Benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitors and their synthesis method and application

Abstract

The present invention belongs to the field of biomedicine technology, and in particular, relates to a benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor and its synthesis method and application. Starting from the dominant skeleton of marine natural products, the present invention designs and synthesizes a benzo[5,6]spiroacetal lactone compound with a general formula (I) structure, which exhibits significant activity in inhibiting NLRP3 inflammasome, and some compounds IC 50 The values ​​are all in the hundreds of nanomolar range, and the results in in vitro experiments are comparable to the inhibitory effects of similar compounds such as glibenclamide and JC124 that have been reported. In addition, in in vivo anti-inflammatory experiments, compounds S6 and S13 can effectively inhibit the activation of NLRP3 inflammasomes and reduce the release of pro-inflammatory factors such as IL-1β and IL-18. They can be used to prepare drugs for the treatment of diseases related to abnormal activation of NLRP3 inflammasomes, such as gout, rheumatoid arthritis, type 2 diabetes, neurodegenerative diseases, and atherosclerosis. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor, a synthesis method and application thereof. Background Art

[0002] The NLRP3 (Nod-like receptor family pyrin domain containing 3) inflammasome is an important component of the body's innate immune system. It mediates inflammatory responses and pyroptosis by recognizing a variety of danger signals inside and outside the cell, such as pathogenic microorganisms, cellular metabolites, and environmental irritants. Activation of the NLRP3 inflammasome triggers the activation of Caspase-1, which in turn promotes the maturation and secretion of inflammatory cytokines IL-1β and IL-18. These inflammatory factors play a key role in a variety of acute and chronic inflammatory diseases, including gout, non-alcoholic fatty liver disease (NAFLD), atherosclerosis, Alzheimer's disease, and colorectal cancer. In addition, excessive activation of the NLRP3 inflammasome is also closely related to autoinflammatory diseases and pathological inflammation associated with infection.

[0003] At present, the treatment strategies for NLRP3 inflammasome mainly focus on inhibiting its activation process. However, the activation mechanism of NLRP3 inflammasome is complex and is regulated by multiple upstream signaling pathways, including potassium ion efflux, accumulation of reactive oxygen species (ROS), and lysosomal rupture, which poses a challenge to its targeted drug development. Although some small molecule inhibitors (such as MCC950) have shown good inhibitory effects, their clinical application is limited by pharmacokinetic properties, insufficient selectivity or potential toxicity issues. Therefore, the development of efficient, specific and safe NLRP3 inflammasome inhibitors has become a hot spot and difficulty in the treatment of inflammatory diseases. It will complement ongoing molecular and genetic studies to accurately define the role of NLRP3 inflammasome in the pathogenesis of related human diseases and develop into a potential treatment method ( J. Med. Chem. 2018, 61 ,5412).

[0004] Spiroacetals are important chemical structures in many natural products and drug molecules. Many marine toxins, such as pinnatoxins and pteriatoxins, are natural compounds with a spiroacetal skeleton in their molecules ( Tetrahedron Lett. 2009, 50 , 1550). In 2015, Jee H. Jung Nemopilema nomurai Derived marine fungi Paecilomyces variotiiThree new benzospiroacetal derivatives were isolated and showed excellent pharmacological activities ( J. Nat. Prod. 2015, 78 , 2832). However, the current research on the activity of spiroacetal skeleton compounds is still focused on the fields of antibacterial, anticancer and antiviral activities. Few studies have used them as NLRP3 inflammasome inhibitors for the treatment of gout, rheumatoid arthritis, type 2 diabetes, neurodegenerative diseases and atherosclerosis. Summary of the invention

[0005] In view of the above problems, the first object of the present invention is to provide a novel benzo[5,6]spiroacetal lactone compound with significant NLRP3 inflammasome inhibitory activity.

[0006] The second object of the present invention is to provide a method for synthesizing benzo[5,6]spiroacetal lactone compounds with readily available raw materials, simple and efficient.

[0007] The third object of the present invention is to provide the use of the above-mentioned benzo[5,6]spiroacetal lactone compounds as NLRP3 inflammasome inhibitors.

[0008] Many marine natural products have spiroacetal skeletons and exhibit excellent pharmacological activities. Inspired by this, the present invention designs and synthesizes a class of NLRP3 inflammasome inhibitors with benzo[5,6]spiroacetal lactone as the parent core, and verifies in vitro and in vivo experiments that such compounds can inhibit the activation of NLRP3 inflammasomes, thereby treating diseases related to NLRP3 inflammasomes.

[0009] In order to achieve the above object, the present invention provides an NLRP3 inflammasome inhibitor, characterized in that it is an acetal lactone compound with a benzo[5,6] spiro ring as a parent core, and the NLRP3 inflammasome inhibitor is a compound shown in the general formula (I):

[0010]

[0011] Wherein, R is phenyl, substituted phenyl or 4-biphenyl, phenylphenyl, 2-naphthyl, 2-furyl, methyl, dimethylacetal, or phenylethynyl.

[0012] The substituted phenyl group is selected from any one of 2-bromophenyl, 2-methylphenyl, 3-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 4-fluorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl and 4-cyanophenyl.

[0013] Preferably, R is 3-methoxyphenyl or styryl.

[0014] In some specific technical solutions, the compound structural formula is as follows:

[0015]

[0016]

[0017] .

[0018] The present invention also provides a method for synthesizing a NLRP3 inflammasome inhibitor benzo[5,6]spiroacetal lactone compound represented by general formula (I), comprising the following steps:

[0019]

[0020] (1) Using the substituted aldehyde compound 1 as the starting material, it first undergoes a Herry reaction with nitromethane and then dehydrates to obtain a nitroolefin compound 2, which is further reacted with a reducing agent to obtain an intermediate 3, which is then subjected to a Herry reaction under base catalysis to obtain a nitroalcohol compound 4.

[0021] (2) Compound 5 is used as a raw material to undergo an esterification reaction under base catalysis to generate compound 6, which is then subjected to an Aldol reaction with formaldehyde or polyformaldehyde and then dehydrated to generate compound 7.

[0022] (3) The nitro alcohol compound 4 and compound 7 undergo a cascade reaction of Michael reaction, hemiketalization and intramolecular ester exchange under base catalysis to finally obtain a benzo[5,6]spiroacetal lactone compound 8 represented by the general formula (I).

[0023] Reaction 1: Preparation of compound 2

[0024] In a corresponding solvent, compound 1 is reacted with nitromethane at -40-110°C for 30-2160 minutes under the catalysis of a base.

[0025] The solvent is one of toluene, nitromethane, methanol and tetrahydrofuran; the preferred solvent is nitromethane.

[0026] The base is an inorganic base, such as one of calcium chloride, ammonium acetate, sodium hydroxide, and potassium hydroxide; or an organic base, such as one of triethylamine, imidazole, lithium aluminum hydride, and potassium tert-butoxide.

[0027] The molar ratio of the compound 1, the base and nitromethane is 1:(0.1-2.0):(3-100); the preferred molar ratio is 1:0.7:75.

[0028] Reaction 2: Preparation of compound 3

[0029] In an organic solvent, compound 2 is reacted with a reducing agent at 0-40° C. for 30-1440 minutes.

[0030] The organic solvent is one of dichloromethane, isopropanol, methanol and tetrahydrofuran; the preferred solvent is isopropanol.

[0031] The reducing agent is one of sodium borohydride and Hantzsch ester.

[0032] The molar ratio of the compound 2 to the reducing agent is 1:(1.1-3.0); the preferred molar ratio is 1:1.2.

[0033] Reaction 3: Preparation of compound 4:

[0034] In a corresponding solvent, compound 3 is reacted with paraformaldehyde at 0-40°C for 60-720 minutes under base catalysis.

[0035] The solvent is one of ethanol, methanol and tetrahydrofuran; preferably the solvent is tetrahydrofuran.

[0036] The alkali is one of sodium hydroxide, potassium hydroxide and sodium acetate.

[0037] The molar ratio of the compound 3, the base and the paraformaldehyde is 1:(0.1-1.1):(0.7-1.2); the preferred molar ratio is 1:0.3:0.9.

[0038] Reaction 4: Preparation of compound 6:

[0039] In an organic solvent, compound 5 is reacted with iodomethane under alkaline conditions at 0-60° C. for 30-300 minutes.

[0040] The organic solvent is one of acetonitrile, DMF and acetone; the preferred solvent is DMF.

[0041] The base is one of potassium carbonate, sodium bicarbonate and triethylamine.

[0042] The molar ratio of the compound 5, the base and methyl iodide is 1:(1.0-2.0):(1.5-4.0); the preferred molar ratio is 1:1.5:3.0.

[0043] Reaction 5: Preparation of compound 7:

[0044] In the corresponding solvent, compound 6 undergoes Aldol reaction with paraformaldehyde under the action of a base, and is dehydrated under acidic conditions at 25-80° C. for 30-1800 minutes to obtain the target compound 7.

[0045] The solvent is one of methanol, ethanol, tetrahydrofuran and DMF; preferably the solvent is tetrahydrofuran.

[0046] The base is one of diisopropylamine, sodium hydroxide, potassium carbonate, iPr2NH•TFA, tripotassium phosphate and diethylamine.

[0047] The acid is one of TFA and hydrochloric acid.

[0048] The molar ratio of the compound 6, base, acid and paraformaldehyde is 1:(0.5-2.0):(0.05-0.2):(2.0-5.0); the preferred molar ratio is 1:1.0:0.1:4.0.

[0049] Reaction 6: Preparation of compound 8:

[0050] In a solvent, in the presence of a basic catalyst, compound 4 reacts with compound 7 at 0-40° C. for 60-1080 minutes to obtain target compound 8.

[0051] The solvent is one of toluene, tetrahydrofuran, ether, dichloromethane, chloroform and cyclopentyl methyl ether; preferably, the solvent is chloroform.

[0052] The alkaline catalyst is one of cesium carbonate, 18-crown-6+ potassium carbonate, potassium hydroxide, triethylamine and sodium acetate.

[0053] The molar ratio of the compound 7, the catalyst and the compound 4 is 1:(0.1-0.5):(1.0-2.0); the preferred molar ratio is 1:1.0:0.2:1.2.

[0054] The present invention also provides a use of a benzo[5,6]spiroacetal lactone compound of the general formula (I) according to any of the above technical solutions in the preparation of an NLRP3 inflammasome inhibitor.

[0055] The present invention also provides an NLRP3 inflammasome inhibitor drug, which is prepared by using the benzo[5,6]spiroacetal lactone compound of general formula (I) described in any of the above technical solutions.

[0056] The present invention also provides the use of the benzo[5,6]spiroacetal lactone compound of the general formula (I) described above in the preparation of a drug for preventing or treating a disease associated with abnormal activation of the NLRP3 inflammasome.

[0057] The diseases associated with abnormal activation of NLRP3 inflammasomes include gout, rheumatoid arthritis, type 2 diabetes, neurodegenerative diseases, inflammatory diseases, and atherosclerosis, etc. The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease; the inflammatory diseases include sepsis, acute peritonitis, acute pancreatitis, etc.

[0058] The present invention has the following advantages:

[0059] (1) In view of the shortcomings of existing NLRP3 inflammasome inhibitors, a novel class of benzo[5,6]spiroacetal lactone compounds was rationally designed and synthesized based on the dominant skeletons in marine natural products, which can serve as NLRP3 inflammasome inhibitors.

[0060] (2) The compounds synthesized in the present invention have significant activity in inducing IL-1β production in RAW264.7, J774A.1 and BMDM macrophages. Among them, the representative compounds S6 and S13 have outstanding inhibitory activity, with IC 50 The value reached less than 0.1 μM. At the same time, S6 and S13 could significantly inhibit the release of IL-1β and IL-18 in LPS-induced acute peritonitis in mice, without affecting the release of another inflammatory factor TNF-α, indicating that S6 and S13 compounds can specifically inhibit NLRP3 inflammasomes and have a certain selectivity.

[0061] (3) The target product can be directly obtained from commercially available raw materials through simple transformation, and multi-step reactions can be continued without purification, which reduces the difficulty of synthesis and is suitable for large-scale preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The graph shows the effects of compounds S6 (10 mg / kg), S13 (10 mg / kg) and MCC950 (10 mg / kg) on ​​LPS-induced IL-1β (A), IL-18 (B) and TNF-α (C) production in C57BL / 6 mice.

[0063] In the figure, A: Effects of compounds S6, S13 and MCC950 on LPS-induced IL-1β production in C57BL / 6 mice; B: Effects of compounds S6, S13 and MCC950 on LPS-induced IL-18 production in C57BL / 6 mice; C: Effects of compounds S6, S13 and MCC950 on LPS-induced TNF-α production in C57BL / 6 mice.

[0064] A: *P<0.05, **P<0.01, NS-not statistically significant. DETAILED DESCRIPTION

[0065] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0066] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. The following abbreviations may be used in this specification.

[0067]

[0068] The synthesis of the compound of general formula (I) in the present invention is shown below, and each substituent is as defined in the summary of the invention.

[0069] Synthesis route of compounds S1-S16:

[0070]

[0071]

[0072]

[0073] Synthesis route of compound S17:

[0074]

[0075]

[0076] Synthesis route of compound S18:

[0077]

[0078]

[0079]

[0080] The present invention is described in detail below through specific synthesis examples:

[0081] Embodiment 1:

[0082] Preparation and data of compound S1:

[0083] In a 50 mL original bottom flask equipped with a magnetic stirring rotor, compound 1 benzaldehyde (530 mg, 5 mmol, 1.0 equiv.) was dissolved in MeNO2 (20 mL), and NH4OAc (270 mg, 3.5 mmol, 0.7 equiv.) was added. The reaction solution was refluxed for 2 h. After the reaction was completed as detected by TLC, the reaction solution was concentrated under vacuum and directly recrystallized with methanol. After filtration, it was dried to obtain a yellow solid compound 2 (685 mg, yield 92%).

[0084] In a 50 mL original bottom flask equipped with a magnetic stirring rotor, Hantzsch ester (1.2 g, 4.8 mmol, 1.2 equiv.) was added to a solution of compound 2 (596 mg, 4 mmol, 1.0 equiv.). i -PrOH (15 mL) solution, stirred at 40 ° C for 12 h, until TLC detection showed the disappearance of compound 2. The solution was concentrated under vacuum, and the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 ~ 10 / 1) to obtain colorless oily compound 3 (513 mg, yield 85%).

[0085] At room temperature, compound 3 (498 mg, 3.3 mmol, 1.0 equiv.), (HCHO) n (90 mg, 3.0 mmol, 0.9 equiv.) and NaOAc (82 mg, 1.0 mmol, 0.3 equiv.) were added to a 25 mL original bottom flask equipped with a magnetic stirring rotor, and then dissolved with THF (12 mL), followed by reaction at room temperature and stirring for 10 h. After the reaction was completed by TLC, 20 mL of water was added to quench the reaction and extracted with ethyl acetate three times. The ethyl acetate organic phase was dried over sodium chloride and sodium sulfate in turn, filtered, and the organic phase solution was concentrated under vacuum. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1) gave compound 4 as a yellow oil (472 mg, yield 79%).

[0086] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, K2CO3 (621 mg, 4.5 mmol, 1.5 equiv.) and MeI (561 μL, 9 mmol, 3.0 equiv.) were added to a DMF solution (10 mL) of 2-acetylbenzoic acid 5 (492 mg, 3 mmol, 1.0 equiv.) and reacted at 25 °C for 3 h. TLC detection was performed until the starting material 5 disappeared. After the reaction was completed, the reaction mixture was diluted with water, and the aqueous phase was extracted three times with ethyl acetate. The organic phase after extraction was washed three times with saturated sodium chloride solution, then dried with sodium chloride and sodium sulfate in turn, filtered, and concentrated under reduced pressure to obtain a colorless oil compound 6 (523 mg, yield 98%), which was used for the next reaction without further purification.

[0087] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, compound 6 (445 mg, 2.5 mmol, 1.0 equiv.) and (HCHO) n(150 mg, 5 mmol, 2.0 equiv.) in dry THF (10 mL) was added i Pr2NH•TFA (538 mg, 2.5 mmol, 1.0 equiv.) and TFA (19 μL, 0.25 mmol, 0.1 equiv.) were added. The reaction mixture was stirred at reflux for 2 h, cooled to room temperature, and HCHO (0.25 mmol, 0.1 equiv.) was added for the second time. n (150 mg, 5 mmol, 2.0 equiv.). Next, the reaction mixture was stirred under reflux for 24 h. Then it was cooled, the solvent was removed under reduced pressure, dissolved in ethyl acetate, and washed three times with 1M hydrochloric acid, 1M sodium hydroxide and saturated sodium chloride solution. The resulting solution was dried with sodium chloride and sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain colorless oily compound 7 (418 mg, yield 88%).

[0088] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, compound 4 (217 mg, 1.2 mmol, 1.2 equiv.) and TEA (28 μL, 0.2 mmol, 0.2 equiv.) were added to a solution of compound 7 (190 mg, 1 mmol, 1.0 equiv.) in CHCl3 (10 mL) in sequence, and the reaction solution was stirred at 25°C. The starting material 7 disappeared after 12 h of reaction according to TLC detection. After the reaction was completed, the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to finally obtain compound S1 (278 mg, yield 82%) as a white solid. 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0Hz, 1H), 7.68 (td, J = 7.5, 1.2 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.45(dt, J = 7.6, 1.0 Hz, 1H), 7.37 – 7.30 (m, 3H), 7.14 – 7.02 (m, 2H), 4.68(dd, J = 12.9, 2.9 Hz, 1H), 4.34 (d, J = 12.9 Hz, 1H), 3.23 (d, J= 14.1 Hz,1H), 3.03 (d, J = 14.1 Hz, 1H), 2.76 – 2.65 (m, 1H), 2.45 – 2.19 (m, 2H), 1.89 (ddd, J = 14.2, 3.9, 2.3 Hz, 1H). 13 C NMR (101 MHz, CDCl3) d 168.0,147.7, 135.0, 132.3, 131.2, 129.8, 129.0, 128.3, 126.1, 125.9, 122.5, 104.7,87.9, 68.8, 43.6, 29.8, 26.9. HRMS: [M+H] + calcd. For C 19 H 18 NO5 + 340.1179;found: 340.1182.

[0089] Embodiment 2:

[0090] Preparation and data of compound S2:

[0091] Referring to the method of Example 1, benzaldehyde was replaced by 2-bromobenzaldehyde to obtain a white solid compound S2 (total yield 44%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.68 (td, J =7.5, 1.1 Hz, 1H), 7.64 – 7.54 (m, 2H), 7.44 (dt, J = 7.6, 1.0 Hz, 1H), 7.32 –7.23 (m, 1H), 7.18 (td, J = 7.7, 1.7 Hz, 1H), 7.04 (dd, J = 7.7, 1.8 Hz, 1H),4.70 (dd, J = 12.9, 3.0 Hz, 1H), 4.43 (d, J = 12.9 Hz, 1H), 3.42 (d, J = 14.4Hz, 1H), 3.37 (d, J= 14.4 Hz, 1H), 2.72 (ddt, J = 14.8, 5.0, 2.6 Hz, 1H),2.56 (ddd, J = 14.8, 13.3, 4.7 Hz, 1H), 2.26 (ddd, J = 14.6, 13.3, 4.8 Hz,1H), 1.90 (ddd, J = 14.6, 4.7, 2.4 Hz, 1H). 13 C NMR (101 MHz, CDCl3) d 168.0,147.7, 134.9, 133.6, 132.7, 131.4, 131.2, 129.9, 128.1, 126.1, 125.8, 125.7,122.5, 104.6, 88.8, 68.5, 41.9, 29.9, 26.7. HRMS: [M+H] + calcd. For C 19 H 17 BrNO5 + 418.0285; found: 418.0281.

[0092] Embodiment 3:

[0093] Preparation and data of compound S3:

[0094] Referring to the method of Example 1, benzaldehyde was replaced by 2-methylbenzaldehyde to obtain a white solid compound S3 (total yield 47%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.68 (td, J = 7.5, 1.1 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.44 (dt, J = 7.5, 0.9Hz, 1H), 7.26 – 7.16 (m, 2H), 7.15 (td, J = 6.6, 5.8, 3.0 Hz, 1H), 7.02 –6.95 (m, 1H), 4.72 (dd, J= 12.8, 3.0 Hz, 1H), 4.34 (d, J = 12.8 Hz, 1H),3.26 (d, J = 14.5 Hz, 1H), 3.16 (d, J = 14.6 Hz, 1H), 2.80 – 2.70 (m, 1H), 2.40 (td, J = 14.2, 13.8, 4.5 Hz, 1H), 2.31 (s, 3H), 2.25 (td, J = 13.7, 4.4Hz, 1H), 1.88 (ddd, J = 14.4, 4.5, 2.4 Hz, 1H), 1.26 (s, 1H). 13 C NMR (101MHz, CDCl3) d 168.0, 147.7, 137.1, 134.9, 131.2, 131.2, 131.0, 130.6, 128.2,126.4, 126.1, 125.8, 122.5, 104.7, 88.4, 68.6, 39.8, 29.9, 26.9, 20.0. HRMS:[M+H] + calcd. For C 20 H 20 NO5 + 354.1336; found: 354.1335.

[0095] Embodiment 4:

[0096] Preparation and data of compound S4:

[0097] Referring to the method of Example 1, benzaldehyde was replaced by 3-bromobenzaldehyde to obtain white solid compound S4 (total yield 55%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.68 (td, J =7.5, 1.1 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.23(t, J = 1.8 Hz, 1H), 7.20 (t,J = 7.9 Hz, 1H), 6.99 (dt, J = 7.8, 1.3 Hz,1H), 4.67 (dd, J = 12.9, 2.9 Hz, 1H), 4.33 (d, J = 12.8 Hz, 1H), 3.19 (d, J =14.1 Hz, 1H), 2.98 (d, J = 14.1 Hz, 1H), 2.76 – 2.63 (m, 1H), 2.43 – 2.23 (m,2H), 1.95 – 1.85 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 167.8, 147.6, 135.0,134.5, 132.8, 131.5, 131.2, 130.5, 128.4, 126.1, 125.9, 123.0, 122.5, 104.6,87.7, 68.7, 42.9, 29.8, 26.9. HRMS: [M+H] + calcd. For C 19 H 17 BrNO5 + 418.0285;found: 418.0288.

[0098] Embodiment 5:

[0099] Preparation and data of compound S5:

[0100] Referring to the method of Example 1, benzaldehyde was replaced by 3-methylbenzaldehyde to obtain a white solid compound S5 (total yield 35%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.68 (td, J = 7.5, 1.1 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.53 – 7.44 (m, 1H), 7.21(t, J = 7.9 Hz, 1H), 7.13 (d, J= 7.5 Hz, 1H), 6.94 – 6.81 (m, 2H), 4.68 (dd, J = 12.9, 3.0 Hz, 1H), 4.32 (d, J = 12.9 Hz, 1H), 3.19 (d, J = 14.0 Hz, 1H),2.99 (d, J = 14.0 Hz, 1H), 2.76 – 2.67 (m, 1H), 2.44 – 2.20 (m, 5H), 1.98 –1.81 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 168.0, 147.7, 138.6, 134.9, 132.2,131.2, 130.5, 129.0, 128.8, 126.8, 126.1, 125.8, 122.5, 104.7, 87.9, 68.8,43.5, 29.8, 26.9, 21.5. HRMS: [M+H] + calcd. For C 20 H 20 NO5 + 354.1336; found:354.1333.

[0101] Embodiment 6:

[0102] Preparation and data of compound S6:

[0103] Referring to the method of Example 1, benzaldehyde was replaced by 3-methoxybenzaldehyde to obtain a white solid compound S6 (total yield 59%). 1 H NMR (400 MHz, CDCl3) d 7.85 (dt, J = 7.6, 1.0 Hz, 1H), 7.68(td, J = 7.5, 1.2 Hz, 1H), 7.58 (td, J = 7.5, 1.0 Hz, 1H), 7.44 (dt, J = 7.7,0.9 Hz, 1H), 7.24 (dd, J = 8.3, 7.5 Hz, 1H), 6.85 (ddd, J= 8.4, 2.6, 1.0 Hz,1H), 6.65 (ddd, J = 7.5, 1.7, 0.9 Hz, 1H), 6.60 (dd, J = 2.5, 1.7 Hz, 1H),4.67 (dd, J = 12.9, 2.9 Hz, 1H), 4.32 (d, J = 12.9 Hz, 1H), 3.79 (s, 3H), 3.20 (d, J = 14.0 Hz, 1H), 3.00 (d, J = 14.0 Hz, 1H), 2.76 – 2.66 (m, 1H), 2.43 – 2.22 (m, 2H), 1.93 – 1.83 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 168.0,159.9, 147.7, 134.9, 133.7, 131.2, 130.0, 126.1, 125.8, 122.5, 122.1, 115.6,113.4, 104.7, 87.9, 68.8, 55.3, 43.6, 29.8, 26.9. HRMS: [M+H] + calcd. ForC 20 H 20 NO6 + 370.1285; found: 370.1285.

[0104] Embodiment 7:

[0105] Preparation and data of compound S7:

[0106] Referring to the method of Example 1, benzaldehyde was replaced by 4-fluorobenzaldehyde to obtain white solid compound S7 (total yield 55%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.68 (td, J =7.5, 1.2 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.44 (dt, J= 7.7, 1.0 Hz,1H), 7.09 – 6.96 (m, 4H), 4.66 (dd, J = 12.9, 2.9 Hz, 1H), 4.33 (d, J = 12.9Hz, 1H), 3.20 (d, J = 14.2 Hz, 1H), 2.99 (d, J = 14.3 Hz, 1H), 2.73 – 2.60 (m, 1H), 2.45 – 2.23 (m, 2H), 2.00 – 1.84 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 167.9, 163.9, 161.5, 147.6, 135.0, 131.4, 131.3, 131.2, 128.1, 128.0, 126.1,125.9, 122.5, 116.1, 115.9, 104.6, 87.9, 68.7, 42.7, 29.8, 26.8. 19 F NMR (376MHz, CDCl3) d -113.8. HRMS: [M+H] + calcd. For C 19 H 17 FNO5 + 358.1085; found:358.1086.

[0107] Embodiment 8:

[0108] Preparation and data of compound S8:

[0109] Referring to the method of Example 1, benzaldehyde was replaced by 4-bromobenzaldehyde to obtain white solid compound S8 (total yield 45%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.69 (td, J =7.5, 1.2 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.50 – 7.41 (m, 3H), 6.98 –6.90 (m, 2H), 4.66 (dd, J= 12.9, 3.0 Hz, 1H), 4.32 (d, J = 12.9 Hz, 1H),3.18 (d, J = 14.1 Hz, 1H), 2.97 (d, J = 14.1 Hz, 1H), 2.74 – 2.63 (m, 1H), 2.41 – 2.22 (m, 2H), 1.97 – 1.84 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 167.9,147.6, 135.0, 132.2, 131.4, 131.2, 131.2, 126.0, 125.9, 122.5, 122.5, 104.6,87.7, 68.7, 42.8, 29.8, 26.8. HRMS: [M+H] + calcd. For C 19 H 17 BrNO5 + 418.0285;found: 418.0282.

[0110] Embodiment 9:

[0111] Preparation and data of compound S9:

[0112] Referring to the method of Example 1, benzaldehyde was replaced by 4-methylbenzaldehyde to obtain white solid compound S9 (total yield 49%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.5, 1.0 Hz, 1H), 7.68 (td, J = 7.5, 1.2 Hz, 1H), 7.58 (td, J = 7.5, 1.0 Hz, 1H), 7.44 (dd, J = 7.6, 1.0Hz, 1H), 7.13 (d, J = 7.8 Hz, 2H), 7.01 – 6.89 (m, 2H), 4.67 (dd, J = 12.9,2.9 Hz, 1H), 4.32 (d, J = 12.9 Hz, 1H), 3.18 (d, J= 14.0 Hz, 1H), 2.99 (d, J = 14.1 Hz, 1H), 2.77 – 2.67 (m, 1H), 2.45 – 2.18 (m, 5H), 1.90 – 1.82 (m,1H). 13 C NMR (101 MHz, CDCl3) d 168.0, 147.7, 138.0, 134.9, 131.1, 129.7,129.6, 129.2, 126.1, 125.8, 122.5, 104.7, 88.0, 68.7, 43.2, 29.8, 26.8, 21.2.HRMS: [M+H] + calcd. For C 20 H 20 NO5 + 354.1336; found: 354.1331.

[0113] Embodiment 10:

[0114] Preparation and data of compound S10:

[0115] Referring to the method of Example 1, benzaldehyde was replaced by 4-methoxybenzaldehyde to obtain a white solid compound S10 (total yield 48%). 1 H NMR (400 MHz, CDCl3) d 7.85 (dt, J = 7.7, 1.0 Hz, 1H), 7.69(dd, J = 7.5, 1.1 Hz, 1H), 7.58 (td, J = 7.5, 1.0 Hz, 1H), 7.44 (dt, J = 7.7,1.0 Hz, 1H), 7.03 – 6.95 (m, 2H), 6.89 – 6.76 (m, 2H), 4.66 (dd, J = 12.9,2.9 Hz, 1H), 4.31 (d, J = 12.9 Hz, 1H), 3.79 (s, 3H), 3.16 (d, J = 14.2 Hz,1H), 2.97 (d, J= 14.2 Hz, 1H), 2.78 – 2.62 (m, 1H), 2.41 – 2.21 (m, 2H), 1.93 – 1.84 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 168.0, 159.5, 147.7, 134.9,131.1, 130.8, 126.1, 125.8, 124.2, 122.5, 114.4, 104.7, 88.1, 68.7, 55.4,42.9, 29.9, 26.8. HRMS: [M+H] + calcd. For C 20 H 20 NO6 + 370.1285; found: 370.1287.

[0116] Embodiment 11:

[0117] Preparation and data of compound S11:

[0118] Referring to the method of Example 1, benzaldehyde was replaced by 4-cyanobenzaldehyde to obtain white solid compound S11 (total yield 53%). 1 H NMR (400 MHz, CDCl3) d 7.87 (dt, J = 7.6, 1.0 Hz, 1H), 7.69 (td, J = 7.5, 1.1 Hz, 1H), 7.66 – 7.62 (m, 2H), 7.60 (td, J = 7.6, 1.0 Hz, 1H), 7.44(dt, J = 7.6, 0.9 Hz, 1H), 7.22 – 7.14 (m, 2H), 3.29 (d, J = 14.0 Hz, 1H),3.06 (d, J = 14.0 Hz, 1H), 2.71 – 2.63 (m, 1H), 2.48 – 2.14 (m, 2H), 1.90(ddd, J = 14.2, 4.0, 2.4 Hz, 1H). 13 C NMR (101 MHz, CDCl3) d167.8, 147.4,137.7, 135.0, 132.7, 131.3, 130.6, 126.0, 125.9, 122.5, 118.4, 112.5, 104.5,87.5, 68.6, 43.3, 29.7, 26.9. HRMS: [M+H] + calcd. For C 20 H 17 N2O5 + 365.1132;found: 365.1129.

[0119] Embodiment 12:

[0120] Preparation and data of compound S12:

[0121] Referring to the method of Example 1, benzaldehyde was replaced by 4-biphenylcarboxaldehyde to obtain white solid compound S12 (total yield 57%). 1 H NMR (400 MHz, CDCl3) d 7.87 (dt, J = 7.6, 1.0 Hz, 1H), 7.69 (td, J =7.5, 1.1 Hz, 1H), 7.64 – 7.52 (m, 5H), 7.49 – 7.40 (m, 3H), 7.40 – 7.32 (m,1H), 7.18 – 7.11 (m, 2H), 4.72 (dd, J = 12.9, 2.9 Hz, 1H), 4.37 (d, J = 12.9Hz, 1H), 3.27 (d, J = 14.0 Hz, 1H), 3.07 (d, J = 14.0 Hz, 1H), 2.75 (ddd, J =13.9, 3.8, 2.3 Hz, 1H), 2.41 (td, J = 13.6, 4.1 Hz, 1H), 2.30 (td, J = 13.6,4.1 Hz, 1H), 1.90 (ddd, J = 14.2, 4.1, 2.3 Hz, 1H) .13 C NMR (101 MHz, CDCl3) d168.0, 147.7, 141.1, 140.4, 134.9, 131.3, 131.2, 130.2, 128.9, 127.6, 127.2,126.1, 125.8, 122.5, 104.7, 88.0, 68.8, 43.2, 29.8, 26.9. HRMS: [M+H] + calcd. For C 25 H 22 NO5 + 416.1492; found: 416.1495.

[0122] Embodiment 13:

[0123] Preparation and data of compound S13:

[0124] Referring to the method of Example 1, benzaldehyde was replaced by cinnamaldehyde to obtain white solid compound S13 (total yield 43%). 1 H NMR (400 MHz, CDCl3) d 7.87 (dt, J = 7.5, 1.0 Hz, 1H), 7.69 (td, J =7.5, 1.2 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.46 (dd, J = 7.6, 0.9 Hz,1H), 7.39 – 7.22 (m, 5H), 6.50 (dt, J = 15.5, 1.3 Hz, 1H), 6.00 (dt, J =15.4, 7.6 Hz, 1H), 4.71 (dd, J = 13.0, 2.9 Hz, 1H), 4.29 (d, J = 13.0 Hz,1H), 2.91 (dq, J = 10.2, 2.1 Hz, 1H), 2.79 (ddd, J = 14.4, 7.6, 1.3 Hz, 1H),2.66 (ddd, J = 14.3, 7.6, 1.3 Hz, 1H), 2.43 – 2.26 (m, 2H), 1.99 – 1.86 (m,1H). 13C NMR (101 MHz, CDCl3) d 167.9, 147.7, 136.5, 136.2, 135.0, 131.2,128.8, 128.2, 126.6, 126.1, 125.8, 122.5, 119.3, 104.7, 87.6, 68.5, 41.0,29.8, 26.9. HRMS: [M+H] + calcd. For C 21 H 20 NO5 + 366.1336; found: 366.1332.

[0125] Embodiment 14:

[0126] Preparation and data of compound S14:

[0127] Referring to the method of Example 1, benzaldehyde was replaced by 2-naphthaldehyde to obtain white solid compound S14 (total yield 46%). 1 H NMR (400 MHz, CDCl3) d 7.90 – 7.78 (m, 4H), 7.67 (td, J = 7.5, 1.1 Hz,1H), 7.62 – 7.54 (m, 2H), 7.54 – 7.47 (m, 2H), 7.44 (dt, J = 7.7, 1.0 Hz,1H), 7.16 (dd, J = 8.4, 1.8 Hz, 1H), 4.73 (dd, J = 12.9, 2.9 Hz, 1H), 4.40(d, J = 12.9 Hz, 1H), 3.40 (d, J = 14.1 Hz, 1H), 3.19 (d, J = 14.1 Hz, 1H),2.74 (ddt, J = 14.6, 4.8, 2.6 Hz, 1H), 2.44 (ddd, J = 14.6, 13.4, 4.6 Hz,1H), 2.29 (ddd, J = 14.5, 13.4, 4.5 Hz, 1H), 1.89 (ddd, J= 14.5, 4.6, 2.4 Hz, 1H). 13 C NMR (101 MHz, CDCl3) d 167.9, 147.7, 134.9, 133.4, 133.0, 131.2,129.8, 129.0, 128.7, 127.9, 127.8, 127.4, 126.6, 126.5, 126.1, 125.8, 122.5,104.7, 88.0, 68.9, 43.7, 29.8, 27.0. HRMS: [M+H] + calcd. For C 23 H 20 NO5 + 390.1336; found: 390.1339.

[0128] Embodiment 15:

[0129] Preparation and data of compound S15:

[0130] Referring to the method of Example 1, benzaldehyde was replaced with 2-furancarboxaldehyde to obtain white solid compound S15 (total yield 50%). 1 H NMR (400 MHz, CDCl3) d 7.86 (dt, J = 7.6, 1.0 Hz, 1H), 7.69 (td, J =7.5, 1.1 Hz, 1H), 7.59 (td, J = 7.5, 1.0 Hz, 1H), 7.45 (dt, J = 7.7, 0.9 Hz,1H), 7.38 (dd, J = 1.9, 0.8 Hz, 1H), 6.33 (dd, J = 3.2, 1.9 Hz, 1H), 6.16(dd, J = 3.2, 0.8 Hz, 1H), 4.67 (dd, J = 13.0, 3.0 Hz, 1H), 4.32 (d, J = 13.1Hz, 1H), 3.26 (d, J = 15.2 Hz, 1H), 3.14 (d, J= 15.2 Hz, 1H), 2.84 – 2.69 (m, 1H), 2.49 – 2.26 (m, 2H), 1.98 – 1.84 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 167.9, 147.7, 146.9, 143.3, 134.9, 131.2, 126.1, 125.8, 122.5, 110.8, 109.9,104.5, 87.2, 68.2, 36.2, 29.8, 27.0. HRMS: [M+H] + calcd. For C 17 H 16 NO6 + 330.0972; found: 330.0968.

[0131] Embodiment 16:

[0132] Preparation and data of compound S16:

[0133] Referring to the method of Example 1, benzaldehyde was replaced by formaldehyde to obtain white solid compound S16 (total yield 43%). 1 H NMR (400 MHz, CDCl3) d 7.89 – 7.78 (m, 1H), 7.69 (td, J = 7.5, 1.1 Hz,1H), 7.59 (td, J = 7.6, 1.1 Hz, 1H), 7.52 – 7.37 (m, 1H), 4.66 (dd, J = 13.0,2.9 Hz, 1H), 4.20 (d, J = 13.0 Hz, 1H), 2.98 – 2.77 (m, 1H), 2.43 – 2.23 (m,2H), 1.96 – 1.80 (m, 1H), 1.58 (s, 3H). 13 C NMR (101 MHz, CDCl3) d 168.0,147.7, 134.9, 131.2, 126.1, 125.8, 122.5, 104.5, 84.4, 69.1, 29.9, 28.8,24.2. 13C NMR (101 MHz, CDCl3) δ 167.9, 147.7, 146.9, 143.3, 134.9, 131.2,126.1, 125.8, 122.5, 110.8, 109.9, 104.5, 87.2, 68.2, 36.2, 29.8, 27.0. HRMS:[M+H] + calcd. For C 13 H 14 NO5 + 264.0866; found: 264.0865.

[0134] Embodiment 17:

[0135] Preparation and data of compound S17:

[0136] In a 50 mL original bottom flask equipped with a magnetic stirring rotor, compound 1 glyoxal-1,1-dimethylacetal solution (452 ​​μL, 5 mmol, 1.0 equiv.) was dissolved in MeNO2 (20 mL), and TEA (69 μL, 0.5 mmol, 0.1 equiv.) was added. The reaction was carried out at room temperature for 2 h. After the reaction was completed as detected by TLC, the reaction solution was concentrated under vacuum to obtain colorless oily compound 1' (800 mg, yield 97%), which was used in the next step without further purification.

[0137] At -40 °C, in a 50 mL original bottom flask equipped with a magnetic stirring rotor, TFAA (686 μL, 4.9 mmol, 1.0 equiv.) and TEA (1.4 mL, 9.9 mmol, 2.1 equiv.) were added to a THF (20 mL) solution of 1' (776 mg, 4.7 mmol, 1.0 equiv.) in sequence, and the mixture was stirred for 16 h. After the reaction was completed by TLC monitoring, water was added to quench the reaction, and the mixture was extracted with ethyl acetate three times. The mixture was then dried with sodium chloride and sodium sulfate, filtered, and concentrated under reduced pressure. The reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain compound 2 (525 mg, 76%) as a yellow oil.

[0138] In a 25 mL original bottom flask equipped with a magnetic stirring rotor, Hantzsch ester (911 mg, 3.6 mmol, 1.2 equiv.) was added to a solution of compound 2 (441 mg, 3 mmol, 1.0 equiv.). i-PrOH (10 mL) solution, stirred at 40 ° C for 12 h until TLC detection showed the disappearance of compound 2. The solution was concentrated under vacuum, and the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 ~ 10 / 1) to obtain colorless oily compound 3 (362 mg, yield 81%).

[0139] At room temperature, compound 3 (328 mg, 2.2 mmol, 1.0 equiv.), (HCHO) n (60 mg, 2.0 mmol, 0.9 equiv.) and NaOAc (54 mg, 0.7 mmol, 0.3 equiv.) were added to a 25 mL original bottom flask equipped with a magnetic stirring rotor, and then dissolved with THF (8 mL), followed by reaction at room temperature and stirring for 10 h. After the reaction was completed by TLC, 20 mL of water was added to quench the reaction and extracted with ethyl acetate three times. The ethyl acetate organic phase was dried over sodium chloride and sodium sulfate in turn, filtered, and the organic phase solution was concentrated under vacuum. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1) gave compound 4 as a yellow oil (339 mg, yield 86%).

[0140] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, K2CO3 (621 mg, 4.5 mmol, 1.5 equiv.) and MeI (561 μL, 9 mmol, 3.0 equiv.) were added to a DMF solution (10 mL) of 2-acetylbenzoic acid 5 (492 mg, 3 mmol, 1.0 equiv.) and reacted at 25 °C for 3 h. TLC detection was performed until the starting material 5 disappeared. After the reaction was completed, the reaction mixture was diluted with water, and the aqueous phase was extracted three times with ethyl acetate. The organic phase after extraction was washed three times with saturated sodium chloride solution, then dried with sodium chloride and sodium sulfate in turn, filtered, and concentrated under reduced pressure to obtain a colorless oil compound 6 (523 mg, yield 98%), which was used for the next reaction without further purification.

[0141] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, compound 6 (445 mg, 2.5 mmol, 1.0 equiv.) and (HCHO) n (150 mg, 5 mmol, 2.0 equiv.) in dry THF (10 mL) was added iPr2NH•TFA (538 mg, 2.5 mmol, 1.0 equiv.) and TFA (19 μL, 0.25 mmol, 0.1 equiv.) were added. The reaction mixture was stirred at reflux for 2 h, cooled to room temperature, and HCHO (0.25 mmol, 0.1 equiv.) was added for the second time. n (150 mg, 5 mmol, 2.0 equiv.). Next, the reaction mixture was stirred under reflux for 24 h. Then it was cooled, the solvent was removed under reduced pressure, dissolved in ethyl acetate, and washed three times with 1M hydrochloric acid, 1M sodium hydroxide and saturated sodium chloride solution. The resulting solution was dried with sodium chloride and sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain colorless oily compound 7 (418 mg, yield 88%).

[0142] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, compound 4 (215 mg, 1.2 mmol, 1.2 equiv.) and TEA (28 μL, 0.2 mmol, 0.2 equiv.) were added to a solution of compound 7 (190 mg, 1 mmol, 1.0 equiv.) in CHCl3 (10 mL) in sequence, and the reaction solution was stirred at 25°C. The starting material 7 disappeared after 12 h of reaction according to TLC detection. After the reaction was completed, the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to finally obtain compound S17 (276 mg, yield 82%) as a white solid. 1 H NMR (400 MHz, CDCl3) d 7.88 – 7.80 (m, 1H),7.68 (td, J = 7.6, 1.2 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.45 (d, J = 7.5Hz, 1H), 4.67 (dd, J = 13.1, 2.9 Hz, 1H), 4.43 (t, J = 5.4 Hz, 1H), 4.24 (d, J = 13.1 Hz, 1H), 3.31 (d, J = 4.3 Hz, 6H), 2.94 – 2.85 (m, 1H), 2.42 – 2.28(m, 2H), 2.22 (dd, J = 14.8, 5.9 Hz, 1H), 2.08 (dd,J = 14.8, 4.8 Hz, 1H),1.91 – 1.84 (m, 1H). 13 C NMR (101 MHz, CDCl3) d 167.9, 147.7, 134.9, 131.2,126.1, 125.8, 122.5, 104.6, 100.6, 85.3, 68.6, 53.9, 53.6, 40.3, 29.7, 27.7.HRMS: [M+H] + calcd. For C 16 H 20 NO7 + 338.1234; found: 338.1232.

[0143] Embodiment 18:

[0144] Preparation and data of compound S18:

[0145] Under N2 protection, LiAlH4 (19 mg, 0.5 mmol, 0.1 equiv.) and dry THF (20 mL) solution were added to a 50 mL original bottom flask equipped with a magnetic stirring rotor at 0°C. After stirring for 2 minutes, compound 1 phenylpropioaldehyde (611 μL, 5 mmol, 1.0 equiv.) and MeNO2 (805 μL, 15 mmol, 3.0 equiv.) were added, and the reaction was carried out at 0°C for 5 h. After the reaction was completed by TLC, 1M hydrochloric acid was added to quench the reaction, and the mixture was extracted with ethyl acetate three times. The mixture was then dried with sodium chloride and sodium sulfate, filtered, concentrated under reduced pressure, and the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain yellow oily compound 1' (621 mg, 65%).

[0146] At -40 °C, in a 25 mL original bottom flask equipped with a magnetic stirring rotor, TFAA (445 μL, 3.2 mmol, 1.0 equiv.) and TEA (874 μL, 6.3 mmol, 2.1 equiv.) were added to a THF (10 mL) solution of 1' (573 mg, 3 mmol, 1.0 equiv.) in sequence, and the mixture was stirred for 16 h. After the reaction was completed by TLC monitoring, water was added to quench the reaction, and the mixture was extracted with ethyl acetate three times. The mixture was then dried with sodium chloride and sodium sulfate, filtered, and concentrated under reduced pressure. The reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain compound 2 (420 mg, 81%) as a yellow oil.

[0147] In a 25 mL original bottom flask equipped with a magnetic stirring rotor, Hantzsch ester (658 mg, 2.6 mmol, 1.2 equiv.) was added to a solution of compound 2 (381 mg, 2.2 mmol, 1.0 equiv.). i -PrOH (8 mL) solution, stirred at 40 ° C for 12 h, until TLC detection showed the disappearance of compound 2. The solution was concentrated under vacuum, and the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1 ~ 10 / 1) to obtain colorless oily compound 3 (335 mg, yield 87%).

[0148] At room temperature, compound 3 (315 mg, 1.8 mmol, 1.0 equiv.), (HCHO) n (49 mg, 1.6 mmol, 0.9 equiv.) and NaOAc (44 mg, 0.5 mmol, 0.3 equiv.) were added to a 25 mL original bottom flask equipped with a magnetic stirring rotor, and then dissolved with THF (8 mL), followed by reaction at room temperature and stirring for 10 h. After the reaction was completed by TLC, 20 mL of water was added to quench the reaction and extracted with ethyl acetate three times. The ethyl acetate organic layer was dried over sodium chloride and sodium sulfate in turn, filtered, and the organic phase solution was concentrated under vacuum. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1) gave compound 4 as a yellow oil (284 mg, yield 77%).

[0149] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, K2CO3 (621 mg, 4.5 mmol, 1.5 equiv.) and MeI (561 μL, 9 mmol, 3.0 equiv.) were added to a DMF solution (10 mL) of 2-acetylbenzoic acid 5 (492 mg, 3 mmol, 1.0 equiv.) and reacted at 25 °C for 3 h. TLC detection was performed until the starting material 5 disappeared. After the reaction was completed, the reaction mixture was diluted with water, and the aqueous phase was extracted three times with ethyl acetate. The organic phase after extraction was washed three times with saturated sodium chloride solution, then dried with sodium chloride and sodium sulfate in turn, filtered, and concentrated under reduced pressure to obtain a colorless oil compound 6 (523 mg, yield 98%), which was used for the next reaction without further purification.

[0150] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, compound 6 (445 mg, 2.5 mmol, 1.0 equiv.) and (HCHO) n(150 mg, 5 mmol, 2.0 equiv.) in dry THF (10 mL) was added i Pr2NH•TFA (538 mg, 2.5 mmol, 1.0 equiv.) and TFA (19 μL, 0.25 mmol, 0.1 equiv.) were added. The reaction mixture was stirred at reflux for 2 h, cooled to room temperature, and HCHO (0.25 mmol, 0.1 equiv.) was added for the second time. n (150 mg, 5 mmol, 2.0 equiv.). Next, the reaction mixture was stirred under reflux for 24 h. Then it was cooled, the solvent was removed under reduced pressure, dissolved in ethyl acetate, and washed three times with 1M hydrochloric acid, 1M sodium hydroxide and saturated sodium chloride solution. The resulting solution was dried with sodium chloride and sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain colorless oily compound 7 (418 mg, yield 88%).

[0151] In a 25 mL round-bottom flask equipped with a magnetic stirring rotor, compound 4 (246 mg, 1.2 mmol, 1.2 equiv.) and TEA (28 μL, 0.2 mmol, 0.2 equiv.) were added to a solution of compound 7 (190 mg, 1 mmol, 1.0 equiv.) in CHCl3 (10 mL) in sequence, and the reaction solution was stirred at 25°C. The starting material 7 disappeared after 12 h of reaction according to TLC detection. After the reaction was completed, the reaction mixture was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to finally obtain compound S18 (312 mg, yield 86%) as a white solid. 1 H NMR (400 MHz,CDCl3) d 7.88 (dt, J = 7.6, 1.0Hz, 1H), 7.71 (td, J = 7.5, 1.1 Hz, 1H), 7.61 (td, J = 7.5, 1.0 Hz, 1H), 7.49(dt, J = 7.6, 1.0 Hz, 1H), 7.46 – 7.39 (m, 2H), 7.36 – 7.28 (m, 3H), 4.77(dd, J = 13.0, 2.9 Hz, 1H), 4.40 (d, J = 13.0 Hz, 1H), 3.11 – 2.92 (m, 3H), 2.52 (td,J = 13.7, 4.3 Hz, 1H), 2.41 (td, J = 13.7, 4.2 Hz, 1H), 1.97 (ddd, J = 14.2, 4.3, 2.3 Hz, 1H). 13 C NMR (101 MHz, CDCl3) d 167.8, 147.6, 135.0,131.9, 131.2, 128.8, 128.5, 126.2, 125.9, 122.5, 122.3, 104.5, 86.2, 86.1,80.1, 68.1, 29.9, 29.0, 26.9. HRMS: [M+H] + calcd. For C 21 H 18 NO5 + 364.1179;found: 364.1180.

[0152] Embodiment 19:

[0153] In vitro, the inhibitory effect of the above-mentioned target compounds on NLRP3 inflammasome activation was verified by detecting the release of the inflammatory factor IL-1β.

[0154] RAW264.7 and J774A.1 cells were plated in 96-well plates at 1 × 10 cells per well. 5 cells. After 12 h, bacterial lipopolysaccharide (LPS) (1 μg / mL) was added and incubated at 37°C for 4.5 h; the above target compound was added and incubated at 37°C for 0.5 h, followed by the addition of 5 mM adenosine triphosphate (ATP). After 30 min, the cell culture supernatant was collected and centrifuged to remove debris (1000 g, 4°C, 10 min). The supernatant was used for ELISA to detect IL-1β. The inhibitory activity of the target compound on NLRP3 inflammasome was calculated. The experimental results are shown in Table 1-2 below:

[0155] Table 1 The activity of target compounds in inhibiting the release of IL-1β from mouse macrophages RAW264.7 induced by LPS / ATP

[0156]

[0157] a Table 1 IC 50Values: +++++ is ≤0.1μM; ++++ is ≤0.5μM; +++ is ≤1μM; ++ is ≤10μM; + is ≤30μM. The data in the table are the average values ​​of three independent experiments.

[0158] Table 2 The activity of the target compounds in inhibiting the release of IL-1β from mouse macrophages J774A.1 induced by LPS / ATP

[0159]

[0160] a Table 2 IC 50 Values: +++++ is ≤0.1μM; ++++ is ≤0.5μM; +++ is ≤1μM; ++ is ≤10μM; + is ≤30μM. The data in the table are the average values ​​of three independent experiments.

[0161] Mouse bone marrow-derived immune cells were extracted from healthy adult C57BL / 6 mice (6-8 weeks old) and cultured in complete medium containing M-CSF (20 ng / mL) to differentiate into BMDM. The differentiated BMDM cells were collected and counted, and the BMDM cells were seeded in a 96-well plate at a density of 1×10 per well. 5 cells. After 12 h, bacterial lipopolysaccharide (LPS) (1 μg / mL) was added and incubated at 37°C for 4.5 h; the above target compound was added and incubated at 37°C for 30 min, followed by the addition of 5 mM adenosine triphosphate (ATP). After 30 min, the cell culture supernatant was collected and centrifuged to remove debris (1000 g, 4°C, 10 min). The supernatant was used for ELISA to detect IL-1β. The inhibitory activity of the target compound on NLRP3 inflammasome was calculated. The experimental results are shown in Table 3:

[0162] Table 3 The activity of the target compounds in inhibiting the release of IL-1β from BMDM macrophages of C57BL / 6 mice

[0163]

[0164] a Table 3 IC 50 Value: +++++ is ≤0.1μM; ++++ is ≤0.5μM; +++ is ≤1μM; ++ is ≤10μM; + is ≤30μM. The data in the table are the average values ​​of three independent experiments.

[0165] From the data in Tables 1-3, it can be seen that the designed compounds all have significant activity in inducing IL-1β production in RAW264.7, J774A.1 and BMDM macrophages. Among them, the IC of the inhibitory activity of most compounds is 50The values ​​were all less than 10 μM. The effects of compounds S6 and S13 in RAW264.7 and J774A.1 macrophages were comparable to those of similar compounds glibenclamide and JC-124 reported previously. In addition, in BMDM macrophages, the drug effects of compounds S6 and S13 were similar to those of the reported MCC950 compound. In addition, the inhibitory activities of representative compounds S6 and S13 were outstanding, with IC values ​​of 50 The values ​​were all lower than 0.1 μM.

[0166] Example 20: Inhibitory experiment of target compounds S6 and S13 on LPS-induced peritonitis in mice.

[0167] Experimental methods:

[0168] (1) 6-8 week old male C57BL / 6 mice were randomly divided into five groups, with 6 mice in each group. The specific grouping and treatment were as follows:

[0169] Group 1: blank, 6 rats; Group 2: blank vehicle was injected intraperitoneally, followed by LPS (35 mg / kg) injection 1 hour later, 6 rats; Groups 3-5: positive drug MCC950 (10 mg / kg), target compounds S6 (10 mg / kg) and S13 (10 mg / kg) were injected intraperitoneally, followed by LPS (35 mg / kg) injection 1 hour later, 6 rats each.

[0170] (2) 2.5 hours after LPS intraperitoneal injection, blood was collected from the eyeball, allowed to stand for 1 hour, and then centrifuged at 3500 r / min for 20 minutes.

[0171] (3) The blood supernatant obtained in step (2) was used to measure the IL-1β, IL-18, and TNF-α contents by ELISA.

[0172] Experimental results (such as Figure 1 In the in vivo anti-inflammatory experiment, the representative compounds S6 and S13 could significantly inhibit the release of IL-1β and IL-18 in LPS-induced acute peritonitis in mice ( Figure 1 Middle A, Figure 1 The experimental results are consistent with the pharmacological activity of the positive group drug MCC950, which can inhibit the expression of IL-1β and IL-18 without affecting the release of another inflammatory factor TNF-α ( Figure 1 (C) shows that S6 and S13 compounds can specifically inhibit NLRP3 inflammasome and have a certain selectivity.

[0173] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor, characterized in that: The structure is shown in general formula (I): ； Wherein, R is one of phenyl, substituted phenyl, 4-biphenyl, styryl, 2-naphthyl, 2-furyl, methyl, dimethylacetal, and phenylethynyl; The substituted phenyl group is selected from any one of 2-bromophenyl, 2-methylphenyl, 3-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 4-fluorophenyl, 4-bromophenyl, 4-methylphenyl, 4-methoxyphenyl and 4-cyanophenyl.

2. The benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor according to claim 1, characterized in that The R is 3-methoxyphenyl or styryl.

3. A method for synthesizing the benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor according to claim 1 or 2, characterized in that: The following steps are involved: ； (1) Using the substituted aldehyde compound 1 as the starting material, it first undergoes a Herry reaction with nitromethane and then undergoes dehydration to obtain a nitroolefin compound 2, which is further reacted with a reducing agent to obtain an intermediate 3, which is then subjected to a Herry reaction under base catalysis to obtain a nitroalcohol compound 4; (2) Compound 5 is used as a raw material to undergo an esterification reaction under base catalysis to generate compound 6, which is then subjected to an Aldol reaction with formaldehyde or paraformaldehyde and then dehydrated to generate compound 7; (3) The nitro alcohol compound 4 and compound 7 undergo a cascade reaction of Michael reaction, hemiketalization and intramolecular ester exchange under base catalysis to finally obtain a benzo[5,6]spiroacetal lactone compound 8 represented by the general formula (I).

4. The method for synthesizing the benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor according to claim 3, characterized in that: In step (1), the reducing agent is one of sodium borohydride and Hantzsch ester.

5. The method for synthesizing the benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor according to claim 3, characterized in that: In step (2), the base is one of potassium carbonate, sodium bicarbonate and triethylamine.

6. The method for synthesizing the benzo[5,6]spiroacetal lactone NLRP3 inflammasome inhibitor according to claim 3, characterized in that: In step (3), the base is one of cesium carbonate, 18-crown-6+ potassium carbonate, potassium hydroxide, triethylamine, and sodium acetate.

7. Use of the benzo[5,6]spiroacetal lactone compound according to claim 1 or 2 in the preparation of NLRP3 inflammasome inhibitors.

8. Use of the benzo[5,6]spiroacetal lactone compound according to claim 1 or 2 in the preparation of a drug for treating diseases associated with abnormal activation of NLRP3 inflammasome.

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