促炎因子IL-1β释放中的应用。
关于本发明的使用术语的定义：除非另有说明，本文中基团或者术语提供的初始定义适用于整篇说明书的该基团或者术语；对于本文没有具体定义的术语，应该根据公开内容和上下文，给出本领域技术人员能够给予它们的含义。
C 1-C 30烷基是指含有一个至十二个碳原子的直链或支链的烃链，可选地被C 1-C 30烷基取代；
取代是指分子中的氢原子被其它不同的原子或分子所替换。
碳氢基团中碳原子含量的最小值和最大值通过前缀表示，例如，前缀(C a～C b)烷基表明任何含“a”至“b”个碳原子的烷基。因此，例如，C 1-C 30烷基是指包含1～30个碳原子的烷基。
芳基表示芳基碳环基团，具有单一环、多个环或多个稠环，其中至少一个是芳族的，C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基等单-、二-、三-、四-、五-取代。
杂芳基表示一个或多个5-、6-或7-元的芳香族环系，至少含有一个选自氮、氧或硫的杂原子，它们可以被卤素、硝基、三氟甲基、腈基、C 1-C 12烷基等单-、二-、三-、四-、五-取代。
C 1-C 30烷基芳基表示含有C 1-C 30的烷基与芳基相连，可选地被C 1-C 30烷基取代。
C 1-C 30烷基杂芳基表示含有C 1-C 30的烷基与杂芳基相连，可选地被C 1-C 30取代。
杂环是指稳定的含杂原子或杂原子团的单环、双环或三环，它们可以是饱和的，部分不饱和或者不饱和的。
烷氧基是指相应的醇少掉一个氢原子而成的基团。
卤素是指氟、氯、溴和碘。
本发明化合物指式I所示的化合物。该术语还包括式I化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物及药学上可接受的载体。
许多化合物可以与溶剂形成复合物，在这种溶剂中，它们进行反应，或它们从其中沉淀或结晶出来，这些复合物被称为“溶剂合物”。例如与水形成的复合物被称为水合物。
药物上可接受的载体是指能够递送本发明有效量活性物质、不干扰活性物质的生物活性并且对宿主或者患者无毒副作用的任何制剂或载体介质。
本发明所述的化合物可以添加药学上可接受的载体制成常见的药用制剂，如片剂，胶囊，粉剂，糖浆，悬浮剂，针剂，可以加入香料，甜味剂，液体或固体填料或稀释剂等常用药用辅料。
本发明所述的化合物在临床上的给药方式可以采用口服、注射等方式。
“治疗有效量”或“有效量”是指无毒的但能达到预期效果的药物或药剂的足够用量。
本发明达到的有益效果：
本发明提供了一种吡啶并咪唑为母核的化合物，如式Ⅰ所示。此类化合物结构新颖，制备方法简单，通过特异性抑制NLRP3炎症小体的活化，对白介素IL-1β的释放具有显著的抑制活性，从而减轻炎性损伤，改善炎症微环境，具有潜在的抗炎活性，同时对THP-1细胞无明显毒性。可用于制备成抗炎药物，并用于许多炎症相关疾病的炎性损伤，这些疾病包括冷吡啉相关周期性综合征，炎性肠道疾病，慢性阻滞性肺部疾病，糖尿病，风湿性关节炎，类风湿性关节炎，痛风，非酒精性脂肪肝病，慢性肾脏疾病，动脉粥样硬化，神经退行性疾病如阿尔兹海默症，帕金森症，亨廷顿病，多发性硬化症。
附图说明
图1为实施例45所得化合物Ⅰ-20的免疫印迹试验(Western Blot)试验结果。
具体实施方式
下面通过具体实施方式对本发明做进一步详细说明，但是并不是对本发明的限制，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过市场购买的常规产品。
仪器和试剂：熔点采用WRS-2微机熔点仪测定，薄层色谱用硅胶GF254购于阿拉丁试剂公司(aladdin，上海晶纯生化科技股份有限公司)；柱色谱用硅胶FCP(200～300目)购于国药集团化学试剂有限公司；其他所用试剂和溶剂均为国产分析纯，根据需要经无水干燥处理后使用。
实施例1 合成化合物III
称量3,4-二氨基吡啶(8g,0.0733mol)于反应瓶，向其依次加入无水乙醇60ml，二硫化碳(15.9ml,0.264mol)，80℃回流反应4h后，TLC监测原料消失。将反应液冷却至室温，抽滤，用乙酸乙酯洗涤滤饼，干燥后得米白色固体10.2g，收率：92.0％。 1H NMR(300MHz,DMSO-d6)δ12.87(s,2H),8.37(d,J＝1.0Hz,1H),8.23(d,J＝5.4Hz,1H),7.16(dd,J＝5.4,1.0Hz,1H).
实施例2 合成化合物IV-1
2-(苄基硫)-3-氢-咪唑并[4,5-c]吡啶(IV-1)
称量化合物III(500mg,3.31mmol)于反应瓶，向其依次加入N,N-二甲基甲酰胺5ml，无水碳酸钠(701.04mg,6.61mmol)，40℃加热反应1h后，缓慢滴加溴苄(412μl,3.47mmol)，继续反应4h，TLC监测原料消失。将反应液冷却至室温，倒入分液漏斗，加大量水稀释，用正丁醇萃取，饱和氯化钠洗涤，无水硫酸钠干燥后，减压浓缩除去溶剂，用二氯甲烷/甲醇柱层析分离纯化，得到白色固体519.6mg，收率：65.11％。
2-(苄基硫)-3-氢-咪唑并[4,5-c]吡啶(IV-1).白色固体，收率：65.11％。 1H NMR(300MHz,DMSO-d6)δ8.75(s,1H),8.17(d,J＝5.7Hz,1H),7.50–7.42(m,3H),7.34–7.19(m,3H),4.60(s,2H).
实施例3 合成化合物IV-2
2-((2-氟苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-2).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，2-氟溴苄(419μl,3.47mmol)为原料，得到白色固体527.6mg，收率61.52％。 1H NMR(300MHz,DMSO-d6)δ13.18(s,1H),8.77(s,1H),8.18(d,J＝5.7Hz,1H),7.58(td,J＝7.7,1.8Hz,1H),7.50(d,J＝4.9Hz,1H),7.39–7.29(m,1H),7.27–7.18(m,1H),7.15(td,J＝7.4,1.3Hz,1H),4.64(s,2H).
实施例4 合成化合物IV-3
2-((3-氟苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-3).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，3-氟溴苄(426μl,3.47mmol)为原料，得到白色固体543.5mg，收率63.38％。 1H NMR(300MHz,DMSO-d6)δ8.77(d,J＝0.9Hz,1H),8.17(d,J＝5.7Hz,1H),7.47(dd,J＝5.7,0.9Hz,1H),7.35–7.26(m,3H),7.09–7.00(m,1H),4.61(s,2H).
实施例5 合成化合物IV-4
2-((4-氟苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-4).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-氟溴苄(418.89μl,3.47mmol)为原料，得到白色固体527.6mg，收率61.52％。 1H NMR(300MHz,DMSO-d6)δ13.10(s,1H),8.74(s,1H),8.16(d,J＝5.7Hz,1H),7.54–7.47(m,2H),7.46(d,J＝5.8Hz,1H),7.17–7.08(m,2H),4.58(s,2H).
实施例6 合成化合物IV-5
2-((4-氯苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-5).合成方法与实施例2 相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-氯溴苄(454μl,3.47mmol)为原料，得到白色固体461.7mg，收率50.60％。 1H NMR(300MHz,DMSO-d6)δ8.75(s,1H),8.18(d,J＝5.7Hz,1H),7.54–7.46(m,3H),7.38(d,J＝8.5Hz,2H),4.60(s,2H).
实施例7 合成化合物IV-6
2-((4-溴苯基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-6).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-溴溴苄(475μl,3.47mmol)为原料，得到白色固体639.4mg，收率60.38％。 1H NMR(300MHz,DMSO-d6)δ8.75(s,1H),8.18(d,J＝5.7Hz,1H),7.54–7.41(m,5H),4.58(s,2H).
实施例8 合成化合物IV-7
4-((3-氢-咪唑[4,5-c]吡啶-2-基)硫代)甲基)苯甲腈(IV-7).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-氰基溴苄(440μl,3.47mmol)为原料，得到白色固体598.3mg，收率67.93％。 1H NMR(300MHz,DMSO-d6)δ8.73(s,1H),8.15(d,J＝5.8Hz,1H),7.76(d,J＝8.2Hz,2H),7.66(d,J＝8.1Hz,2H),7.46(d,J＝5.7Hz,1H),4.66(s,2H).
实施例9 合成化合物IV-8
2-((4-硝基苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-8).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-硝基溴苄(454μl,3.47mmol)为原料，得到黄色固体611.2mg，收率64.55％。 1H NMR(300MHz,DMSO-d6)δ8.72(s,1H),8.19–8.10(m,3H),7.74(d,J＝8.7Hz,2H),7.47(d,J＝5.8Hz,1H),4.71(s,2H).
实施例10 合成化合物IV-9
2-((4-(三氟甲基)苄基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-9).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-三氟甲基溴苄(537μl,3.47mmol)为原料，得到白色固体652.3mg，收率63.77％。 1H NMR(300MHz,DMSO-d6)δ8.73(d,J＝0.9Hz,1H),8.15(d,J＝5.8Hz,1H),7.72–7.62(m,4H),7.47(dd,J＝5.8,0.9Hz,1H),4.67(s,2H).
实施例11 合成化合物IV-10
2-((4-甲基苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-10).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-甲基溴苄(485μl,3.47mmol)为原料，得到白色固体609.5mg，收率72.18％。 1H NMR(300MHz,DMSO-d6)δ8.74(d,J＝0.9Hz,1H),8.17(d,J＝5.7Hz,1H),7.45(dd,J＝5.6,1.0Hz,1H),7.33(d,J＝8.1Hz,2H),7.10(d,J＝7.8Hz,2H),4.55(s,2H),2.24(s,3H).
实施例12 合成化合物IV-11
2-((4-甲氧基苄基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-11).合成方法与实施 例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-甲氧基溴苄(506μl,3.47mmol)为原料，得到白色固体589.7mg，收率65.72％。 1H NMR(300MHz,DMSO-d6)δ8.76(s,1H),8.18(d,J＝5.6Hz,1H),7.46(dd,J＝5.7,1.0Hz,1H),7.37(d,J＝8.6Hz,2H),6.85(d,J＝8.7Hz,2H),4.54(s,2H),3.69(s,3H).
实施例13 合成化合物IV-12
2-((3-甲基苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-12).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，3-甲基溴苄(469μl,3.47mmol)为原料，得到白色固体577.4mg，收率68.38％。 1H NMR(300MHz,DMSO-d6)δ13.07(s,1H),8.74(s,1H),8.17(d,J＝5.7Hz,1H),7.45(d,J＝5.7Hz,1H),7.28–7.14(m,3H),7.06(d,J＝7.2Hz,1H),4.55(s,2H),2.26(s,3H).
实施例14 合成化合物IV-13
2-((3-硝基苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-13).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，3-硝基溴苄(445μl,3.47mmol)为原料，得到黄色固体632.7mg，收率66.82％。 1H NMR(300MHz,DMSO-d6)δ8.74(d,J＝0.9Hz,1H),8.37(t,J＝2.0Hz,1H),8.14(d,J＝5.8Hz,1H),8.04(ddd,J＝8.3,2.4,1.1Hz,1H),7.91(dt,J＝7.8,1.3Hz,1H),7.54(t,J＝8.0Hz,1H),7.47(dd,J＝5.8,0.9Hz,1H),4.70(s,2H).
实施例15 合成化合物IV-14
2-((3,5-二氟苄基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-14).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，3,5-二氟溴苄(449μl,3.47mmol)为原料，得到白色固体601.8mg，收率65.62％。 1H NMR(300MHz,DMSO-d6)δ8.76(d,J＝0.9Hz,1H),8.17(d,J＝5.8Hz,1H),7.50(dd,J＝5.8,0.9Hz,1H),7.28–7.17(m,2H),7.10(tt,J＝9.5,2.4Hz,1H),4.61(s,2H).
实施例16 合成化合物IV-15
2-((3,5-二甲基苄基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-15).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，3,5-二甲基溴苄(534μl,3.47mmol)为原料，得到白色固体587.7mg，收率65.97％。 1H NMR(300MHz,DMSO-d6)δ8.77(d,J＝1.0Hz,1H),8.19(d,J＝5.6Hz,1H),7.48(dd,J＝5.6,1.0Hz,1H),7.06(s,2H),6.88(s,1H),4.53(s,2H),2.22(s,6H).
实施例17 合成化合物IV-16
2-((6-氟吡啶-3-基)甲基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-16).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，5-溴甲基-2-氟吡啶氢溴酸盐(941mg,3.47mmol)为原料，得到紫红色固体324.5mg，收率37.70％。 1H NMR(300MHz,DMSO-d6)δ8.73(s,1H),8.34(d,J＝2.5Hz,1H),8.19–8.05(m,2H),7.47(d,J＝5.8Hz,1H),7.12(dd,J＝ 8.5,2.9Hz,1H),4.59(s,2H).
实施例18 合成化合物IV-17
2-((吡啶-4-基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-17).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，4-溴甲基吡啶氢溴酸盐(878mg,3.47mmol)为原料，得到紫红色固体304.6mg，收率38.01％。 1H NMR(300MHz,DMSO-d6)δ8.73(d,J＝0.9Hz,1H),8.52–8.46(m,2H),8.15(d,J＝5.8Hz,1H),7.50–7.45(m,3H),4.60(s,2H).
实施例19 合成化合物IV-18
2-((吡啶-3-基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-18).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，3-溴甲基吡啶氢溴酸盐(878mg,3.47mmol)为原料，得到紫红色固体298.3mg，收率37.23％。
实施例20 合成化合物IV-19
2-((吡啶-2-基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-19).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，2-溴甲基吡啶氢溴酸盐(878mg,3.47mmol)为原料，得到紫红色固体337.1mg，收率42.07％。
实施例21 合成化合物IV-20
2-((环己基甲基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-20).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，溴甲基环己烷(485μl,3.47mmol)为原料，得到白色固体703.7mg，收率86.02％。 1H NMR(300MHz,DMSO-d6)δ8.72(s,1H),8.18(d,J＝5.6Hz,1H),7.44(d,J＝5.6Hz,1H),3.24(d,J＝6.8Hz,2H),1.84(d,J＝12.5Hz,2H),1.73–1.56(m,4H),1.26–0.98(m,5H).
实施例22 合成化合物IV-21
2-((环戊基甲基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-21).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，溴甲基环戊烷(445μl,3.47mmol)为原料，得到白色固体534.1mg，收率69.21％。 1H NMR(300MHz,DMSO-d6)δ12.98(s,1H),8.72(s,1H),8.18(d,J＝5.6Hz,1H),7.43(d,J＝5.6Hz,1H),2.25(hept,J＝7.5Hz,1H),1.86–1.75(m,2H),1.68–1.45(m,5H),1.37–1.21(m,3H).
实施例23 合成化合物IV-22
2-((环丁基甲基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-22).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，溴甲基环丁烷(407μl,3.47mmol)为原料，得到白色固体550.9mg，收率75.96％。 1H NMR(300MHz,DMSO-d6)δ12.98(s,1H),8.73(s,2H),8.18(d,J＝5.6Hz,2H),7.44(d,J＝5.6Hz,2H),3.39(d,J＝7.6Hz,7H),2.67(hept,J＝7.9Hz,2H),2.13–1.98(m,5H),1.87–1.69(m,9H).
实施例24 合成化合物IV-23
2-((环丙基甲基)硫代)-3-氢-咪唑并[4,5-c]吡啶(IV-23).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，溴甲基环丙烷(337μl,3.47mmol)为原料，得到白色固体516.2mg，收率76.04％。 1H NMR(300MHz,DMSO-d6)δ12.97(s,1H),8.70(s,2H),8.17(d,J＝5.6Hz,2H),7.42(d,J＝5.6Hz,2H),3.26(d,J＝7.2Hz,4H),1.26–1.17(m,2H),0.58–0.51(m,4H),0.36–0.28(m,4H).
实施例25 合成化合物IV-24
2-((3-甲基-2-烯-1-基)硫代)-3-氢-咪唑[4,5-c]吡啶(IV-24).合成方法与实施例2相同，以化合物III(500mg,3.31mmol)，无水碳酸钠(701.04mg,6.61mmol)，1-溴-3-甲基-2-丁烯(401μl,3.47mmol)为原料，得到白色固体537.8mg，收率74.15％。 1H NMR(300MHz,DMSO-d6)δ8.71(s,1H),8.17(d,J＝5.6Hz,1H),7.43(d,J＝5.6Hz,1H),5.39(tt,J＝8.0,1.6Hz,1H),3.96(d,J＝7.8Hz,2H),1.69(d,J＝4.8Hz,6H).
实施例26 合成化合物I-1
4-((2-(苄基硫基)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-1)
以I-1为例，称量化合物IV-1(400mg,1.66mmol)于反应瓶，向其依次加入N,N-二甲基甲酰胺5ml，氢化钠(60％,69.61mg,1.74mmol)，室温反应0.5h后，缓慢加入4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(474.36mg,1.66mmol)，继续反应4h，TLC监测原料消失。将反应液冷却至室温，倒入分液漏斗，加大量水稀释，正丁醇萃取，饱和氯化钠洗涤，无水硫酸钠干燥后，减压浓缩除去溶剂。用二氯甲烷/甲醇柱层析分离纯化，得到白色固体418.7mg，收率56.56％。
4-((2-(苄基硫基)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-1).白色固体，收率56.56％。m.p.131.3-132.1℃. 1H NMR(300MHz,DMSO-d6)δ8.95(d,J＝1.5Hz,1H),8.52(t,J＝5.6Hz,1H),8.18(dd,J＝6.8,1.6Hz,1H),7.87(d,J＝8.3Hz,2H),7.59(d,J＝6.8Hz,1H),7.53–7.45(m,4H),7.34–7.20(m,3H),5.69(s,2H),4.58(s,2H),3.39–3.27(m,4H),3.23(s,3H),1.76(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ173.33,166.18,156.45,145.68,139.73,139.20,135.21,131.73,129.28,129.15,128.83,128.25,128.21,127.41,110.74,70.16,61.11,58.36,37.05,35.20,29.66.HRMS(ESI)calcd for C 25H 26N 4O 2S[M+H] +447.1849,found 447.1855.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.34min,>99.15％.
实施例27 合成化合物I-2
4-((2-((2-氟苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-2).合成方法与实施例3相同，以化合物Ⅳ-2(400mg,1.54mmol)，氢化钠(64.78mg,1.62mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(441.44mg,1.54mmol)为原料，得到白色固体348.6mg，收率48.64％。 m.p.177.4-177.1℃. 1H NMR(300MHz,DMSO-d6)δ8.96(d,J＝1.5Hz,1H),8.50(t,J＝5.6Hz,1H),8.18(dd,J＝6.8,1.6Hz,1H),7.86(d,J＝8.3Hz,2H),7.71–7.66(m,1H),7.59(d,J＝6.8Hz,1H),7.53–7.45(m,3H),7.33–7.23(m,2H),5.69(s,2H),4.67(s,2H),3.33(dt,J＝20.1,6.8Hz,4H),3.23(s,3H),1.75(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.76,166.13,156.44,145.65,139.72,136.46,135.22,133.70,131.78,131.61,129.81,129.50,129.32,128.24,128.21,127.73,110.84,70.16,61.12,58.37,37.04,33.18,29.67.HRMS(ESI)calcd for C 25H 25FN 4O 2S[M+H] +465.1755,found 465.1751.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝13.21min,>97.64％.
实施例28 合成化合物I-3
4-((2-((3-氟苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-3).合成方法与实施例3相同，以化合物Ⅳ-3(400mg,1.54mmol)，氢化钠(64.78mg,1.62mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(441.44mg,1.54mmol)为原料，得到白色固体377.1mg，收率52.62％。m.p.147.6-148.1℃. 1H NMR(300MHz,DMSO-d6)δ8.98(d,J＝1.5Hz,1H),8.52(t,J＝5.6Hz,1H),8.19(dd,J＝6.8,1.6Hz,1H),7.88(d,J＝8.4Hz,2H),7.61(d,J＝6.7Hz,1H),7.52(d,J＝8.3Hz,2H),7.38–7.30(m,3H),7.12–7.02(m,1H),5.70(s,2H),4.60(s,2H),3.40–3.28(m,4H),3.23(s,3H),1.76(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.97,166.16,164.07,160.85,156.46,145.68,142.54,142.44,139.71,135.23,131.77,130.74,130.62,129.29,128.25,128.21,125.38,125.34,116.09,115.81,114.32,114.04,110.82,70.16,61.12,58.35,37.05,34.52,29.67.HRMS(ESI)calcd for C 25H 25FN 4O 2S[M+H] +465.1755,found 465.1761.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.85min,>99.29％.
实施例29 合成化合物I-4
4-((2-((4-氟苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-4).合成方法与实施例3相同，以化合物Ⅳ-4(400mg,1.54mmol)，氢化钠(64.78mg,1.62mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(441.44mg,1.54mmol)为原料，得到白色固体359.6mg，收率50.18％。m.p.171.8-173.2℃. 1H NMR(300MHz,DMSO-d6)δ8.98(d,J＝1.5Hz,1H),8.52(t,J＝5.6Hz,1H),8.19(dd,J＝6.8,1.6Hz,1H),7.88(d,J＝8.4Hz,2H),7.61(d,J＝6.7Hz,1H),7.52(d,J＝8.3Hz,2H),7.38–7.30(m,3H),7.12–7.02(m,1H),5.70(s,2H),4.60(s,2H),3.40–3.28(m,4H),3.23(s,3H),1.76(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.97,166.16,164.07,160.85,156.46,145.68,142.54,142.44,139.71,135.23,131.77,130.74,130.62,129.29,128.25,128.21,125.38,125.34,116.09,115.81,114.32,114.04,110.82,70.16,61.12,58.35,37.05,34.52,29.67.HRMS(ESI)calcd for C 25H 25FN 4O 2S[M+H] +465.1755,found 465.1761.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝18.05min,>99.41％.
实施例30 合成化合物I-5
4-((2-((4-氯苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-5).合成方法与实施例3相同，以化合物Ⅳ-5(400mg,1.45mmol)，氢化钠(60.92mg,1.52mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(415.11mg,1.45mmol)为原料，得到白色固体406.3mg，收率58.23％。m.p.150.0-151.0℃ 1H NMR(300MHz,DMSO-d6)δ8.91(d,J＝1.5Hz,1H),8.45(s,1H),8.15(dd,J＝6.8,1.6Hz,1H),7.82(d,J＝8.3Hz,2H),7.55(d,J＝6.7Hz,1H),7.49(t,J＝8.5Hz,4H),7.10(s,2H),5.65(s,2H),4.52(s,2H),3.21(s,3H),1.71(s,2H). 13C NMR(75MHz,DMSO)δ172.95,166.14,156.40,145.64,139.71,138.63,135.22,131.98,131.79,131.09,129.27,128.74,128.25,128.22,110.80,70.16,61.12,58.36,37.04,34.31,29.66.HRMS(ESI)calcd for C 25H 25ClN 4O 2S[M+H] +481.1460,found 481.1465.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝13.50min,>97.31％.
实施例31 合成化合物I-6
4-((2-((4-溴苯基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-6).合成方法与实施例3相同，以化合物Ⅳ-6(400mg,1.25mmol)，氢化钠(52.46mg,1.31mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(357.48mg,1.25mmol)为原料，得到白色固体377.5mg，收率57.51％。m.p.155.4-156.6℃ 1H NMR(300MHz,DMSO-d6)δ8.97(d,J＝1.5Hz,1H),8.55(t,J＝5.6Hz,1H),8.20(dd,J＝6.8,1.6Hz,1H),7.90(d,J＝8.3Hz,2H),7.61(d,J＝6.7Hz,1H),7.55–7.43(m,6H),5.71(s,2H),4.56(s,2H),3.41–3.29(m,4H),3.24(s,3H),1.77(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ173.00,166.16,156.46,145.69,139.70,139.07,135.23,131.75,131.65,131.45,129.24,128.26,128.22,120.48,110.81,70.17,61.13,58.37,37.06,34.38,29.68.HRMS(ESI)calcd for C 25H 25BrN 4O 2S[M+H] +525.0954,found 525.0956.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.051min,>99.99％.
实施例32 合成化合物I-7
4-((2-((4-氰基苯)硫基)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-7).合成方法与实施例3相同，以化合物Ⅳ-7(400mg,1.50mmol)，氢化钠(63.08mg,1.58mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(429.81mg,1.50mmol)为原料，得到白色固体413.6mg，收率58.39％。m.p.168.2-169.2℃ 1H NMR(300MHz,DMSO-d6)δ8.96(d,J＝1.5Hz,1H),8.50(t,J＝5.6Hz,1H),8.19(dd,J＝6.8,1.6Hz,1H),7.87(d,J＝8.3Hz,2H),7.77(d,J＝8.4Hz,2H),7.69(d,J＝8.4Hz,2H),7.59(d,J＝6.7Hz,1H),7.51(d,J＝8.3Hz,2H),5.69(s,2H),4.64(s,2H),3.39–3.26(m,4H),3.23(s,3H),1.75(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.61,166.13,156.45,145.80,145.65,139.69,135.22,132.71,131.80,130.20,129.38,128.24,119.32,110.89,110.08,70.16,61.12,58.36,37.04,34.61,29.66.HRMS(ESI)calcd for C 26H 25N 5O 2S[M+H] +472.1802,found 472.1808.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝10.12 min,>99.23％.
实施例33 合成化合物I-8
N-(3-甲氧基丙基)-4-((2-((4-硝基苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-8).合成方法与实施例3相同，以化合物Ⅳ-8(400mg,1.47mmol)，氢化钠(61.91mg,1.55mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(421.86mg,1.47mmol)为原料，得到淡黄色固体405.6mg，收率57.73％。m.p.161.9-163.0℃ 1H NMR(300MHz,DMSO-d6)δ8.97(d,J＝1.5Hz,1H),8.51(t,J＝5.7Hz,1H),8.23–8.13(m,3H),7.87(d,J＝8.3Hz,2H),7.77(d,J＝8.7Hz,2H),7.60(d,J＝6.7Hz,1H),7.51(d,J＝8.2Hz,2H),5.70(s,2H),4.70(s,2H),3.39–3.27(m,4H),3.23(s,3H),1.76(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.50,166.12,156.46,148.03,146.85,145.66,139.68,135.22,131.81,130.41,129.41,128.23,123.92,110.91,70.16,61.13,58.36,37.04,34.33,29.66.HRMS(ESI)calcd for C 25H 25N 5O 4S[M+H] +492.1700,found 492.1697.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝12.59min,>99.27％.
实施例34 合成化合物I-9
N-(3-甲氧基丙基)-4-((2-((4-(三氟甲基)苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-9).合成方法与实施例3相同，以化合物Ⅳ-9(400mg,1.29mmol)，氢化钠(54.31mg,1.36mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(370.07mg,1.29mmol)为原料，得到白色固体379.4mg，收率57.02％。m.p.132.9-134.1℃ 1H NMR(300MHz,DMSO-d6)δ8.97(d,J＝1.6Hz,1H),8.52(t,J＝5.6Hz,1H),8.20(dd,J＝6.8,1.6Hz,1H),7.91–7.83(m,2H),7.76–7.63(m,4H),7.60(d,J＝6.7Hz,1H),7.55–7.48(m,2H),5.70(s,2H),4.66(s,2H),3.40–3.28(m,4H),3.23(s,3H),1.76(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.77,166.13,156.47,145.68,144.66,144.64,139.70,135.23,131.78,129.98,129.34,128.24,128.22,127.77,126.55,125.71,125.66,125.61,125.56,122.95,110.86,70.16,61.12,58.34,37.04,34.48,29.66.HRMS(ESI)calcd for C 26H 25F 3N 4O 2S[M+H] +515.1723,found 515.1724.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.74min,>99.28％.
实施例35 合成化合物I-10
N-(3-甲氧基丙基)-4-((2-((4-甲基苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-10).合成方法与实施例3相同，以化合物Ⅳ-10(400mg,1.57mmol)，氢化钠(65.79mg,1.64mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(448.30mg,1.57mmol)为原料，得到白色固体388.3mg，收率53.82％。m.p.152.7-152.8℃ 1H NMR(300MHz,DMSO-d6)δ8.93(d,J＝1.5Hz,1H),8.49(t,J＝5.6Hz,1H),8.16(dd,J＝6.8,1.6Hz,1H),7.85(d,J＝8.4Hz,2H),7.56(d,J＝6.8Hz,1H),7.49(d,J＝8.3Hz,2H),7.34(d,J＝8.1Hz,2H),7.10(d,J＝7.6Hz,2H),5.67(s,2H),4.51(s,2H),3.37–3.25(m,4H),3.22(s,3H),2.26(s,3H),1.74(p,J＝6.7Hz,2H). 13C NMR(75MHz,DMSO)δ173.43,166.12,156.43,145.68, 139.76,136.54,136.02,135.20,131.69,129.39,129.28,129.19,129.09,128.23,128.20,110.69,70.16,61.06,58.37,37.03,34.98,29.66,21.15.HRMS(ESI)calcd for C 26H 28N 4O 2S[M+H] +461.2006,found 461.2004.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝20.68min,>99.99％.
实施例36 合成化合物I-11
4-((2-((4-甲氧基苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-11).合成方法与实施例3相同，以化合物Ⅳ-11(400mg,1.47mmol)，氢化钠(61.91mg,1.55mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(421.86mg,1.47mmol)为原料，得到白色固体376.5mg，收率53.59％。m.p.144.2-144.4℃ 1H NMR(300MHz,DMSO-d6)δ8.93(d,J＝1.5Hz,1H),8.53(t,J＝5.6Hz,1H),8.16(dd,J＝6.8,1.5Hz,1H),7.91–7.83(m,2H),7.58(d,J＝6.7Hz,1H),7.50(d,J＝8.3Hz,2H),7.39(d,J＝8.6Hz,2H),6.85(d,J＝8.7Hz,2H),5.68(s,2H),4.52(s,2H),3.71(s,3H),3.38–3.27(m,4H),3.21(s,3H),1.75(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ173.55,166.21,158.76,156.44,145.70,139.73,135.22,131.72,130.90,130.45,129.07,128.26,128.20,114.23,110.69,70.17,61.12,58.35,55.47,37.07,34.74,29.66.HRMS(ESI)calcd for C 26H 28N 4O 3S[M+H] +477.1955,found 477.1953.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.49min,>99.21％.
实施例37 合成化合物I-12
N-(3-甲氧基丙基)-4-((2-((3-甲基苄基)硫代)-3-氢-咪唑并[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-12).合成方法与实施例3相同，以化合物Ⅳ-12(400mg,1.57mmol)，氢化钠(65.79mg,1.64mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(448.3mg,1.57mmol)为原料，得到白色固体401.8mg，收率55.69％。m.p.130.6-131.7℃ 1H NMR(300MHz,DMSO-d6)δ8.93(d,J＝1.6Hz,1H),8.47(t,J＝5.6Hz,1H),8.16(dd,J＝6.7,1.6Hz,1H),7.84(d,J＝8.3Hz,2H),7.57(d,J＝6.7Hz,1H),7.49(d,J＝8.2Hz,2H),7.25(d,J＝7.7Hz,2H),7.18(t,J＝7.9Hz,1H),7.04(d,J＝7.4Hz,1H),5.67(s,2H),4.52(s,2H),3.36–3.24(m,4H),3.22(s,3H),2.27(s,3H),1.74(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ173.39,166.15,156.45,145.69,139.78,138.92,137.96,135.22,131.74,129.89,129.16,128.78,128.25,128.22,128.11,126.41,110.74,70.17,61.10,58.39,37.03,35.24,29.67,21.44.HRMS(ESI)calcd for C 26H 28N 4O 2S[M+H] +461.2006,found 461.2002.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝13.32min,>97.13％.
实施例38 合成化合物I-13
N-(3-甲氧基丙基)-4-((2-((3-硝基苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-13).合成方法与实施例3相同，以化合物Ⅳ-13(400mg,1.40mmol)，氢化钠(58.67mg,1.47mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(399.80mg,1.40mmol)为原料，得到淡黄色固体397.1mg，收率58.72％。m.p.170.8-171.3℃ 1H NMR(300MHz,DMSO-d6)δ8.99(d,J＝1.6Hz,1H),8.49 (t,J＝5.6Hz,1H),8.41(t,J＝2.0Hz,1H),8.21(dd,J＝6.8,1.6Hz,1H),8.11(ddd,J＝8.3,2.4,1.1Hz,1H),7.97(dt,J＝7.7,1.3Hz,1H),7.87(d,J＝8.3Hz,2H),7.65–7.57(m,2H),7.52(d,J＝8.2Hz,2H),5.71(s,2H),4.70(s,2H),3.38–3.27(m,4H),3.25(s,3H),1.76(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.47,166.12,156.34,148.11,145.58,142.33,139.68,136.05,135.23,130.26,129.51,128.24,123.85,122.36,110.96,70.16,61.14,58.37,37.02,34.12,29.65.HRMS(ESI)calcd for C 25H 25N 5O 4S[M+H] +492.1700,found 492.1696.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.51min,>96.62％.
实施例39 合成化合物I-14
4-((2-((3,5-二氟苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-14).合成方法与实施例3相同，以化合物Ⅳ-14(400mg,1.44mmol)，氢化钠(60.58mg,1.51mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(412.80mg,1.44mmol)为原料，得到白色固体389.7mg，收率55.98％。m.p.146.1-146.6℃ 1H NMR(300MHz,DMSO-d6)δ8.95(d,J＝1.6Hz,1H),8.47(t,J＝5.6Hz,1H),8.16(dd,J＝6.8,1.6Hz,1H),7.84(d,J＝8.4Hz,2H),7.57(d,J＝6.7Hz,1H),7.48(d,J＝8.2Hz,2H),7.26–7.17(m,2H),7.06(tt,J＝9.4,2.4Hz,1H),5.67(s,2H),4.55(s,2H),3.36–3.25(m,4H),3.20(s,3H),1.73(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.62,166.16,164.32,164.15,161.07,160.89,156.45,145.65,144.48,144.36,139.70,135.24,131.82,129.44,128.26,128.23,112.52,112.29,112.19,110.90,103.20,102.86,102.52,70.16,61.13,58.35,37.04,34.20,29.66.HRMS(ESI)calcd for C 25H 24F 2N 4O 2S[M+H] +483.1661,found 483.1659.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝12.38min,>97.79％.
实施例40 合成化合物I-15
4-((2-((3,5-二甲基苄基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-15).合成方法与实施例3相同，以化合物Ⅳ-15(400mg,1.48mmol)，氢化钠(62.36mg,1.48mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(424.95mg,1.48mmol)为原料，得到白色固体411.2mg，收率58.34％。m.p.170.7-170.9℃ 1H NMR(300MHz,DMSO-d6)δ8.96(d,J＝1.5Hz,1H),8.52(t,J＝5.6Hz,1H),8.18(dd,J＝6.7,1.6Hz,1H),7.89(d,J＝8.3Hz,2H),7.60(d,J＝6.8Hz,1H),7.52(d,J＝8.2Hz,2H),7.08(s,2H),6.88(s,1H),5.70(s,2H),4.52(s,2H),3.41–3.29(m,4H),3.25(s,3H),2.25(s,6H),1.77(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ173.52,166.15,156.48,145.73,139.76,138.61,137.83,135.22,131.69,129.10,128.91,128.25,128.19,127.04,110.70,70.17,61.09,58.37,37.05,35.30,29.68,21.32.HRMS(ESI)calcd for C 27H 30N 4O 2S[M+H] +475.2162,found 475.2159.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝14.79min,>98.62％.
实施例41 合成化合物I-16
4-((2-((6-氟吡啶-3-基)甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-16).合成方法与实施例3相同，以化合物Ⅳ-16(400mg,1.54mmol)，氢化钠(64.54mg,1.61mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(439.77mg,1.54mmol)为原料，得到淡黄色固体314.2mg，收率43.92％。m.p.178.8-180.3℃ 1H NMR(300MHz,DMSO-d6)δ8.96(d,J＝1.5Hz,1H),8.49(t,J＝5.6Hz,1H),8.36(d,J＝2.9Hz,1H),8.18(dd,J＝6.8,1.6Hz,1H),8.10(td,J＝8.2,2.6Hz,1H),7.85(d,J＝8.3Hz,2H),7.59(d,J＝6.8Hz,1H),7.50(d,J＝8.4Hz,2H),7.12(ddd,J＝8.4,2.9,0.6Hz,1H),5.68(s,2H),4.56(s,2H),3.37–3.26(m,4H),3.23(s,3H),1.74(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.53,166.12,164.04,160.93,156.39,147.92,147.72,145.61,142.97,142.86,139.70,135.22,134.03,133.97,131.82,129.42,128.23,110.89,109.93,109.43,70.15,61.11,58.36,37.02,31.16,29.65.HRMS(ESI)calcd for C 24H 24FN 5O 2S[M+H] +466.1708,found 466.1706.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝8.20min,>98.93％.
实施例42 合成化合物I-17
N-(3-甲氧基丙基)-4-((2-((吡啶-4-基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-17).合成方法与实施例3相同，以化合物Ⅳ-17(400mg,1.65mmol)，氢化钠(69.33mg,1.73mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(472.42mg,1.65mmol)为原料，得到黄色固体297.4mg，收率40.25％。m.p.183.2-184.8℃ 1H NMR(300MHz,DMSO-d6)δ8.95(d,J＝1.5Hz,1H),8.54–8.42(m,3H),8.17(dd,J＝6.8,1.6Hz,1H),7.84(d,J＝8.2Hz,2H),7.58(d,J＝6.7Hz,1H),7.51–7.44(m,4H),5.67(s,2H),4.54(s,2H),3.36–3.24(m,4H),3.19(s,3H),1.72(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ172.36,166.17,156.29,150.01,148.65,145.55,139.66,135.25,131.92,129.50,128.26,124.33,110.91,70.17,61.17,58.36,37.05,33.85,29.66.HRMS(ESI)calcd for C 24H 25N 5O 2S[M+H] +448.1802,found 448.1798.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝14.68min,>97.11％.
实施例43 合成化合物I-18
N-(3-甲氧基丙基)-4-((2-((吡啶-3-基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-18).合成方法与实施例3相同，以化合物Ⅳ-18(400mg,1.65mmol)，氢化钠(69.33mg,1.73mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(472.42mg,1.65mmol)为原料，得到淡黄色固体278.4mg，收率37.68％。m.p.180.1-182.7℃ 1H NMR(300MHz,DMSO-d6)δ9.58(d,J＝1.3Hz,1H),8.95(s,1H),8.76(dd,J＝6.8,1.3Hz,1H),8.68(s,1H),8.56(t,J＝5.6Hz,1H),8.35(d,J＝8.1Hz,1H),8.08(d,J＝6.7Hz,1H),7.87(d,J＝8.3Hz,2H),7.70(dd,J＝8.0,5.2Hz,1H),7.57(d,J＝8.1Hz,2H),5.89(s,2H),4.78(s,2H),3.30(dt,J＝17.7,6.4Hz,4H),3.19(s,3H),1.72(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ165.98,162.36,159.58,159.12,147.72,146.37,145.12,142.02,140.04, 138.46,137.31,136.17,135.53,133.70,128.64,128.32,125.82,111.20,70.13,62.47,58.29,37.03,32.14,29.62.HRMS(ESI)calcd for C 24H 25N 5O 2S[M+H] +448.1802,found 448.1809.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝16.65min,>99.52％.
实施例44 合成化合物I-19
N-(3-甲氧基丙基)-4-((2-((吡啶-2-基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)苯甲酰胺(I-19).合成方法与实施例3相同，以化合物Ⅳ-19(400mg,1.65mmol)，氢化钠(69.33mg,1.73mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(472.42mg,1.65mmol)为原料，得到黄色固体296.7mg，收率40.16％。m.p.182.9.-183.9℃ 1H NMR(300MHz,DMSO-d6)δ9.57(d,J＝1.3Hz,1H),8.76(dd,J＝6.8,1.4Hz,1H),8.59–8.50(m,2H),8.08(d,J＝6.7Hz,1H),7.91–7.78(m,3H),7.63(dt,J＝7.9,1.1Hz,1H),7.56(d,J＝8.2Hz,2H),7.36(ddd,J＝7.6,4.9,1.2Hz,1H),5.89(s,2H),4.83(s,2H),3.30(dt,J＝17.7,6.5Hz,4H),3.19(s,3H),1.72(p,J＝6.6Hz,2H). 13C NMR(75MHz,DMSO)δ165.98,162.96,159.40,158.95,155.84,148.99,147.71,140.03,138.50,138.46,137.28,135.52,133.55,128.62,128.32,124.03,123.54,118.68,114.77,111.14,70.14,62.45,58.31,37.03,29.63.HRMS(ESI)calcd for C 24H 25N 5O 2S[M+H] +448.1802,found 448.1807.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝15.32min,>99.60％.
实施例45 合成化合物I-20
4-((2-((环己基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-20).合成方法与实施例3相同，以化合物Ⅳ-20(400mg,1.62mmol)，氢化钠(67.91mg,1.70mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(462.76mg,1.62mmol)为原料，得到白色固体567.9mg，收率77.59％。m.p.156.6-157.1℃ 1H NMR(300MHz,DMSO-d6)δ8.89(d,J＝1.6Hz,1H),8.50(t,J＝5.6Hz,1H),8.15(dd,J＝6.8,1.6Hz,1H),7.86(d,J＝8.3Hz,2H),7.52(dd,J＝11.7,7.5Hz,3H),5.66(s,2H),3.39–3.26(m,4H),3.23(s,3H),3.18(d,J＝6.8Hz,2H),1.90–1.80(m,2H),1.75(p,J＝6.7Hz,2H),1.70–1.53(m,4H),1.26–1.07(m,3H),1.07–0.92(m,2H). 13C NMR(75MHz,DMSO)δ174.14,166.11,156.45,145.71,139.76,135.18,131.54,128.73,128.22,128.19,110.42,70.16,61.03,58.35,38.11,38.02,37.03,32.56,29.67,26.38,26.00.HRMS(ESI)calcd for C 25H 32N 4O 2S[M+H] +453.2319,found 453.2319.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝14.35min,>99.33％.
实施例46 合成化合物I-21
4-((2-((环戊基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-21).合成方法与实施例3相同，以化合物Ⅳ-21(400mg,1.71mmol)，氢化钠(71.99mg,1.80mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(490.58mg,1.71mmol)为原料，得到白色固体525.3mg，收率69.8％。 m.p.144.3-145.0℃ 1H NMR(300MHz,DMSO-d6)δ8.92(d,J＝1.5Hz,1H),8.56(t,J＝5.6Hz,1H),8.17(dd,J＝6.8,1.6Hz,1H),7.89(d,J＝8.4Hz,2H),7.56(d,J＝6.7Hz,1H),7.51(d,J＝8.4Hz,2H),5.69(s,2H),3.39–3.27(m,6H),3.23(s,3H),2.26(hept,J＝7.5Hz,1H),1.84–1.70(m,4H),1.65–1.43(m,4H),1.36–1.24(m,2H). 13C NMR(75MHz,DMSO)δ174.10,166.14,156.43,145.72,139.75,135.19,131.56,128.74,128.23,128.17,110.43,70.17,61.06,58.34,37.05,36.95,32.23,29.67,25.24.HRMS(ESI)calcd for C 24H 30N 4O 2S[M+H] +439.2162,found 439.2158.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝12.61min,>97.66％.
实施例47 合成化合物I-22
4-((2-((环丁基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-22).合成方法与实施例3相同，以化合物Ⅳ-22(400mg,1.82mmol)，氢化钠(76.60mg,1.92mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(521.95mg,1.82mmol)为原料，得到白色固体495.4mg，收率63.97％。m.p.145.3-146.7℃ 1H NMR(300MHz,DMSO-d6)δ8.73(s,1H),8.34(t,J＝5.6Hz,1H),7.98(dd,J＝6.8,1.6Hz,1H),7.69(d,J＝8.2Hz,2H),7.39(d,J＝6.7Hz,1H),7.32(d,J＝8.3Hz,2H),5.50(s,2H),3.20–3.09(m,6H),3.04(s,3H),2.59–2.39(m,1H),1.92–1.78(m,2H),1.64–1.48(m,6H). 13C NMR(75MHz,DMSO)δ173.82,166.15,156.36,145.66,139.75,135.20,131.66,128.85,128.24,128.19,110.51,70.16,61.09,58.35,37.36,37.04,35.25,29.66,27.51,17.83.HRMS(ESI)calcd for C 23H 28N 4O 2S[M+H] +425.2006,found 425.2001.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝11.05min,>99.58％.
实施例48 合成化合物I-23
4-((2-((环丙基甲基)硫代)-3-氢-咪唑[4,5-c]吡啶-3-基)甲基)-N-(3-甲氧基丙基)苯甲酰胺(I-23).合成方法与实施例3相同，以化合物Ⅳ-23(400mg,1.95mmol)，氢化钠(81.83mg,2.05mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(557.62mg,1.95mmol)为原料，得到白色固体486.2mg，收率60.78％。m.p.147.9-148.4℃ 1H NMR(300MHz,DMSO-d6)δ8.89(d,J＝1.6Hz,1H),8.57(t,J＝5.6Hz,1H),8.14(dd,J＝6.7,1.6Hz,1H),7.88(d,J＝8.0Hz,2H),7.55(d,J＝6.7Hz,1H),7.48(d,J＝8.0Hz,2H),5.68(s,2H),3.37–3.27(m,4H),3.23–3.17(m,5H),1.74(p,J＝6.6Hz,2H),1.30–1.13(m,1H),0.54–0.46(m,2H),0.32–0.24(m,2H). 13C NMR(75MHz,DMSO)δ174.08,166.28,156.41,145.71,139.70,135.19,131.67,128.77,128.26,128.16,110.49,70.16,61.15,58.31,49.08,37.10,36.74,29.64,11.77,6.05.HRMS(ESI)calcd for C 22H 26N 4O 2S[M+H] +411.1849,found 411.1847.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝8.89min,>99.49％.
实施例49 合成化合物I-24
N-(3-甲氧基丙基)-4-((2-((3-甲基-2-烯-1-基)硫代)-3-氢-咪唑并[4,5-c] 吡啶-3-基)甲基)苯甲酰胺(I-24).合成方法与实施例3相同，以化合物Ⅳ-24(400mg,1.82mmol)，氢化钠(76.60mg,1.92mmol)，4-(溴甲基)-N-(3-甲氧基丙基)苯甲酰胺(521.95mg,1.82mmol)为原料，得到白色固体517.6mg，收率66.84％。m.p.148.5-150.2℃ 1H NMR(300MHz,DMSO-d6)δ8.85(d,J＝1.5Hz,1H),8.48(t,J＝5.6Hz,1H),8.12(dd,J＝6.8,1.6Hz,1H),7.82(d,J＝8.3Hz,2H),7.52(d,J＝6.8Hz,1H),7.45(d,J＝8.3Hz,2H),5.65(s,2H),5.40(tt,J＝7.7,1.4Hz,1H),3.88(d,J＝7.8Hz,2H),3.35–3.23(m,4H),3.19(s,3H),1.76–1.65(m,8H). 13C NMR(75MHz,DMSO)δ173.68,166.20,156.39,145.66,139.75,135.74,135.15,131.70,128.92,128.22,128.17,120.51,110.57,70.15,61.08,58.35,49.07,37.04,29.62,29.27,25.83,18.12.HRMS(ESI)calcd for C 23H 28N 4O 2S[M+H] +425.2006,found 425.2000.HPLC(20％–60％acetonitrile in water,with 0.1％TFA),t R＝20.46min,>97.17％.
性能测试
下面是本发明化合物的药理学实验结果，该部分的化合物代号所对应的结构式等同于实施例部分的代号所对应的结构式。
一、用于测定IL-1β含量的ELISA实验
细胞培养上清中IL-1β的分泌使用双抗体夹心法ELISA检测。新鲜收集的血清样本室温静置20min后，于4℃3000g离心10min，上清即可用于检测分析。将样品与标准品分别加入已包被抗体的96孔板中，加入生物素化抗体后置于37℃摇床孵育1h后，洗板5次。加入酶结合工作液，37℃避光孵育30min后，洗板5次。加入显色底物，37℃避光孵育15min。加入终止液终止反应。10min内用全波长酶标仪(Thermo)于450nm波长处测量吸光度值，根据标准曲线换算检测样品浓度。化合物对白介素IL-1β的抑制率计算如下：
抑制率(％)＝1-(药物孔OD值-空白孔OD值)/(药物孔OD值-空白孔OD值)，以不加药物和诱导因子的细胞孔为空白对照，以不加药物，加LPS和ATP的细胞孔为阴性对照。
本发明以MCC950为阳性对照，DMSO组为阴性对照，细胞裂解液为空白背景，设置3个复孔，化合物对IL-1β抑制率结果表示为为各组数据平均值±SEM。本发明中部分化合物对白介素IL-1β的抑制率如表1所示。
表1：
Figure PCTCN2022086781-appb-000012
Figure PCTCN2022086781-appb-000013
由表1数据可知，本发明的化合物对白介素IL-1β具有抑制活性，这为开发高效安全结构新颖特异性强的NLRP3抑制剂，及治疗NLRP3介导的炎症相关疾病如风湿性关节炎，类风湿性关节炎，炎性肠道疾病，痛风，动脉粥样硬化，非酒精性脂肪肝病，冷吡啉相关周期性综合征，慢性肾脏疾病，糖尿病和神经退行性疾病等的药物提供了基础。
二细胞毒性评价
通过使用MTT测定确定细胞毒性。MTT购自Sigma(St.Louis，MO)。将其溶解在磷酸盐缓冲盐水(PBS)中至储备溶液浓度为5mg/mL并储存在-20℃。在用测试化合物或DMSO的密度梯度处理细胞24小时后，将20.0μLMTT溶液(5mg/mL)加入到96孔板的每个孔中并孵育4小时。然后，除去溶液并向每个孔中加入150.0μL DMSO以溶解水溶性MTT-甲臢晶体。通过Elx800吸光度酶标仪(BioTek，Vermont，USA)在570nm记录吸光度值(OD值)。化合物对细胞生长抑制率的计算如下：
抑制率(％)＝1-(药物孔OD值-空白孔OD值)/(药物孔OD值-空白孔OD值)，以不加药物和诱导因子的细胞孔为空白对照。本发明部分化合物的细胞毒性评价如表2所示。
表2：
Figure PCTCN2022086781-appb-000014
Figure PCTCN2022086781-appb-000015
由表2数据可知，本发明的绝大多数化合物IC 50>100μM，细胞毒性小。
三.Western Blot监测化合物对目的蛋白的影响
细胞蛋白制备：吸取六孔板细胞上清，经冰预冷PBS缓冲液漂洗两次后用细胞刮刀将细胞从六孔板中刮下并收集。1000g离心5min，按照1：5(体细胞压积：裂解液体积)比例加入RIPA蛋白裂解液，冰上裂解60min，16000g离心10min，吸取上清即为全细胞蛋白，取4μL进行BCA法测定蛋白浓度，其余蛋白上清按体积加入5×上样缓冲液后沸水浴中变性10min，分装后-20℃保存。
根据BCA定量结果，取40-80μg样品/泳道上样，依据目的蛋白的分子量采用不同胶浓度的SDS-PAGE分离，进行恒压(80V)浓缩25min，恒压(120V)凝胶电泳55min。200mA恒流湿转电转90min使蛋白转移至PVDF膜(Millipore，USA)。将PVDF膜置于3％BSA-TBST室温震荡封闭1h后，加入5％BSA-TBST配置的一抗：人抗IL-1β(1:1000)，人抗GAPDH(1:1000，sigma，USA)，4℃过夜。TBST漂洗10min×3次后，加入HRP标记的山羊抗 兔二抗(1:4000)，室温震荡孵育1.5h，TBST漂洗10min×3次后加入ECL(Pierce)发光底物显色。Image Quant LAS 4000mini(GE)显影。将目的蛋白灰度值与内参GAPDH灰度值之比进行半定量分析(图1)。
由western blot实验表明，本发明中的化合物I-20可明显抑制NLRP3蛋白的表达，具有浓度依赖性。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (12)

  1. 一种母核为吡啶并咪唑的化合物，其特征在于，结构如式Ⅰ所示：
    Figure PCTCN2022086781-appb-100001
    式中，R 1为C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基；
    R 2为氢、C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基。
  2. 根据权利要求1所述的母核为吡啶并咪唑的化合物，其特征在于：所述的R 1为
    Figure PCTCN2022086781-appb-100002
    其中R 3-R 7分别独立地为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
    优选：R 3-R 7中有一个或两个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基，且其余为H；
    进一步优选：R 3-R 7中有一个或两个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基，且其余为H；
    或：所述的R 1为
    Figure PCTCN2022086781-appb-100003
    其中Y 1-Y 5分别独立地为CH、N，R 8为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
    优选：Y 1-Y 3中有一个或两个为N，其余为CH，R 8为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
    进一步优选：Y 1-Y 3中有一个为N，其余为CH，R 8为H、烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、氨基、硝基、羟基、酯基、三氟甲基；
    最优选：Y 1-Y 3中有一个为N，其余为CH，R 8为H、F；
    或：所述的R 1为C 1-C 30烷基、卤代烷基、环烷基、卤代环烷基、杂环烷基；
    优选：R 1为C 1-C 10烷基、卤代烷基、环烷基、卤代环烷基、杂环烷基；
    进一步优选：R 1为C 3-C 6烷基、环烷基；
    最优选：R 1为C 3-C 6环烷基、
    Figure PCTCN2022086781-appb-100004
  3. 根据权利要求1所述的母核为吡啶并咪唑的化合物，其特征在于：所述的R 2为
    Figure PCTCN2022086781-appb-100005
    其中Z 1-Z 2分别独立地为CH、N，R 9-R 10有且仅有一个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、亚胺基、硝基、羟基、酯基、三氟甲基，且另一个为H；
    优选：Z 1-Z 2有且仅有一个为N，另一个为CH，R 9-R 10有且仅有一个为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、亚胺基、硝基、羟基、酯基、三氟甲基，且另一个为H；
    进一步优选：Z 1-Z 2为CH，R 9为H，R 10为烷基、烷氧基、卤素、氰基、羰基、羧基、酰基、磺酰基、亚胺基、硝基、羟基、酯基、三氟甲基；
    最优选：Z 1-Z 2为CH，R 9为H，R 10为酰基、亚胺基。
  4. 根据权利要求1所述的化合物，其特征在于，所述的化合物选自：
    Figure PCTCN2022086781-appb-100006
    Figure PCTCN2022086781-appb-100007
    Figure PCTCN2022086781-appb-100008
  5. 权利要求1所述的母核为吡啶并咪唑的化合物的制备方法，包括如下步骤：
    步骤1，化合物Ⅱ成环，得到化合物Ⅲ；
    步骤2，将所述化合物Ⅲ在碱性条件下发生亲核取代反应，得到化合物Ⅳ；
    步骤3，将所述化合物Ⅳ在碱性条件下发生亲核取代反应，得到化合物I；
    Figure PCTCN2022086781-appb-100009
    式中，R 1为C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基；
    R 2为氢、C 1～C 30的烷基、芳基、杂芳基、C 1～C 30烷基芳基、C 1～C 30烷基杂芳基或C 1～C 12杂环；该C 1～C 12杂环被以下一个或多个相同或不同的取代基取代：C 1～C 30的烷基，烷氧基，烷氨基，酰胺基，硝基，卤素，苄基，腈基，氨基，羧基，羰基，三氟甲基。
  6. 根据权利要求5所述的化合物I的制备方法，其特征在于，所述步骤1为，i.将化合物II与二硫化碳加入乙醇中，加热回流4h，得到化合物III；
  7. 根据权利要求5所述的化合物I的制备方法，其特征在于，所述步骤2为，ii.将化合物III与无水碳酸钠加入N,N-二甲基甲酰胺中，40℃加热反应1h；iii.向反应液中缓慢加入
    Figure PCTCN2022086781-appb-100010
    继续反应4h得到化合物IV。
  8. 根据权利要求5所述的化合物I的制备方法，其特征在于，所述步骤3为，iv.将化合物IV溶解于N,N-二甲基甲酰胺中，边搅边缓慢加入氢化钠，室温反应0.5h；v.向反应液中缓慢加入
    Figure PCTCN2022086781-appb-100011
    继续反应4h，得到化合物I。
  9. 一种药物组合物，其特征在于：该组合物包括：含有治疗有效量的权利要求1所述的母核为吡啶并咪唑的化合物、药学上可接受的盐、水合物或溶剂合物及药学上可接受的载体。
  10. 权利要求1所述的母核为吡啶并咪唑的化合物或者其药学上可接受的盐，或者权利要求9所述的药物组合物在制备NLRP3炎症小体抑制剂中的应用。
  11. 权利要求1所述的母核为吡啶并咪唑的化合物或者其药学上可接受的盐，或者权利要求9所述的药物组合物在制备治疗炎症相关疾病的药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述炎症相关疾病选自风湿性关节炎，类风湿性关节炎，炎性肠道疾病，痛风，动脉粥样硬化，非酒精性脂肪肝病，冷吡啉相关周期性综合征，慢性肾脏疾病，糖尿病和神经退行性疾病等，所述神经退行性疾病选自帕金森症，多发性硬化症，阿尔兹海默症，亨廷顿病。
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