Cis-alkenyl amide-pyrazole derivative and application thereof in anti-inflammatory drugs

Abstract

The invention discloses a cis-alkenyl amide-pyrazole derivative and application of the cis-alkenyl amide-pyrazole derivative in anti-inflammatory drugs. The compound has the effects of resisting inflammation, treating lung injury and chronic obstructive pulmonary diseases and the like.

Description

Technical Field

[0001] The present application belongs to the field of medical technology, and specifically relates to cis-enamide-pyrazole derivatives, which have anti-inflammatory, pneumonia-treating and lung injury effects. Background Art

[0002] Pneumonia is an important symptom associated with many lung diseases such as chronic obstructive pulmonary disease and acute respiratory distress syndrome. It is mainly manifested by diffuse inflammation in the lungs. Pathological manifestations include damage to alveolar epithelial cells and capillary endothelial cells, as well as diffuse pulmonary interstitial edema and alveolar edema. The main causes of pneumonia include infections by bacteria, viruses, rickettsia, mycoplasma, fungi, etc. When pollutants or bacteria enter the lungs through the respiratory tract, they will be deposited in alveolar cells, phagocytosed by macrophages and release cytokines to attract immune cells, neutrophils and other inflammatory cells, thereby stimulating lung epithelial cells to release inflammatory factors such as IL-6, TNF-α, IL-1β, etc. to cause inflammation. Continuous inflammatory response further damages lung cells, leading to decreased lung function and inducing a pro-inflammatory cascade, which leads to lung damage.

[0003] The lead compound N-methyl-N-cis-styrene-cinnamamide (lansiumamide B) is a cis-enamide compound isolated from the kernel of the yellow peel fruit, which has multiple pharmacological activities such as treating obesity, anti-diabetes, antibacterial, and anti-inflammatory. However, the cis-trans configuration of the enamide in the structure of lansiumamide B affects its biological activity, and the biological activity of the cis structure is significantly stronger than that of the trans structure. Pyrazole, also known as Pyrazole in English, is a nitrogen-containing aromatic compound that is often used as a building block of drug molecules in medicinal chemistry. Pyrazole and its derivatives have multiple pharmacological activities such as antibacterial, antiviral, antitumor, anti-inflammatory, and antidepressant, and are therefore widely used in the medical field. For example, pyrazole compounds have potential application value in the treatment of cancer, cardiovascular disease, dementia, diabetes, etc. This application aims to combine the cis-enamide structure with the common pyrazole structure in medicinal chemistry to develop drugs for anti-inflammatory, pneumonia, and lung injury. Summary of the invention

[0004] The purpose of the present invention is to provide cis-enamide-pyrazole derivatives and their use in preparing anti-inflammatory drugs.

[0005] One or more embodiments of the present application provide a compound of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof.

[0006]

[0007] Wherein, R1 is C6-14 aryl, five to fourteen membered heteroaryl, C6-10 cycloalkyl, five to fourteen membered heterocyclyl, C1-C6 alkyl, C1-C6 cycloalkyl, halogen, cyano; Optionally, the C6-14 aryl, five to fourteen membered heteroaryl, C6-10 cycloalkyl, or five to fourteen membered heterocyclyl is substituted by one or more substituents selected from halogen, halogenated C1-C6 alkyl, carbonyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, carboxyl, cyano, hydroxyl, carbonyl, phenyl-SO2-, nitro, C1-C6 alkyloxycarbonyl; the five to fourteen membered heteroaryl or five to fourteen membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; Preferably, the halogen is F, Cl, Br or I;

[0008] R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 haloalkyl, C1-C6 haloalkoxy;

[0009] In one or more embodiments, when the amide group is located at the 5-position of the pyrazole, R1 is located at the 3-position of the pyrazole.

[0010] In one or more embodiments, when the amide group is located at the 3-position of the pyrazole, R1 is located at the 5-position of the pyrazole.

[0011] In one or more embodiments, Formula I is Wherein, R1 is independently tert-butyl, methyl, or phenyl, and R2 is hydrogen, fluorine, chlorine, bromine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

[0012] In one or more embodiments, Formula I is Wherein, R1 is independently methyl, tert-butyl, or phenyl, and R2 is hydrogen, fluorine, chlorine, bromine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

[0013] In one or more embodiments, Formula I is R1 is independent of

[0014] One or more embodiments of the present application provide a compound or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated substance thereof:

[0015]

[0016] One or more embodiments of the present application provide a method for preparing a compound of formula I,

[0017]

[0018] Wherein, R1 and R2 are as described above;

[0019] The preparation method comprises: (1)

[0021]

[0022] a) adding the compounds of formula I and formula II to a sodium ethoxide solution at -20°C-40°C, and reacting at 20°C-50°C for 8-12 hours;

[0023] b) adding the product compound of formula III obtained in step a) to an anhydrous methanol solution of the compound of formula IV at 0°C, heating the mixture to room temperature and stirring for 1-2 hours, and then reacting at 60°C-90°C for 2-4 hours;

[0024] c) dissolving the product compound of formula V obtained in step b) in an ammonia methanol solution and reacting at 20° C.-50° C. for 2-8 days;

[0025] d) reacting the product compound of formula VI obtained in step c) with the compound of formula VII at 40° C.-80° C. for 0.5-3 hours;

[0026] e) reacting the product compound of formula VIII obtained in step d) with sodium hydride at -20°C-5°C with stirring for 0.5-1 hour, then adding iodomethane at this temperature, and finally raising the temperature to 20°C-50°C with stirring for 2-6 hours to obtain a compound of formula IX; (2)

[0028]

[0029] f) adding the product compound of formula III obtained in step a) to an anhydrous ethanol solution of the compound of formula IV at 0°C, heating the mixture to room temperature and stirring for 1-2 hours, and then reacting at 60°C-90°C for 5-10 hours;

[0030] g) dissolving the product compound of formula X obtained in step f) in an ammonia methanol solution and reacting at 20° C.-50° C. for 3-9 days;

[0031] h) reacting the product compound of formula XI obtained in step g) with the compound of formula VII at 40° C.-80° C. for 1-4 hours;

[0032] i) reacting the product compound of formula XII obtained in step h) with sodium hydride at -20°C-10°C with stirring for 0.5-1 hour, then adding iodomethane at this temperature, and finally raising the temperature to 20°C-50°C with stirring for 3-7 hours to obtain a compound of formula XIII; (3)

[0034]

[0035] j) stirring the compound of formula IV, the compound of formula XIV and methyl propiolate at 0°C for 5 minutes, then adding potassium carbonate, potassium bromide and potassium peroxymonosulfonate double salt (Oxone), and the reaction temperature was raised to 20°C-40°C and stirred for 9-12 hours;

[0036] k) dissolving the product compound of formula XV obtained in step j) in an ammonia methanol solution and reacting at 20° C.-50° C. for 3-10 days;

[0037] l) reacting the product compound of formula XVI obtained in step k) with the compound of formula VII at 40° C.-80° C. for 1-4 hours;

[0038] m) reacting the product compound of formula XVII obtained in step l) with sodium hydride at -20°C-10°C with stirring for 0.5-1 hour, then adding iodomethane at this temperature, and finally raising the temperature to 20°C-50°C with stirring for 3-7 hours to obtain a compound of formula XVIII;

[0039] Wherein, R1 and R2 are as described in any one of claims 1-5.

[0040] One or more embodiments of the present application provide a pharmaceutical composition comprising a compound of the present application and a pharmaceutically acceptable adjuvant or excipient.

[0041] One or more embodiments of the present application provide use of the compound of the present application or the pharmaceutical composition of the present application in the preparation of a drug for preventing and / or treating inflammatory diseases and respiratory diseases.

[0042] One or more embodiments of the present application provide a compound of the present application for use as a medicament.

[0043] One or more embodiments of the present application provide the pharmaceutical composition of the present application, which is used as a medicament.

[0044] One or more embodiments of the present application provide a compound or composition of the present application for preventing and / or treating inflammatory diseases and respiratory diseases.

[0045] One or more embodiments of the present application provide a compound or composition of the present application for preventing and / or treating diseases mediated by IL-1β, IL-6, or TNF-α.

[0046] One or more embodiments of the present application provide a compound or composition of the present application for inhibiting IL-1β, IL-6, and TNF-α.

[0047] One or more embodiments of the present application provide a method for preventing and / or treating inflammatory diseases and respiratory diseases, which comprises administering the compound or composition of the present application to a subject in need thereof.

[0048] One or more embodiments of the present application provide a method for preventing and / or treating diseases mediated by IL-1β, IL-6, and TNF-α, which comprises administering a compound or composition of the present application to a subject in need thereof.

[0049] One or more embodiments of the present application provide a method for inhibiting IL-1β, IL-6, and TNF-α, which comprises administering a compound or composition of the present application to a subject in need thereof.

[0050] In one or more embodiments, the inflammatory disease is an inflammatory lung disease.

[0051] In one or more embodiments, the respiratory disease is acute lung injury or pneumonia.

[0052] One or more embodiments of the present application provide the use of the compound of the present application or the pharmaceutical composition of the present application in the preparation of a drug or IL-1β, IL-6, TNF-α inhibitor for preventing and / or treating IL-1β, IL-6, TNF-α mediated diseases.

[0053] In one or more embodiments, the compounds of the present application have anti-inflammatory and psoriasis treatment uses. Biological experiments have shown that the compounds of the present application can significantly reduce the number of inflammatory cells in an inflammatory model, or reduce the levels of IL-1β, IL-6, and TNF-α in cells.

[0054] In one or more embodiments, the results of the lung injury model mouse experiment show that the compounds of the present application can significantly improve the severity of alveolar atrophy, inflammatory infiltration and fibrous collagen formation in mice, indicating that the compounds of the present application can reduce the release of proinflammatory factors and the production of proinflammatory mediators to inhibit the occurrence and development of inflammatory responses, and can also protect the lungs of mice and alleviate lung injury.

[0055] The following is an explanation of the terms used in the technical solution of the present application. As used in the specification and the appended claims, unless otherwise specifically stated, the terms of the present application have the following meanings:

[0056] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0057] The term "amino" refers to -NH2.

[0058] The term "hydroxy" refers to -OH.

[0059] "Alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group of 1 to 20 carbon atoms, preferably an alkyl group of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) carbon atoms, more preferably an alkyl group of 1 to 6 carbon atoms, and further preferably an alkyl group of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and various branched isomers thereof; when the alkyl group is substituted, it may be optionally further substituted by one or more substituents.

[0060] "Alkoxy" refers to a group formed by replacing at least one carbon atom in an alkyl group with an oxygen atom. Non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexyloxy, cyclopropyloxy and cyclobutyloxy. The definition of alkyl is the same as that of "alkyl" described above.

[0061] "Aryl" refers to a substituted or unsubstituted aromatic ring, which can be, for example, a monocyclic ring of 6 to 8 carbon atoms (e.g., 6, 7, 8 carbon atoms), a bicyclic ring of 6 to 12 carbon atoms (e.g., 6, 7, 8, 9, 10, 11, 12 carbon atoms), or a tricyclic ring system of 10 to 14 carbon atoms (e.g., 10, 11, 12, 13, 14 carbon atoms), which can be a bridged ring or a spirocyclic ring, and non-limiting examples include phenyl and naphthyl. The aryl group can be optionally further substituted by one or more substituents.

[0062] "Heteroaryl" refers to a substituted or unsubstituted aromatic ring, which can be, for example, a 5- to 8-membered (e.g., 5, 6, 7, 8-membered) monocyclic ring, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10- to 14-membered (e.g., 10, 11, 12, 13, 14-membered) tricyclic ring system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O or S, preferably a 5- to 8-membered heteroaryl. The 1 to 4 (e.g., 1, 2, 3, 4) N and S optionally substituted in the heteroaryl ring can be oxidized to various oxidation states. Heteroaryl can be connected to a heteroatom or a carbon atom, and can be a bridged ring or a spiro ring, and non-limiting examples include cyclopyridyl, furanyl, thienyl, pyranyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinyl benzimidazolyl, benzopyridinyl, pyrrolopyridinyl. Heteroaryl is optionally further substituted by one or more substituents.

[0063] "Heterocyclyl" or "heterocycle" refers to a saturated or unsaturated non-aromatic heterocycle, which can be, for example, a 5- to 10-membered (e.g., 5, 6, 7, 8, 9, 10-membered) monocyclic ring, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10- to 14-membered (e.g., 10, 11, 12, 13, 14-membered) tricyclic ring system, and contains 1 to 4 (e.g., 1, 2, 3, 4) heteroatoms selected from N, O, or S, preferably a 5- to 8-membered heterocyclyl. The 1 to 4 (e.g., 1, 2, 3, 4) N and S optionally substituted in the ring of the "heterocyclyl" or "heterocycle" can be oxidized to various oxidation states; the "heterocyclyl" or "heterocycle" can be attached to a heteroatom or a carbon atom; the "heterocyclyl" or "heterocycle" can be a bridged ring or a spiro ring. Non-limiting examples of “heterocyclyl” or “heterocycle” include oxirane, oxirane, aziridine, oxetanyl, azetidinyl, thietanyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxhexacyclyl, azepanyl, oxetanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, pyridinyl, piperidinyl, homopiperidinyl, furanyl, pyridinyl, piperidinyl, homopiperidinyl, furanyl, pyridinyl, piperidinyl, piperidinyl, homopiperidinyl, pyrid ... pyranyl, thiophene, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, 1,3-dithianyl, dihydrofuranyl, dithiolanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl oxazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuranyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-dioxolane, pyrazolinyl, dithianyl, dithiolanyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 3-azabicyclo[3 .1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolylquinolizinyl, N-pyridylurea, 1,1-dioxothiomorpholinyl, azabicyclo[3.2.1]octanyl, azabicyclo[5.2.0]nonanyl, oxatricyclo[5.3.1.1]dodecyl, azaadamantyl and oxaspiro[3.3]heptanyl. The "heterocyclyl" or "heterocycle" may be optionally further substituted by one or more substituents.

[0064] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group, which can be, for example, a monocyclic, bicyclic or polycyclic ring of 6 to 10 carbon atoms (e.g., 6, 7, 8, 9, 10 carbon atoms), preferably 6 to 8 carbon atoms. Non-limiting examples of "cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. When the cycloalkyl is substituted, it can be optionally further substituted by one or more substituents.

[0065] "Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" refers to a salt of the compound of the present invention that retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, and the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

[0066] "Pharmaceutical composition" refers to a mixture of one or more compounds described herein, their pharmaceutically acceptable salts or prodrugs and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable carriers, excipients and / or one or more other therapeutic agents.

[0067] "Carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0068] "Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, and disintegrants.

[0069] "Prodrug" refers to a compound of the present invention that can be converted into a biologically active compound through in vivo metabolism. The prodrug of the present invention is prepared by modifying the amino or carboxyl group in the compound of the present invention, and the modification can be removed by conventional operations or in vivo to obtain the parent compound. When the prodrug of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free amino or carboxyl group.

[0070] "Co-crystal" refers to a crystal formed by the active pharmaceutical ingredient (API) and the co-crystal former (CCF) under the action of hydrogen bonds or other non-covalent bonds, in which the pure state of API and CCF are solid at room temperature and there is a fixed stoichiometric ratio between the components. Co-crystal is a multi-component crystal, including binary eutectics formed between two neutral solids and multi-component eutectics formed between neutral solids and salts or solvates.

[0071] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformational isomers.

[0072] "Optional" or "optionally" or "selective" or "selectively" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "heterocyclyl optionally substituted with alkyl" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclyl group is substituted with alkyl group and instances where the heterocyclyl group is not substituted with alkyl group. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The inhibitory effects of the cis-enamide-pyrazole derivatives in Example 1 on the cytotoxicity of Raw264.7 cells and on the mRNA levels of NO, IL-6, and IL-1β inflammatory factors were compared with those of the control group. * P<0.05, ** P<0.01, *** P<0.001; compared with the model group # P<0.05, ## P<0.01, ### P<0.001.

[0074] Figure 2 The compound in Example 2 showed the inhibitory effect on the protein levels of inflammatory factors IL-6 and IL-1β in the Raw264.7 cell model, compared with the control group. **** P<0.0001; compared with LPS group ## P<0.01, #### P<0.0001.

[0075] Figure 3 It shows the change of body weight of mice in Activity Example 3.

[0076] Figure 4 The total number of inflammatory cells in the alveolar lavage fluid of mice and the total number of leukocytes in the whole blood in Example 3 were compared with those in the control group. **** P<0.0001; compared with the model group # P<0.05, ## P<0.01, #### P<0.0001.

[0077] Figure 5 The HE staining pathological picture of the mouse lung tissue in Example 3 is shown, magnification: 200×; Scalebar=200 μm.

[0078] Figure 6 The Masson staining image of mouse lung tissue in Example 3 shows the activity, magnification: 200×; Scale bar = 200 μm.

[0079] Figure 7 The figure shows the immunohistochemical staining of macrophage iNOS, Arg-1 and neutrophil LY6G markers in mouse lung tissue in Example 3. Specific embodiments

[0080] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only for the purpose of explanation and are not intended to limit the scope and essence of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0081] Example 1 Synthesis of Series A Compounds

[0082] (1) Synthesis of Compound 2

[0083]

[0084] Take 100mL sealed tube and add anhydrous ethanol (6mL), slowly add 20% sodium ethoxide solution (2.1mL, 10mmol), and cool in an ice-water bath. Pinacolone (1.0g, 10mmol) and diethyl oxalate (1.46g, 10mmol) are premixed, and then the precooled sodium ethoxide solution is added dropwise. After the addition is complete, return to room temperature and continue the reaction for 12h. After TLC monitoring and tracking until the raw material disappears or there is no change, acidify with 20% dilute sulfuric acid in an ice-water bath to pH = 3, filter with diatomaceous earth, and wash with dichloromethane (3×20mL), and extract the aqueous phase with dichloromethane (3×20mL). After combining the organic phases, they were washed with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate for 30 minutes, and concentrated under reduced pressure. The crude product was separated and purified using a silica gel chromatography column with petroleum ether and ethyl acetate (V / V=5:1) as the eluent to obtain 1.4 g of a yellow liquid product (Compound 2) with a yield of 70%.

[0085] (2) Synthesis of Compound 3

[0086]

[0087] Add anhydrous methanol (5mL) to a 15mL sealed tube, then add phenylhydrazine (0.324g, 3mmol), slowly add 5,5-dimethyl-2,4-dioxohexanoic acid methyl ester (0.60g, 3.0mmol) under ice bath cooling, return to room temperature and stir to react for 1h, then reflux to react for 2h. After monitoring by TLC until the raw material disappears or remains unchanged, remove the oil bath, wait for the reaction liquid to cool to room temperature naturally, collect the organic phase with ethyl acetate, concentrate under reduced pressure, separate and purify the obtained crude product on a silica gel chromatography column, and use petroleum ether and ethyl acetate (V / V=10:1) as the eluent to obtain 0.416g of a light yellow solid product with a yield of 51%.

[0088] (3) Synthesis of Compound 4

[0089]

[0090] Add 5-tert-butyl-1-phenylpyrazole-3-carboxylic acid ethyl ester (0.544 g, 2 mmol) and 7 mol / L ammonia methanol solution (4 mL) to a 15 mL sealed tube, and stir at room temperature for 7 days. After TLC monitoring until the raw material disappears or remains unchanged, collect the organic phase with acetone, concentrate under reduced pressure, and separate and purify the obtained crude product on a silica gel chromatography column using petroleum ether and acetone (V / V=1:1) as eluents to obtain 0.46 g of a white solid product with a yield of 94%.

[0091] (4) Synthesis of Compound 5

[0092]

[0093] Argon was filled into a dry 10mL Schlenk tube, compound 4 (243mg, 1mmol), cuprous iodide (9.5mg, 0.05mmol), cesium carbonate (407mg, 1.25mmol) were added, (Z)-2-iodovinylbenzene (253mg, 1.1mmol) was diluted with dry anhydrous tetrahydrofuran (5mL) and added to the reaction tube, and N,N'-dimethylethylenediamine (DMEDA, 10.8μL, 0.1mmol) was added dropwise, and the reaction was continued in a 65℃ oil bath under sealed conditions for 3h. After natural cooling, the mixture was filtered with diatomaceous earth, washed with ethyl acetate (3×20mL), the organic phase was dried over anhydrous sodium sulfate for 30min and filtered out, and the filtrate was concentrated under reduced pressure. The crude product was chromatographed on a silica gel column to obtain 259mg of a light yellow solid with a yield of 75%.

[0094] (5) Synthesis of Compound A1

[0095]

[0096] Argon was filled into a dry two-necked flask, and compound 5 (173 mg, 0.5 mmol) dissolved in dry anhydrous N,N-dimethylformamide (2 mL) was added, cooled in an ice-water bath, 60% sodium hydride (32 mg, 0.8 mmol) was added, and the mixture was reacted at the same temperature for 30 min, and then iodomethane (142 mg, 1 mmol) was added and reacted for 3 h. After the reaction, ice water was added, and the mixture was extracted with ethyl acetate (3×20 mL), washed with saturated sodium chloride solution (20 mL), and the organic phase was dried over anhydrous sodium sulfate for 30 min and filtered out, and the filtrate was concentrated under reduced pressure. The crude product was chromatographed on a silica gel column to obtain 160 mg of a yellow oil (compound A1), with a yield of 89%.

[0097] The obtained compound A1 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were:

[0098] Main conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.44-7.37(m,3H),7.33-7.30(m,2H),7.26-7.20(m,4H),7.17-7.13( m,1H),6.87(d,J=8.8Hz,1H),6.43(s,1H),5.86(d,J=8.8Hz,1H),3.02(s,3H),1.09(s,9H). 13 C NMR (100 MHz, CDCl3) δ: 164.6, 153.7, 145.7, 141.9, 135.6, 131.3, 129.5 (2×C), 128.8 (2×C), 128.7 (2×C), 128.4 (2×C), 127.3 (2×C), 119.2, 106.5, 35.8, 32.1, 30.7 (3×C). Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.44-7.37(m,3H),7.33-7.30(m,2H),7.26-7.20(m,4H),7.1 7-7.13(m,1H),6.93(br,1H),6.67(s,1H),6.05(br,1H),3.22(s,3H),1.09(s,9H). 13 C NMR (100 MHz, CDCl3) δ: 164.6, 153.7, 145.7, 141.9, 135.6, 130.2, 129.5 (2×C), 128.8 (2×C), 128.7 (2×C), 128.4 (2×C), 127.3 (2×C), 118.9, 107.3, 38.8, 32.1, 30.7 (3×C). HRMS (ESI) m / z calculated value is C 23 H 26 ON3 + [M+H] + 360.2070, the measured value is 360.2074.

[0099] By selecting different methyl ketones and aryl hydrazines, compounds A1-A10 can be obtained. The compounds A1-A10 are arranged in the following order:

[0100]

[0101] Example 2 Synthesis of Compound A2:

[0102] Synthesis steps refer to Example 1, the hydrazine reagent in the second step reaction is selected as p-fluorophenylhydrazine to obtain compound A2, yield: 88%; main conformational isomers: 1H NMR (400MHz, CDCl3) δ: 7.38-7.35 (m, 2H), 7.33-7.26 (m, 4H), 7.24-7.21 (m, 1H), 7.15 (t, J = 8. 4Hz,2H),6.90(d,J=8.8Hz,1H),6.50(s,1H),5.94(d,J=8.8Hz,1H),3.10(s,3H),1.16(s,9H). 13 C NMR (100MHz, CDCl3) δ: 164.4, 162.9 (d, J = 248Hz), 153.9, 145.8, 137.8 (d, J = 3.4Hz), 135.5, 131.2, 130.5 (d, J =8.8Hz,2×C),128.7(2×C),128.4(2×C),127.3,119.5,115.7(d,J=23Hz,2×C),106.5,35.7,32.0,30.6(3×C). 19 F NMR (376MHz, CDCl3) δ: -111.1. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.38-7.35(m,2H),7.33-7.26(m,4H),7.24-7.21(m,1H),7.15 (t,J=8.4Hz,2H),7.01(br,1H),6.73(s,1H),6.13(br,1H),3.28(s,3H),1.16(s,9H). 13 C NMR (100MHz, CDCl3) δ: 164.4, 162.9 (d, J = 248Hz), 153.9, 145.8, 137.8 (d, J = 3.4Hz), 135.5, 131.2, 130.5 (d, J =8.8Hz,2×C),128.7(2×C),128.4(2×C),127.3,118.9,115.7(d,J=23Hz,2×C),107.4,38.8,32.0,26.9(3×C). 19 F NMR (376 MHz, CDCl3) δ: -111.1. HRMS (ESI) m / z calculated value is C 23 H 25 ON3F + [M+H] + 378.1976, the measured value is 378.1981.

[0103] Example 3 Synthesis of Compound A3:

[0104] Synthesis steps: Refer to Example 1, the hydrazine reagent in the second step reaction is p-chlorophenylhydrazine to obtain compound A3, yield: 86%; main conformational isomers: 1 H NMR (400MHz, CDCl3)δ:7.46-7.44(m,2H),7.34-7.22(m,7H),6.90(d,J=8.8Hz,1H),6.51(s,1H),5.94(d,J=8.8Hz,1H),3.10(s,3H),1.17(s,9H). 13 CNMR (100MHz,CDCl3)δ:164.3,153.9,146.0,140.4,135.5,131.2,130.1(3×C),129.0(2×C),128.7(2×C),128.4(2×C),127.3,119.6,106.7,35.8,32.0,30.7(3×C).Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.46-7.44(m,2H),7.34-7.22(m,7H),7.01(br,1H),6.75(s,1H),6.13(br,1H),3.28(s,3H),1.17(s,9H). 13 C NMR (100 MHz, CDCl3) δ: 164.3, 153.9, 146.0, 140.4, 135.5, 131.2, 130.1 (3×C), 129.0 (2×C), 128.7 (2×C), 128.4 (2×C), 127.3, 119.1, 107.6, 38.8, 32.0, 27.0 (3×C). HRMS (ESI) m / z calculated value is C 23 H 25 ON3Cl + [M+H] + 394.1681, the measured value is 394.1684.

[0105] Example 4 Synthesis of Compound A4:

[0106] Synthesis steps: Refer to Example 1, the hydrazine reagent in the second step reaction is p-bromophenylhydrazine to obtain compound A4, yield: 87%; main conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.61 (d, J = 8.4Hz, 2H), 7.34-7.22 (m, 7H), 6.91 (d, J = 8.8Hz, 1H), 6.52 (s, 1H), 5.95 (d, J = 8.8Hz, 1H), 3.10 (s, 3H), 1.18 (s, 9H). 13C NMR (100MHz, CDCl3) δ: 164.4, 153.9, 146.1, 141.0, 135.6, 132.0 (2×C), 131.2, 130.4 (2×C), 128.8 (2×C), 128.5 (2×C), 127.4, 123.6, 119.6, 106.8, 35.8, 32.1, 30.7 (3×C). Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.61 (d, J = 8.4Hz, 2H), 7.34-7.22 (m, 7H), 7.02 (br, 1H), 6.75 (s, 1H), 6.14 (br, 1H), 3.28 (s, 3H), 1.18 (s, 9H). 13 C NMR (100 MHz, CDCl3) δ: 164.4, 153.9, 146.1, 141.0, 135.6, 132.0 (2×C), 131.2, 130.4 (2×C), 128.8 (2×C), 128.5 (2×C), 127.4, 123.6, 119.1, 107.6, 38.9, 32.1, 30.7 (3×C). HRMS (ESI) m / z calculated value is C 23 H 25 ON3B + [M+H] + 438.1176, measured value is 438.1187.

[0107] Example 5 Synthesis of Compound A5:

[0108] Synthesis steps: Refer to Example 1, the hydrazine reagent in the second step reaction is p-toluhydrazine to obtain compound A5, yield: 84%; main conformational isomers: 1 H NMR (400MHz, CDCl3)δ:7.34-7.22(m,9H),6.97(d,J=8.8Hz,1H),6.51(s,1H),5.93(d,J=8.8Hz,1H),3.10(s,3H),2.45(s,3H),1.17(s,9H). 13 C NMR (100 MHz, CDCl3) δ: 164.7, 153.7, 145.6, 139.6, 139.4, 135.7, 131.4, 129.3 (2×C), 128.8 (2×C), 128.5 (2×C), 128.4 (2×C), 127.2, 119.1, 106.5, 35.8, 32.1, 30.7 (3×C), 21.4. Minor conformational isomers: 1H NMR (400MHz, CDCl3) δ: 7.34-7.22 (m, 9H), 6.97 (d, J = 8.8Hz, 1H), 6.74 (s, 1H), 6.14 (br, 1H), 3.30 (s, 3H), 2.45 (s, 3H), 1.17 (s, 9H). 13 C NMR (100 MHz, CDCl3) δ: 164.7, 153.7, 145.6, 139.6, 139.4, 135.7, 130.2, 129.3 (2×C), 128.8 (2×C), 128.5 (2×C), 128.4 (2×C), 127.2, 120.4, 107.3, 39.0, 32.1, 30.7 (3×C), 21.4. HRMS (ESI) m / z calculated for C 24 H 28 ON3 + [M+H] + 374.2227, the measured value is 374.2229.

[0109] Example 6 Synthesis of Compound A6:

[0110] Synthesis steps: Refer to Example 1, the hydrazine reagent in the second step reaction is p-methoxyphenylhydrazine to obtain compound A6, yield: 96%; main conformational isomers: 1 H NMR (400MHz, CDCl3)δ:7.34-7.21(m,7H),6.96(d,J=8.8Hz,3H),6.50(s,1H),5.93(d,J=8.8Hz,1H),3.88(s,3H),3.10(s,3H),1.17(s,9H). 13 CNMR (100MHz,CDCl3)δ:164.7,160.2,153.9,145.6,135.7,134.7(2×C),131.4,129.9(2×C),128.8(2×C),128.4(2×C),127.2,119.1,113.8,106.4,55.7,35.8,32.1,30.7(3×C).Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.34-7.21 (m, 7H), 6.96 (d, J = 8.8Hz, 3H), 6.73 (s, 1H), 6.13 (br, 1H), 3.88 (s, 3H), 3.30 (s, 3H), 1.17 (s, 9H). 13C NMR (100 MHz, CDCl3) δ: 164.7, 160.2, 153.9, 145.6, 135.7, 134.7 (2×C), 130.2, 129.9 (2×C), 128.8 (2×C), 128.4 (2×C), 127.2, 118.9, 113.8, 107.2, 55.7, 38.8, 32.1, 30.7 (3×C). HRMS (ESI) m / z calculated value is C 24 H 28 O2N3 + [M+H] + 390.2176, the measured value is 390.2177.

[0111] Example 7 Synthesis of Compound A7:

[0112] Synthesis steps refer to Example 1, the hydrazine reagent in the second step reaction is selected as trifluoromethylphenylhydrazine to obtain compound A7, yield: 85%; main conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.76(d,J=8.0Hz,2H),7.54(d,J=8.0Hz,2H),7.35-7.22(m,5H) ,6.89(d,J=8.8Hz,1H),6.55(s,1H),5.96(d,J=8.8Hz,1H),3.11(s,3H),1.19(s,9H). 13 C NMR (100MHz, CDCl3) δ: 164.3, 154.1, 146.4, 145.0, 135.5, 131.7 (q, J = 32.7Hz), 131.1, 129.3 (2×C), 12 8.8(2×C),128.5(2×C),127.4,126.1,126.0,123.7(q,J=270Hz),119.8,107.0,35.8,32.1,30.8(3×C). 19 F NMR (376MHz,CDCl3)δ:-62.6(3×F).Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.76(d,J=8.0Hz,2H),7.54(d,J=8.0Hz,2H),7.35-7.2 2(m,5H),7.02(br,1H),6.79(s,1H),6.16(br,1H),3.29(s,3H),1.19(s,9H). 13C NMR (100MHz, CDCl3) δ: 164.3, 154.3, 146.6, 145.0, 135.5, 131.7 (q, J = 32.7Hz), 130.1, 129.3 (2×C), 12 8.8(2×C),128.5(2×C),127.4,126.1,126.0,123.7(q,J=270Hz),119.2,107.8,38.9,32.1,30.8(3×C). 19 F NMR (376 MHz, CDCl3) δ: -62.6 (3×F). HRMS (ESI) m / z calculated value is C 24 H 25 ON3F3 + [M+H] + 428.1944, the measured value is 428.1947.

[0113] Example 8 Synthesis of Compound A8:

[0114] Synthesis steps: Referring to Example 1, the hydrazine reagent in the second step reaction is p-trifluoromethoxyphenylhydrazine to obtain compound A8 with a yield of 87%; the main conformational isomers are: 1 H NMR (400MHz, CDCl3) δ: 7.41 (d, J = 8.8Hz, 2H), 7.31-7.20 (m, 7H), 6.87 (d, J = 8.8Hz, 1H), 6.49 (s, 1H), 5.93 (d, J = 8.8Hz, 1H), 3.09 (s, 3H), 1.15 (s, 9H). 13 C NMR(100MHz, CDCl3)δ:164.4,154.0,149.7,146.2,140.3,135.5,131.2,130.4(2×C),128.8(2× C),128.5(2×C),127.4,121.1(2×C),120.4(q,J=257Hz),119.7,106.7,35.8,32.1,30.7(3×C). 19 F NMR (376MHz,CDCl3)δ:-57.9(3×F).Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.41 (d, J = 8.8Hz, 2H), 7.31-7.20 (m, 7H), 7.00 (br, 1H), 6.74 (s, 1H), 6.12 (br, 1H), 3.26 (s, 3H), 1.15 (s, 9H). 13C NMR(100MHz, CDCl3)δ:164.4,154.0,149.7,146.2,140.3,135.8,130.1,130.4(2×C),128.8(2× C),128.5(2×C),127.4,121.1(2×C),120.4(q,J=257Hz),119.7,107.6,38.8,32.1,30.7(3×C). 19 F NMR (376 MHz, CDCl3) δ: -57.9 (3×F). HRMS (ESI) m / z calculated value is C 24 H 25 O2N3F3 + [M+H] + 444.1893, the measured value is 444.1903.

[0115] Example 9 Synthesis of Compound A9:

[0116] The synthesis steps are as shown in Example 1. The methyl ketone reagent in the first step is acetone, and the hydrazine reagent in the second step is phenylhydrazine to obtain compound A9 with a yield of 87%. The main conformational isomers are: 1 H NMR(400MHz, CDCl3)δ:7.49-7.44(m,4H),7.42-7.38(m,1H),7.34-7.29(m,4H),7.24-7.20( m,1H),6.96(d,J=8.8Hz,1H),6.62(s,1H),5.98(d,J=8.8Hz,1H),3.09(s,3H),2.34(s,3H). 13 C NMR (100MHz, CDCl3) δ: 164.4, 146.9, 139.5, 135.5, 131.5, 129.2 (2×C), 128.8 (2×C), 128.4 (2×C), 128.2, 127.4, 125.0 (3×C), 119.2, 109.5, 35.8, 12.5. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.49-7.44(m,4H),7.42-7.38(m,1H),7.34-7.29(m,4H),7.2 4-7.20(m,1H),7.03(br,1H),6.73(s,1H),6.13(br,1H),3.34(s,3H),2.34(s,3H). 13C NMR (100 MHz, CDCl3) δ: 164.4, 146.9, 139.5, 135.8, 130.1, 129.2 (2×C), 128.8 (2×C), 128.4 (2×C), 128.2, 127.4, 125.0 (3×C), 119.0, 110.1, 39.0, 12.5. HRMS (ESI) m / z calculated value is C 20 H 20 ON3 + [M+H] + 318.1601, the measured value is 318.1611.

[0117] Example 10 Synthesis of Compound A10:

[0118] The synthesis steps are as shown in Example 1. The methyl ketone reagent in the first step is acetophenone, and the hydrazine reagent in the second step is phenylhydrazine to obtain compound A10 with a yield of 95%. The main conformational isomers are: 1 H NMR (400MHz, CDCl3) δ: 7.37-7.32 (m, 12H), 7.28-7.25 (m, 3H), 7.04 (d, J = 8.8Hz, 1H), 6.92 (s, 1H), 6.06 (d, J = 8.8Hz, 1H), 3.16 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ: 164.1, 147.4, 143.6, 139.7, 135.5, 131.4, 129.9, 129.0 (2×C), 128.9 (2×C), 128.8 (2×C), 128.7 (2×C), 128.6 (2×C), 128.5, 128.0, 127.5, 125.3 (2×C), 119.8, 110.2, 35.9. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.37-7.32 (m, 12H), 7.28-7.25 (m, 3H), 7.08 (s, 1H), 7.04 (d, J = 8.8Hz, 1H), 6.19 (br, 1H), 3.45 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ: 164.4, 147.4, 143.6, 139.7, 135.8, 131.4, 130.1, 129.0 (2×C), 128.9 (2×C), 128.8 (2×C), 128.7 (2×C), 128.6 (2×C), 128.5, 128.0, 127.5, 125.3 (2×C), 119.2, 111.0, 39.1. HRMS (ESI) m / z calculated for C 25H 22 ON3 + [M+H] + 380.1757, the measured value is 380.1768.

[0119] Example 11 Synthesis of B series compounds

[0120] (1) Synthesis of Compound 6

[0121]

[0122] Take 100mL sealed tube and add anhydrous ethanol (6mL), slowly add 20% sodium ethoxide solution (2.1mL, 10mmol), and cool in an ice-water bath. After premixing acetone (0.58g, 10mmol) and diethyl oxalate (1.46g, 10mmol), add precooled sodium ethoxide solution dropwise. After the addition is complete, return to room temperature and continue the reaction for 12h. After TLC monitoring and tracking until the raw material disappears or there is no change, acidify with 20% dilute sulfuric acid in an ice-water bath to pH = 3, filter with diatomaceous earth, and wash with dichloromethane (3×20mL), and extract the aqueous phase with dichloromethane (3×20mL). After the organic phases were combined, they were washed with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate for 30 minutes, and concentrated under reduced pressure. The crude product was separated and purified using a silica gel chromatography column with petroleum ether and ethyl acetate (V / V=5:1) as the eluent to obtain 1.14 g of a yellow liquid product (Compound 6) with a yield of 72%.

[0123] (2) Synthesis of Compound 7

[0124]

[0125] Anhydrous ethanol (5 mL) and phenylhydrazine (324 mg, 3 mmol) were added to a 15 mL sealed tube, and compound 6 (475 mg, 3 mmol) was slowly added. The reaction was continued for 5 h and the oil bath temperature was maintained at 80° C. After TLC monitoring until the raw material disappeared or remained unchanged, the oil bath was removed, and the reaction solution was cooled to room temperature naturally. The organic phase was collected with ethyl acetate and concentrated under reduced pressure. The crude product was separated and purified on a silica gel chromatography column using petroleum ether and ethyl acetate (V / V=10:1) as eluents to obtain 317 mg of a light yellow solid product with a yield of 46%.

[0126] (3) Synthesis of Compound 8

[0127]

[0128] Compound 7 (460 mg, 2 mmol) and 7 mol / L methanolic ammonia solution (4 mL) were added to a 15 mL sealed tube and stirred at room temperature for 7 days. After TLC monitoring until the starting material disappeared or remained unchanged, the organic phase was collected with acetone and concentrated under reduced pressure. The obtained crude product was separated and purified on a silica gel chromatography column using petroleum ether and acetone (V / V=1:1) as eluents to obtain 370 mg of a white solid product with a yield of 92%.

[0129] (4) Synthesis of Compound 9

[0130]

[0131] Argon was filled into a dry 10mL Schlenk tube, compound 8 (201mg, 1mmol), cuprous iodide (9.5mg, 0.05mmol), cesium carbonate (407mg, 1.25mmol) were added, (Z)-2-iodovinylbenzene (253mg, 1.1mmol) was diluted with dry anhydrous tetrahydrofuran (5mL) and added to the reaction tube, and DMEDA (10.8μL, 0.1mmol) was added dropwise, and the reaction was continued in a 65℃ oil bath under sealed conditions for 3h. After natural cooling, the mixture was filtered with diatomaceous earth, washed with ethyl acetate (3×20mL), the organic phase was dried over anhydrous sodium sulfate for 30min and filtered out, and the filtrate was concentrated under reduced pressure. The crude product was chromatographed on a silica gel column to obtain 170mg of a light yellow solid with a yield of 56%.

[0132] (5) Synthesis of Compound B1

[0133]

[0134] Argon was filled into a dry two-necked flask, compound 9 (152 mg, 0.5 mmol) dissolved in dry anhydrous N,N-dimethylformamide (2 mL) was added, cooled in an ice-water bath, 60% sodium hydride (32 mg, 0.8 mmol) was added, reacted at the same temperature for 30 min, and then iodomethane (142 mg, 1 mmol) was added to react for 3 h. After the reaction, ice water was added, extracted with ethyl acetate (3×20 mL), washed with saturated sodium chloride solution (20 mL), the organic phase was dried over anhydrous sodium sulfate for 30 min and filtered out, the filtrate was concentrated under reduced pressure, and the crude product was subjected to silica gel column chromatography to obtain 137 mg of yellow oil (compound B1), with a yield of 86%.

[0135] The obtained compound B1 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were:

[0136] Main conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.54-7.37(m,4H),7.33-7.30(m,1H),7.25-7.09(m,3H),6.86-6.79( m,2H),6.31(s,1H),6.01(d,J=9.2Hz,1H),5.82(d,J=9.2Hz,1H),2.93(s,3H),2.32(s,3H). 13 C NMR (100MHz, CDCl3) δ: 163.1, 149.5, 140.0, 137.3, 134.6, 129.3 (2×C), 128.6 (2×C), 128.4 (3×C), 127.9, 127.8, 123.8 (2×C), 121.2, 109.0, 34.9, 13.6. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.54-7.37(m,4H),7.33-7.30(m,1H),7.25-7.09(m,3H),6.95(d,J=9 .6Hz,1H),6.86-6.79(m,2H),6.42(s,1H),6.09(d,J=9.6Hz,1H),2.51(s,3H),2.37(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 164.3, 150.2, 139.9, 137.1, 135.1, 129.8 (2×C), 128.9 (2×C), 128.3 (3×C), 127.9, 127.4, 123.2 (2×C), 119.7, 108.0, 37.7, 13.6. HRMS (ESI) m / z calculated value is C 20 H 20 ON3 + [M+H] + 318.1601, the measured value is 318.1604.

[0137] By selecting different methyl ketones and aryl hydrazines, compounds B1-B10 can be obtained.

[0138] The compounds B1-B10 are arranged in the following order:

[0139]

[0140] Example 12 Synthesis of Compound B2:

[0141] Synthesis steps: Refer to Example 11, and the hydrazine reagent in the second step reaction is p-fluorophenylhydrazine to obtain compound B2 with a yield of 80%; 1H NMR(400MHz, CDCl3)δ:7.41-7.38(m,2H),7.31-7.25(m,3H),7.12-7.07(m,2H),6.95-6.93( m,2H),6.36(s,1H),6.11(d,J=8.8Hz,1H),5.90(d,J=8.8Hz,1H),2.98(s,3H),2.33(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.8, 162.0 (d, J = 246Hz), 149.4, 137.3, 136.2 (d, J = 2.7Hz), 134.4, 128.6 (2×C), 128.5(2×C),128.4,128.0,125.9(d,J=8.6Hz,2×C),121.7,116.1(d,J=22.8Hz,2×C),109.1,35.0,13.5. 19 F NMR (376 MHz, CDCl3) δ: -113.8. HRMS (ESI) m / z calculated value is C 20 H 19 ON3F + [M+H] + 336.1507, the measured value is 336.1513.

[0142] Example 13 Synthesis of Compound B3:

[0143] Synthesis steps: Refer to Example 11, the hydrazine reagent in the second step reaction is p-chlorophenylhydrazine to obtain compound B3, yield: 93%; main conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.37(s,4H),7.31-7.20(m,3H),6.92-6.90(m,2H),6.35 (s,1H),6.07(d,J=8.8Hz,1H),5.89(d,J=8.8Hz,1H),2.99(s,3H),2.33(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 162.7, 149.7, 138.5, 137.2, 134.4, 133.6, 129.4 (2×C), 128.5 (4×C), 128.3, 128.0, 125.1 (2×C), 121.9, 109.3, 35.0, 13.5. Minor conformational isomers: 1HNMR (400MHz, CDCl3) δ: 7.51 (s, 4H), 7.31-7.20 (m, 3H), 6.95 (d, J = 8.8Hz, 1H), 6 .92-6.90(m,2H),6.44(s,1H),6.15(d,J=8.8Hz,1H),2.56(s,3H),2.38(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 164.0, 150.4, 138.5, 137.2, 135.1, 133.6, 129.8 (2×C), 128.5 (4×C), 127.9, 127.7, 124.4 (2×C), 120.1, 108.4, 37.7, 13.5. HRMS (ESI) m / z calculated value is C 20 H 19 ON3Cl + [M+H] + 352.1211, the measured value is 352.1215.

[0144] Example 14 Synthesis of Compound B4:

[0145] Synthesis steps: Refer to Example 11, the hydrazine reagent in the second step reaction is p-bromophenylhydrazine to obtain compound B4, yield: 93%; main conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.52 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.31-7.19 (m, 3H), 6.91-6. 89(m,2H),6.35(s,1H),6.06(d,J=8.8Hz,1H),5.88(d,J=8.8Hz,1H),2.98(s,3H),2.33(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 162.7, 149.8, 139.0, 137.2, 134.3, 132.3 (2×C), 128.6 (2×C), 128.5 (2×C), 128.3, 128.0, 125.3 (2×C), 121.9, 121.5, 109.4, 34.9, 13.5. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.66 (d, J = 8.4Hz, 2H), 7.45 (d, J = 8.4Hz, 2H), 7.31-7.19 (m, 3H), 6.91-6. 89(m,2H),6.44(s,1H),6.95(d,J=9.6Hz,1H),6.15(d,J=9.6Hz,1H),2.55(s,3H),2.38(s,3H).13 C NMR (100 MHz, CDCl3) δ: 163.9, 150.4, 138.3, 137.0, 135.1, 132.8 (2×C), 128.4 (4×C), 127.8, 127.7, 124.6 (2×C), 121.9, 120.1, 108.5, 37.7, 13.5. HRMS (ESI) m / z calculated value is C 20 H 19 ON3B + [M+H] + 396.0706, the measured value is 396.0710.

[0146] Example 15 Synthesis of Compound B5:

[0147] Synthesis steps: Refer to Example 11, the hydrazine reagent in the second step reaction is p-methylphenylhydrazine to obtain compound B5, yield: 97%; main conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.43-7.08(m,7H),6.83-6.78(m,2H),6.30(s,1H),6.01 (d,J=8.8Hz,1H),5.80(d,J=8.8Hz,1H),2.92(s,3H),2.34(s,3H),2.32(s,3H). 13 C NMR (100MHz, CDCl3)δ:163.2,149.2,137.7,137.6,137.2,134.5,129.7(2×C),128.5(2×C),128.4,128.2(2×C),127.6,123.6(2×C),120.9,108.5,34.8,21.1,13.5.Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.43-7.08(m,7H),6.95(d,J=9.2Hz,1H),6.83-6.78(m, 2H),6.40(s,1H),6.08(d,J=9.2Hz,1H),2.51(s,3H),2.46(s,3H),2.36(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 164.3, 149.7, 137.7, 137.4, 136.9, 135.1, 130.2 (2×C), 128.7 (2×C), 128.1 (2×C), 127.9, 127.4, 123.0 (2×C), 119.5, 107.6, 37.6, 26.9, 13.5. HRMS (ESI) m / z calculated value is C 21 H22 ON3 + [M+H] + 332.1757, the measured value is 332.1764.

[0148] Example 16 Synthesis of Compound B6:

[0149] Synthesis steps: Refer to Example 11, the hydrazine reagent in the second step reaction is p-methoxyphenylhydrazine to obtain compound B6, yield: 85%; main conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.30 (d, J = 8.8Hz, 2H), 7.21-7.06 (m, 3H), 6.92-6.76 (m, 4H), 6.28 (s ,1H),6.01(d,J=8.8Hz,1H),5.79(d,J=8.8Hz,1H),3.73(s,3H),2.90(s,3H),2.28(s,3H). 13 C NMR (100MHz, CDCl3) δ: 163.0, 159.1, 148.8, 137.2, 134.4, 133.1, 128.5 (2×C), 128.4, 128.2 (2×C), 127.6, 125.2 (2×C), 120.9, 114.2 (2×C), 108.2, 55.4, 34.7, 13.4. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.43(d,J=8.8Hz,2H),7.21-7.06(m,3H),6.99(d,J=8.4Hz,1H) ,6.92-6.76(m,4H),6.36(s,1H),6.06(br,1H),3.82(s,3H),2.49(s,3H),2.32(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 164.2, 159.1, 149.5, 137.0, 135.0, 132.9, 128.6 (2×C), 128.0 (2×C), 127.8, 127.3, 124.7 (2×C), 119.5, 114.6 (2×C), 107.3, 55.5, 37.5, 26.8. HRMS (ESI) m / z calculated value is C 21 H 22 O2N3 + [M+H] + 348.1707, measured value is 348.1714.

[0150] Example 17 Synthesis of Compound B7:

[0151] Synthesis steps: Referring to Example 11, the hydrazine reagent in the second step reaction is p-trifluoromethylphenylhydrazine to obtain compound B7 with a yield of 85%; the main conformational isomers are: 1 H NMR (400MHz, CDCl3) δ: 7.65 (d, J = 8.4Hz, 2H), 7.53 (d, J = 8.4Hz, 2H), 7.28-7.20 (m, 3H), 6.96-6. 88(m,2H),6.38(s,1H),6.07(d,J=8.8Hz,1H),5.88(d,J=8.8Hz,1H),2.99(s,3H),2.33(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.6, 150.3, 142.7, 137.3, 134.3, 129.6 (q, J = 32.7Hz), 128.6 (2×C), 128.5 (2× C),128.2,128.1,126.4(q,J=3.6Hz,2×C),123.9(q,J=270Hz),123.7(2×C),122.1,110.1,35.0,13.6. 19 F NMR (376MHz,CDCl3)δ:-62.4(3×F).Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.77 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 7.6 Hz, 2H), 7.18-7.12 (m, 3H), 6.96-6. 88(m,2H),6.95(d,J=8.8Hz,1H),6.45(s,1H),6.16(d,J=8.8Hz,1H),2.58(s,3H),2.37(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.6, 150.6, 142.2, 137.0, 134.3, 129.6 (q, J = 32.7Hz), 128.6 (2×C), 128.5 (2× C),128.2,128.1,126.4(q,J=3.6Hz,2×C),123.9(q,J=270Hz),122.9(2×C),120.2,109.1,37.8,27.0. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 21 H 19 ON3F3 + [M+H] + 386.1475, the measured value is 386.1479.

[0152] Example 18 Synthesis of Compound B8:

[0153] Synthesis steps: Referring to Example 11, the hydrazine reagent in the second step reaction is p-trifluoromethoxyphenylhydrazine to obtain compound B8 with a yield of 88%; the main conformational isomers are: 1 H NMR(400MHz, CDCl3)δ:7.36(d,J=8.8Hz,2H),7.29-7.15(m,5H),6.87-6.85(m,2H), 6.29(s,1H),6.02(d,J=8.8Hz,1H),5.81(d,J=8.8Hz,1H),2.90(s,3H),2.25(s,3H). 13 C NMR(100MHz, CDCl3)δ:162.6,149.8,148.3,138.5,137.2,134.4,128.6(2×C),128.5(2×C ),128.4,128.0,125.2(2×C),121.8,121.6(2×C),240.9(q,J=256Hz),109.6,35.0,13.5. 19 F NMR (376MHz,CDCl3)δ:-57.9(3×F).Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.51(d,J=7.2Hz,2H),7.29-7.15(m,5H),7.10(br,1H),6 .87-6.85(m,2H),6.34(s,1H),6.08(d,J=9.2Hz,1H),2.53(s,3H),2.29(s,3H). 13 C NMR(100MHz, CDCl3)δ:163.9,149.8,148.3,138.5,137.2,134.4,128.9(2×C),128.5(2×C ),127.8,127.7,124.5(2×C),122.0(2×C),120.1,240.9(q,J=256Hz),108.5,37.8,13.5. 19 F NMR (376 MHz, CDCl3) δ: -57.9 (3×F). HRMS (ESI) m / z calculated value is C 21 H 19 O2N3F3 + [M+H] + 402.1424, measured value is 402.1428.

[0154] Example 19 Synthesis of Compound B9:

[0155] Synthesis steps: Refer to Example 11, the methyl ketone reagent in the first step reaction is pinacolone, and the hydrazine reagent in the second step reaction is phenylhydrazine to obtain compound B9 with a yield of 98%; the main conformational isomers are: 1 H NMR(400MHz, CDCl3)δ:7.56-7.28(m,5H),7.25-7.09(m,3H),6.86-6.80(m,2H),6. 30(s,1H),5.90(d,J=8.8Hz,1H),5.80(d,J=8.8Hz,1H),2.94(s,3H),1.31(s,9H). 13 C NMR (100MHz, CDCl3) δ: 163.5, 162.6, 140.3. 136.8, 134.7, 129.3 (2×C), 128.6 (2×C), 128.4 (2×C), 128.3, 127.8, 127.7, 123.8, 121.3, 105.9, 34.9, 32.4, 30.6 (3×C). Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.56-7.28(m,5H),7.25-7.09(m,3H),6.96(d,J=9.6Hz,1H) ,6.86-6.80(m,2H),6.49(s,1H),6.09(d,J=9.6Hz,1H),2.49(s,3H),1.36(s,9H). 13 C NMR (100 MHz, CDCl3) δ: 164.7, 163.2, 140.1, 136.5, 135.2, 129.7 (2×C), 128.6 (2×C), 128.4 (2×C), 128.3, 128.0, 127.4, 123.3, 119.6, 105.0, 37.7, 32.5, 30.6 (3×C). HRMS (ESI) m / z calculated value is C 23 H 26 ON3 + [M+H] + 360.2070, measured value is 360.2081.

[0156] Example 20 Synthesis of Compound B10:

[0157] Synthesis steps: Referring to Example 11, the methyl ketone reagent in the first step reaction is acetophenone, and the hydrazine reagent in the second step reaction is phenylhydrazine, to obtain compound B10, with a yield of 86%; the main conformational isomers are: 1H NMR (400MHz, CDCl3) δ: 7.83 (d, J = 7.2Hz, 2H), 7.68-7.33 (m, 8H), 7.24-7.10 (m, 3H), 6.8 8-6.81(m,2H),6.76(s,1H),6.01(d,J=8.8Hz,1H),5.85(d,J=8.8Hz,1H),2.98(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 162.8, 152.0, 140.1, 138.1, 134.5, 132.4, 129.4 (2×C), 128.9, 128.8 (2×C), 128.6 (2×C), 128.5 (2×C), 128.4, 128.2, 127.9, 126.0 (2×C), 123.9 (2×C), 121.9, 106.3, 35.0. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.92-7.88(m,2H),7.68-7.33(m,8H),7.24-7.10(m,3H),6.98 (d,J=9.2Hz,1H),6.95(s,1H),6.88-6.81(m,2H),6.13(d,J=9.2Hz,1H),2.56(s,3H). 13 C NMR (100 MHz, CDCl3) δ: 164.1, 152.6, 139.9, 137.8, 135.1, 132.4, 129.8 (2×C), 129.0, 128.9 (2×C), 128.6 (2×C), 128.5 (2×C), 128.4, 128.3, 127.5, 126.0 (2×C), 123.4 (2×C), 119.9, 105.4, 37.7. HRMS (ESI) m / z calculated for C 25 H 22 ON3 + [M+H] + 380.1757, the measured value is 380.1767.

[0158] Example 21 Synthesis of C series compounds

[0159] (1) Synthesis of Compound 10

[0160]

[0161] To a 25 mL round-bottom flask were added p-fluorobenzaldehyde (205 mg, 1.65 mmol), 5 mL of acetonitrile and 0.5 mL of pure water, potassium carbonate (725 mg, 5.25 mmol), p-trifluoromethylphenylhydrazine hydrochloride (319 mg, 1.5 mmol), potassium bromide (214 mg, 1.8 mmol), methyl propiolate (252 mg, 3 mmol), and potassium peroxymonosulfonate double salt (1.56 g, 4.5 mmol) was added under ice bath cooling. After keeping for 10 minutes, the ice bath was removed and the reaction was stirred at room temperature. After TLC monitoring until the raw material was consumed, the reaction was quenched with saturated sodium sulfite, extracted with ethyl acetate (3×20 mL), washed with saturated sodium chloride solution (20 mL), and the organic phase was dried over anhydrous sodium sulfate for 30 min and filtered out. The filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 350 mg of a yellow solid product (Compound 10) with a yield of 64%. The reaction was repeated 3 times until a sufficient amount of compound 10 was obtained.

[0162] (2) Synthesis of Compound 11

[0163]

[0164] Compound 10 (730 mg, 2 mmol) and 7 mol / L methanolic ammonia solution (4 mL) were added to a 15 mL sealed tube and stirred at room temperature for 7 days. After TLC monitoring until the starting material disappeared or remained unchanged, the organic phase was collected with acetone and concentrated under reduced pressure. The crude product was separated and purified on a silica gel chromatography column using petroleum ether and acetone (V / V=1:1) as eluents to obtain 649 mg of a white solid product with a yield of 93%.

[0165] (3) Synthesis of Compound 12

[0166]

[0167] Argon was filled into a dry 10mL Schlenk tube, compound 11 (349mg, 1mmol), cuprous iodide (9.5mg, 0.05mmol), cesium carbonate (407mg, 1.25mmol) were added, (Z)-2-iodovinylbenzene (253mg, 1.1mmol) was diluted with dry anhydrous tetrahydrofuran (5mL) and added to the reaction tube, and DMEDA (10.8μL, 0.1mmol) was added dropwise, and the reaction was continued in a 65℃ oil bath under sealed conditions for 3h. After natural cooling, the mixture was filtered with diatomaceous earth, washed with ethyl acetate (3×20mL), the organic phase was dried over anhydrous sodium sulfate for 30min and filtered out, and the filtrate was concentrated under reduced pressure. The crude product was chromatographed on a silica gel column to obtain 325mg of a light yellow oil with a yield of 72%.

[0168] (4) Synthesis of Compound C1

[0169]

[0170] Argon was filled into a dry two-necked flask, and compound 12 (226 mg, 0.5 mmol) dissolved in dry anhydrous N,N-dimethylformamide (2 mL) was added, cooled in an ice-water bath, 60% sodium hydride (32 mg, 0.8 mmol) was added, and the mixture was reacted at the same temperature for 30 min, and then iodomethane (142 mg, 1 mmol) was added and reacted for 3 h. After the reaction, ice water was added, extracted with ethyl acetate (3×20 mL), washed with saturated sodium chloride solution (20 mL), the organic phase was dried over anhydrous sodium sulfate for 30 min, filtered out, and the filtrate was concentrated under reduced pressure. The crude product was chromatographed on a silica gel column to obtain 200 mg of a colorless oil (compound C1), with a yield of 86%.

[0171] The obtained compound C1 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were:

[0172] Main conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.84-7.80(m,2H),7.72(d,J=8.4Hz,2H),7.65(d,J=8.4Hz,2H),7.28-7.26(m,3H),7.1 4(t,J=8.8Hz,2H),6.94-6.92(m,2H),6.79(s,1H),6.10(d,J=8.8Hz,1H),5.95(d,J=8.8Hz,1H),3.07(s,3H). 13 C NMR (100MHz, CDCl3) δ: 163.1 (d, J=246Hz), 162.2, 151.7, 142.6, 138.2, 134.2, 130.0 (q, J=32.8Hz), 128.6 (2×C), 128.4 (2×C), 128.2, 128.0 (d, J= 30.0Hz, 2×C), 127.9, 127.7 (d, J=8.2Hz), 126.5 (q, J=3.6Hz, 2×C), 123.9 (2×C),123.8(q,J=270Hz),122.9,115.8(d,J=21.6Hz,2×C),107.0,35.0. 19 F NMR (376MHz, CDCl3)δ: -62.4 (3×F), -112.9. Minor conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.90-7.80(m,6H),7.28-7.26(m,3H),7.14(t,J=8.8Hz,2H),6.9 4-6.92(m,2H),7.02(s,1H),7.00(d,J=9.2Hz,1H),6.22(d,J=9.2Hz,1H),2.66(s,3H). 13 C NMR (100MHz, CDCl3) δ: 163.1 (d, J=246Hz), 162.2, 152.3, 142.6, 137.8, 134.2, 130.0 (q, J=32.8Hz), 128.5 (2×C), 128.3 (2×C), 128.2, 128.0 (d, J= 30.0Hz, 2×C), 127.9, 127.7 (d, J=8.2Hz), 126.5 (q, J=3.6Hz, 2×C), 123.9 (2×C),123.8(q,J=270Hz),123.1,115.8(d,J=21.6Hz,2×C),106.1,37.8. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F), -112.9. HRMS (ESI) m / z calculated value is C 26 H 20 ON3F4 + [M+H] + 466.1537, the measured value is 466.1542.

[0173] By selecting different aldehydes, compounds C1-C22 can be obtained. The compounds C1-C22 are arranged in the following order:

[0174]

[0175] Example 22 Synthesis of Compound C2:

[0176] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is m-fluorobenzaldehyde to obtain compound C2 with a yield of 87%; the main conformational isomers are: 1 H NMR(400MHz, CDCl3)δ:7.72(d,J=8.4Hz,2H),7.64(d,J=8.4Hz,2H),7.84-7.54(m,2H),7.47-7.38(m,1H),7 .28-7.06(m,4H),6.95-6.92(m,2H),6.80(s,1H),6.08(d,J=8.8Hz,1H),5.96(d,J=8.8Hz,1H),3.07(s,3H). 13C NMR (100MHz, CDCl3) δ: 163.3 (d, J = 244Hz), 162.1, 151.4 (d, J = 2.4Hz), 142.6, 13 8.3,134.2(d,J=8.1Hz),134.1,130.6(2×C),130.5(q,J=8.3Hz),130.0(2×C),12 9.0,128.3,127.9,126.6(q,J=3.6Hz,2×C),124.0(2×C),123.8(q,J=271Hz),12 3.2, 121.7 (d, J = 2.7Hz), 115.6 (d, J = 21.2Hz), 112.9 (d, J = 22.7Hz), 107.3, 35.1. 19 F NMR (376MHz, CDCl3)δ: -62.4 (3×F), -112.7. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.84-7.54(m,6H),7.47-7.38(m,1H),7.28-7.06(m,4H),7.02 (s,1H),6.95-6.92(m,2H),6.23(d,J=8.8Hz,1H),6.05(d,J=8.8Hz,1H),3.40(s,3H). 13 C NMR (100MHz, CDCl3) δ: 164.6, 163.3 (d, J = 244Hz), 151.8 (d, J = 3.2Hz), 142.1, 13 8.3,134.1,134.0(d,J=8.5Hz),130.5(q,J=8.3Hz),129.6,128.6(2×C),128.4(2 ×C),128.1,127.3,126.6(q,J=3.6Hz,2×C),125.2(2×C),123.8(q,J=271Hz),12 3.4, 121.8 (d, J = 3.3Hz), 115.8 (d, J = 23.1Hz), 113.0 (d, J = 22.8Hz), 107.7, 30.7. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F), -112.7. HRMS (ESI) m / z calculated value is C 26 H 20 ON3F4 + [M+H] + 466.1537, the measured value is 466.1543.

[0177] Example 23 Synthesis of Compound C3:

[0178] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is o-fluorobenzaldehyde to obtain compound C3 with a yield of 84%. 1 H NMR(400MHz, CDCl3)δ:8.10(td,J=7.6,1.6Hz,1H),7.71(d,J=8.4Hz,2H),7.64(d,J=8.4Hz,2H),7.39-7.34(m,1H),7 .27-7.15(m,5H),7.08(d,J=3.6Hz,1H),6.94-6.92(m,2H),6.18(d,J=8.8Hz,1H),5.96(d,J=8.8Hz,1H),3.06(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.2, 160.4 (d, J = 248Hz), 147.3, 142.6, 137.7, 134.2, 13 0.2(d,J=8.4Hz),130.1(q,J=32.6Hz),128.7(2×C),128.6,128.5(2×C),128.2,1 28.1,126.5(q,J=3.6Hz,2×C),124.5(d,J=3.2Hz),124.1(2×C),123.9(q,J=271H z), 122.8, 119.9 (d, J = 11.5Hz), 116.3 (d, J = 21.9Hz), 110.6 (d, J = 10.6Hz), 35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F), -115.4. HRMS (ESI) m / z calculated value is C 26 H 20 ON3F4 + [M+H] + 466.1537, the measured value is 466.1544.

[0179] Example 24 Synthesis of Compound C4:

[0180] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is p-chlorobenzaldehyde to obtain compound C4 with a yield of 86%. 1H NMR (400MHz, CDCl3) δ: 7.75 (d, J = 8.4Hz, 2H), 7.70 (d, J = 8.4Hz, 2H), 7.62 (d, J = 8.4Hz, 2H), 7.39 (d, J = 8.4Hz, 2H) ),7.26-7.24(m,3H),6.92-6.89(m,2H),6.77(s,1H),6.06(d,J=8.8Hz,1H),5.92(d,J=8.8Hz,1H),3.04(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.1, 151.5, 142.6, 138.3, 134.6, 134.2, 130.5, 130.1 (q, J = 32.8Hz), 129.1 (2×C), 128.7 (2×C) ,128.4(2×C),128.2,127.9,127.2(2×C),126.5(q,J=3.6Hz,2×C),123.9(2×C),128.8(q,J=271Hz),123.1,107.1,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 20 ON3F3Cl + [M+H] + 482.1242, measured value is 482.1249.

[0181] Example 25 Synthesis of Compound C5:

[0182] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is m-chlorobenzaldehyde to obtain compound C5 with a yield of 82%. 1 H NMR (400MHz, CDCl3) δ: 7.82 (s, 1H), 7.70 (d, J = 8.4Hz, 2H), 7.68-7.66 (m, 1H), 7.61 (d, J = 8.4Hz, 2H), 7.37-7.32 (m, 2H),7.26-7.24(m,3H),6.93-6.90(m,2H),6.78(s,1H),6.06(d,J=8.8Hz,1H),5.94(d,J=8.8Hz,1H),3.05(s,3H). 13C NMR (100MHz, CDCl3) δ: 162.0, 151.2, 142.5, 138.3, 134.9, 134.2, 133.8, 130.2, 130.1 (q, J = 32.8Hz), 128.7 (3×C), 12 8.5(2×C),128.3,127.8,126.5(q,J=3.6Hz,2×C),126.1,124.1,124.0(2×C),123.8(q,J=271Hz),123.3,107.3,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 20 ON3F3Cl + [M+H] + 482.1242, the measured value is 482.1246.

[0183] Example 26 Synthesis of Compound C6:

[0184] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is o-chlorobenzaldehyde to obtain compound C6 with a yield of 88%; 1 H NMR(400MHz, CDCl3)δ:7.87-7.85(m,1H),7.69(d,J=8.4Hz,2H),7.60(d,J=8.4Hz,2H),7.48-7.45(m,1H),7.36-7.29 (m,2H),7.26-7.23(m,3H),7.16(s,1H),6.96-6.94(m,2H),6.20(d,J=8.8Hz,1H),5.98(d,J=8.8Hz,1H),3.05(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.1, 150.2, 142.6, 137.0, 134.2, 132.6, 131.0, 130.8, 130.5, 130.1 (q, J = 32.8Hz), 129.8, 128. 7(2×C),128.5(2×C),128.2(2×C),127.1,126.4(q,J=3.6Hz,2×C),124.2(2×C),123.9(q,J=271Hz),123.1,111.4,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 20 ON3F3Cl + [M+H]+ 482.1242, measured value is 482.1251.

[0185] Example 27 Synthesis of Compound C7:

[0186] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is p-bromobenzaldehyde to obtain compound C7 with a yield of 78%; 1 H NMR (400MHz, CDCl3) δ: 7.70 (d, J = 8.4Hz, 2H), 7.69 (d, J = 8.4Hz, 2H), 7.62 (d, J = 8.4Hz, 2H), 7.55 (d, J = 8.4Hz, 2H ),7.26-7.24(m,3H),6.91-6.89(m,2H),6.77(s,1H),6.05(d,J=8.8Hz,1H),5.92(d,J=8.8Hz,1H),3.04(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.1, 151.5, 142.6, 138.3, 134.2, 132.0 (2×C), 130.9, 130.1 (q, J = 32.8Hz), 128.7 (2×C), 128.5 (2×C),128.2,127.9,127.5(2×C),126.5(q,J=3.6Hz,2×C),123.9(2×C),123.8(q,J=271Hz),123.1,122.8,107.1,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 20 ON3F3Br + [M+H] + 526.0736, the measured value is 526.0719.

[0187] Example 28 Synthesis of Compound C8:

[0188] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is m-bromobenzaldehyde to obtain compound C8 with a yield of 82%. 1H NMR (400MHz, CDCl3) δ: 7.98 (s, 1H), 7.81-7.69 (m, 4H), 7.61 (d, J = 8.4Hz, 2H), 7.49 (d, J = 8.0Hz, 1H), 7.31 -7.25(m,3H),6.93-6.90(m,2H),6.77(s,1H),6.05(d,J=8.8Hz,1H),5.94(d,J=8.8Hz,1H),3.05(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.0, 151.1, 142.5, 138.3, 134.2, 134.0, 131.6, 130.4, 130.1 (q, J = 32.8Hz), 129.0, 128.7 (2×C) ,128.5(2×C),128.3,127.8,126.6(q,J=3.6Hz,2×C),124.6,124.0(2×C),123.8(q,J=271Hz),123.3,123.1,107.3,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 20 ON3F3Br + [M+H] + 526.0736, the measured value is 526.0729.

[0189] Example 29 Synthesis of Compound C9:

[0190] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is o-bromobenzaldehyde to obtain compound C9 with a yield of 74%; 1 H NMR(400MHz, CDCl3)δ:7.76(dd,J=7.6,1.6Hz,1H),7.68(d,J=8.4Hz,2H),7.68-7.66(m,1H),7.59(d,J=8.4Hz,2H),7.38(t, J=7.6Hz,1H),7.26-7.21(m,4H),7.14(s,1H),6.98-6.96(m,2H),6.21(d,J=8.8Hz,1H),5.99(d,J=8.8Hz,1H),3.06(s,3H). 13C NMR (100MHz, CDCl3) δ: 162.0, 151.6, 142.6, 136.8, 134.2, 133.8, 133.1, 131.3, 130.1 (q, J = 32.7Hz), 130.0, 128.7 (2×C) ,128.6(2×C),128.3,128.2,127.7,126.4(q,J=3.6Hz,2×C),124.2(2×C),123.9(q,J=271Hz),123.2,122.2,111.4,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 20 ON3F3Br + [M+H] + 526.0736, the measured value is 526.0745.

[0191] Example 30 Synthesis of Compound C10:

[0192] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is p-methylbenzaldehyde to obtain compound C10 with a yield of 88%; 1 H NMR(400MHz, CDCl3)δ:7.72(d,J=8.0Hz,2H),7.69(d,J=8.4Hz,2H),7.63(d,J=8.4Hz,2H),7.26-7.22(m,5 H),6.92-6.89(m,2H),6.81(s,1H),6.10(d,J=8.8Hz,1H),5.91(d,J=8.8Hz,1H),3.03(s,3H),2.39(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.4, 152.7, 142.7, 138.7, 138.0, 134.3, 129.8 (q, J = 32.8Hz), 129.6 (2×C), 129.1, 128.6 (2×C), 12 8.5(2×C),128.1,128.0,126.5(q,J=3.6Hz,2×C),125.9(2×C),123.9(2×C),123.8(q,J=271Hz),122.6,107.1,35.1,21.5. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 27 H 23 ON3F3 + [M+H]+ 462.1788, the measured value is 462.1788.

[0193] Example 31 Synthesis of Compound C11:

[0194] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is m-methylbenzaldehyde to obtain compound C11 with a yield of 92%. 1 H NMR(400MHz, CDCl3)δ:7.71-7.68(m,3H),7.64-7.59(m,3H),7.32(t,J=7.6Hz,1H),7.26-7.24(m,3H),7.19(d,J=7 .6Hz,1H),6.93-6.91(m,2H),6.84(s,1H),6.11(d,J=8.8Hz,1H),5.92(d,J=8.8Hz,1H),3.04(s,3H),2.41(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.4, 152.7, 142.7, 138.6, 138.0, 134.3, 131.8, 129.9 (q, J = 32.8Hz), 129.6, 128.8, 128.6 (2×C), 12 8.5(2×C),128.2,128.1,126.6,126.5(q,J=3.7Hz,2×C),124.0(2×C),123.9(q,J=271Hz),123.2,122.7,107.3,35.1,21.6. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 27 H 23 ON3F3 + [M+H] + 462.1788, the measured value is 462.1797.

[0195] Example 32 Synthesis of Compound C12:

[0196] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is o-methylbenzaldehyde to obtain compound C12 with a yield of 84%; 1H NMR (400MHz, CDCl3) δ: 7.69 (d, J = 8.4Hz, 2H), 7.63 (d, J = 8.4Hz, 2H), 7.58 (d, J = 6.4Hz, 1H), 7.28-7.25 (m, 6 H),6.97-6.94(m,2H),6.76(s,1H),6.13(d,J=8.8Hz,1H),5.95(d,J=8.8Hz,1H),3.06(s,3H),2.51(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.4, 153.0, 142.7, 137.0, 136.4, 134.2, 131.6, 131.2, 129.8 (q, J = 32.8Hz), 129.4, 128.7 (2×C), 12 8.6,128.5(2×C),128.2,128.1,126.4(q,J=3.7Hz,2×C),126.1,123.9(q,J=271Hz),123.8(2×C),122.9,110.4,35.1,21.4. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 27 H 23 ON3F3 + [M+H] + 462.1788, the measured value is 462.1788.

[0197] Example 33 Synthesis of Compound C13:

[0198] Synthesis steps Referring to Example 21, the aldehyde in the first step reaction was p-trifluoromethylbenzaldehyde to obtain compound C13 with a yield of 81%; 1 H NMR(400MHz, CDCl3)δ:7.92(d,J=8.0Hz,2H),7.71(d,J=8.4Hz,2H),7.68(d,J=8.4Hz,2H),7.63(d,J=8.4Hz,2H ),7.26-7.22(m,3H),6.93-6.90(m,2H),6.82(s,1H),6.04(d,J=8.8Hz,1H),5.94(d,J=8.8Hz,1H),3.06(s,3H). 13C NMR(100MHz, CDCl3)δ:161.9,151.1,142.5,138.5,135.4,134.1,130.5(q,J=32.2Hz),130.3(q,J=32.8Hz),128.7(2×C),128.5(2×C),128.3,1 27.8,126.6(q,J=3.6Hz,2×C),126.2(2×C),125.9(q,J=3.7Hz,2×C),12 4.2(q,J=270Hz),124.0(2×C),123.8(q,J=271Hz),123.5,107.5,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F), -62.6 (3×F). HRMS (ESI) m / z calculated value is C 27 H 20 ON3F6 + [M+H] + 516.1505, the measured value is 516.1496.

[0199] Example 34 Synthesis of Compound C14:

[0200] Synthesis steps Referring to Example 21, the aldehyde in the first step reaction was selected as m-trifluoromethylbenzaldehyde to obtain compound C14 with a yield of 85%; 1 H NMR (400MHz, CDCl3) δ: 8.06 (s, 1H), 7.97 (d, J = 7.6Hz, 1H), 7.71 (d, J = 8.4Hz, 2H), 7.63 (d, J = 8.4Hz, 2H), 7.63-7.61 (m, 1H), 7.54 (t,J=7.6Hz,1H),7.26-7.24(m,3H),6.92-6.90(m,2H),6.78(s,1H),6.02(d,J=8.8Hz,1H),5.94(d,J=8.8Hz,1H),3.05(s,3H). 13C NMR(100MHz, CDCl3)δ:162.0,151.1,142.5,138.5,134.2,132.8,131.4(q, J=32.2Hz),130.2(q,J=32.8Hz),129.4,129.2,128.7(2×C),128.5(2×C),12 8.3,127.7,126.9(q,J=271Hz),126.6(q,J=3.6Hz,2×C),125.3(q,J=3.6Hz ),124.0(2×C),123.8(q,J=271Hz),123.6,122.8(q,J=3.4Hz),107.2,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.5 (3×F), -62.7 (3×F). HRMS (ESI) m / z calculated value is C 27 H 20 ON3F6 + [M+H] + 516.1505, the measured value is 516.1509.

[0201] Example 35 Synthesis of Compound C15:

[0202] Synthesis steps Referring to Example 21, the aldehyde in the first step reaction was o-trifluoromethylbenzaldehyde to obtain compound C15 with a yield of 80%; 1 H NMR(400MHz, CDCl3)δ:7.78(d,J=7.6Hz,1H),7.72-7.66(m,3H),7.63-7.56(m,3H),7.52(t,J=7.6Hz,1H),7 .24-7.22(m,3H),6.95-6.92(m,2H),6.85(s,1H),6.17(d,J=8.8Hz,1H),6.00(d,J=8.8Hz,1H),3.06(s,3H). 13 C NMR(100MHz, CDCl3)δ:162.0,150.6,142.5,137.1,134.2,132.0,131.9,13 1.6,130.0(q,J=32.8Hz),129.6(q,J=32.8Hz),128.8,128.6(2×C),128.5(2 ×C),128.2,128.1,126.5(q,J=5.5Hz),126.4(q,J=3.6Hz,2×C),124.2(2×C ), 124.1 (q, J = 272Hz), 123.8 (q, J = 271Hz), 123.6, 110.9 (q, J = 3.2Hz), 35.1.19 F NMR (376 MHz, CDCl3) δ: -57.7 (3×F), -62.4 (3×F). HRMS (ESI) m / z calculated value is C 27 H 20 ON3F6 + [M+H] + 516.1505, the measured value is 516.1506.

[0203] Example 36 Synthesis of Compound C16:

[0204] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction was benzaldehyde to obtain compound C16 with a yield of 92%. 1 H NMR(400MHz, CDCl3)δ:7.85-7.83(m,2H),7.70(d,J=8.4Hz,2H),7.64(d,J=8.4Hz,2H),7.45-7.42(m,2H),7.39-7.35 (m,1H),7.26-7.24(m,3H),6.92-6.90(m,2H),6.85(s,1H),6.10(d,J=8.8Hz,1H),5.92(d,J=8.8Hz,1H),3.04(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.3, 152.6, 142.7, 138.1, 134.2, 131.9, 129.9 (q, J = 32.8Hz), 128.9 (2×C), 128.8, 128.6 (2×C) ,128.5(2×C),128.1,128.0,126.5(q,J=3.6Hz,2×C),126.0(2×C),124.0(q,J=271Hz),123.9(2×C),122.7,107.3,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 21 ON3F3 + [M+H] + 448.1631, the measured value is 448.1628.

[0205] Example 37 Synthesis of Compound C17:

[0206] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is 1-naphthaldehyde to obtain compound C17 with a yield of 86%. 1H NMR(400MHz, CDCl3)δ:8.49-8.46(m,1H),7.92-7.89(m,2H),7.74-7.68(m,5H),7.56-7.52(m,3H),7.28 -7.24(m,3H),6.99-6.97(m,2H),6.89(s,1H),6.16(d,J=8.8Hz,1H),5.98(d,J=8.8Hz,1H),3.09(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.3, 152.5, 142.7, 137.3, 134.2, 134.1, 131.2, 130.0 (q, J = 32.7Hz), 129.8, 129.3, 128.7 (2×C), 128.6, 128.5 (2×C),128.3,128.0,127.6,126.8,126.5(q,J=3.6Hz,2×C),126.1,125.8,125.4,124.0(2×C),123.9(q,J=271Hz),123.2,111.1,35.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 30 H 23 ON3F3 + [M+H] + 498.1788, the measured value is 498.1785.

[0207] Example 38 Synthesis of Compound C18:

[0208] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is cyclohexylcarboxaldehyde to obtain compound C18 with a yield of 82%; 1 H NMR (400MHz, CDCl3) δ: 7.64 (d, J = 8.4Hz, 2H), 7.53 (d, J = 8.4Hz, 2H), 7.25-7.22 (m, 3H), 6.90-6.88 (m, 2H), 6.37 (s, 1H), 6.03 (d, J = 8.8Hz, 1H), 5.8 7(d,J=8.8Hz,1H),2.99(s,3H),2.71-2.65(m,1H),2.07-1.95(m,2H),1. 86-1.80(m,2H),1.74-1.70(m,1H),1.46-1.32(m,4H),1.29-1.21(m,1H). 13C NMR (100MHz, CDCl3) δ: 162.9, 159.7, 142.8, 136.9, 134.4, 129.5 (q, J = 32.6Hz), 128.6 (2×C), 128.5 (2×C), 128.2, 128 .1,126.4(q,J=3.6Hz,2×C),123.9(q,J=271Hz),123.8(2×C),122.0,107.6,37.6,35.0,33.1(2×C),26.3(2×C),26.1. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 26 H 27 ON3F3 + [M+H] + 454.2101, the measured value is 454.2101.

[0209] Example 39 Synthesis of Compound C19:

[0210] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is n-propanal to obtain compound C19 with a yield of 82%; 1 H NMR(400MHz, CDCl3)δ:7.65(d,J=8.4Hz,2H),7.54(d,J=8.4Hz,2H),7.25-7.22(m,3H),6.89-6.87(m,2H),6.39 (s,1H),6.04(d,J=8.8Hz,1H),5.87(d,J=8.8Hz,1H),2.99(s,3H),2.69(q,J=7.6Hz,2H),1.26(t,J=7.6Hz,3H). 13 C NMR (100MHz, CDCl3) δ: 162.8, 156.2, 142.7, 137.2, 134.3, 129.6 (q, J = 32.7Hz), 128.6 (2×C), 128.5 (2×C) ,128.1,128.0,126.5(q,J=3.6Hz,2×C),123.9(q,J=271Hz),123.8(2×C),122.1,108.7,35.0,21.5,13.7. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 22 H 21 ON3F3 + [M+H] + 400.1631, the measured value is 400.1636.

[0211] Example 40 Synthesis of Compound C20:

[0212] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is n-butyraldehyde to obtain compound C20 with a yield of 90%; 1 H NMR(400MHz, CDCl3)δ:7.65(d,J=8.4Hz,2H),7.53(d,J=8.4Hz,2H),7.25-7.22(m,3H),6.90-6.88(m,2H),6.38(s,1H),6 .04(d,J=8.8Hz,1H),5.88(d,J=8.8Hz,1H),2.99(s,3H),2.64(t,J=7.6Hz,2H),1.73-1.64(m,2H),0.98(t,J=7.2Hz,3H). 13 C NMR (100MHz, CDCl3) δ: 162.8, 154.8, 142.7, 137.1, 134.3, 129.6 (q, J = 32.7Hz), 128.6 (2×C), 128.5 (2×C), 12 8.2,128.1,126.4(q,J=3.6Hz,2×C),123.9(q,J=270Hz),123.8(2×C),122.2,109.2,35.0,30.2,22.8,14.0. 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 23 H 23 ON3F3 + [M+H] + 414.1788, the measured value is 414.1780.

[0213] Example 41 Synthesis of Compound C21:

[0214] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is isobutylaldehyde to obtain compound C21 with a yield of 82%. 1 H NMR(400MHz, CDCl3)δ:7.65(d,J=8.4Hz,2H),7.54(d,J=8.4Hz,2H),7.25-7.22(m,3H),6.89-6.87(m,2H),6.37(s ,1H),6.01(d,J=8.8Hz,1H),5.87(d,J=8.8Hz,1H),3.02(hept,J=6.8Hz,1H),2.99(s,3H),1.27(d,J=6.8Hz,6H). 13C NMR (100MHz, CDCl3) δ: 162.8, 160.5, 142.8, 137.1, 134.4, 129.5 (q, J = 32.7Hz), 128.6 (2×C), 128.5 (2×C), 1 28.1(2×C),126.4(q,J=3.6Hz,2×C),123.9(q,J=270Hz),123.8(2×C),122.1,107.3,35.0,28.0,22.7(2×C). 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 23 H 23 ON3F3 + [M+H] + 414.1788, the measured value is 414.1785.

[0215] Example 42 Synthesis of Compound C22:

[0216] Synthesis steps: Referring to Example 21, the aldehyde in the first step reaction is 3,3-dimethylbutyraldehyde to obtain compound C22 with a yield of 91%; 1 H NMR (400MHz, CDCl3) δ: 7.64 (d, J = 8.4Hz, 2H), 7.52 (d, J = 8.4Hz, 2H), 7.26-7.22 (m, 3H), 6.93-6.90 (m, 2H),6.40(s,1H),6.06(d,J=8.8Hz,1H),5.89(d,J=8.8Hz,1H),3.00(s,3H),2.55(s,2H),0.96(s,9H). 13 C NMR (100MHz, CDCl3) δ: 162.7, 152.4, 142.7, 136.7, 134.3, 129.6 (q, J = 32.7Hz), 128.6 (2×C), 128.5 (2×C), 128. 2,128.1,126.4(q,J=3.6Hz,2×C),124.0(q,J=270Hz),123.9(2×C),122.3,110.9,42.2,35.1,31.3,29.6(3×C). 19 F NMR (376 MHz, CDCl3) δ: -62.4 (3×F). HRMS (ESI) m / z calculated value is C 25 H 27 ON3F3 + [M+H] + 442.2011, the measured value is 442.2107.

[0217] Active Examples

[0218] Activity Example 1 Screening of cytotoxicity and anti-inflammatory activity of cis-enamide-pyrazole derivatives.

[0219] 1. Cell Culture

[0220] Mouse mononuclear macrophage Raw 264.7 cells were cultured in DMEM high glucose medium (Gibco, Catalog No.: 11995065) supplemented with 10% (V / V) FBS (Gibco, Catalog No.: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, Catalog No.: 15140122). The culture conditions were 37°C, 5% CO2, and the cells were passaged when they reached 80% confluence.

[0221] 2. Cytotoxicity Assay

[0222] Mouse mononuclear macrophage Raw 264.7 cells were used and 2×10 4 / mL, inoculated in a 96-well plate at a density of 100μL / well. When the cell coverage rate was about 80%, all synthetic compounds (10μM) were added, with 3 replicates in each group, and a blank control group was set up. After continued cultivation for 24h, 10μL of CCK8 solution was added to each well, and the cells were continued to be incubated in a 37℃, 5% CO2 incubator for 2h. The absorbance OD value was measured at a wavelength of 450nm with an enzyme reader and the cell inhibition rate was calculated.

[0223] 3. Detection of the Effect of Compounds on NO Expression Levels

[0224] Raw 264.7 cells were cultured at 2×10 5 / mL, 1mL / well density was inoculated in a 12-well plate. When the cell coverage rate was about 80%, the positive control drug (dexamethasone, final concentration 1μM) and the candidate compound (DMSO solvent control, candidate compound 10μM) were added. After 1h, LPS was added at a final concentration of 100ng / mL. LPS continued to stimulate for 24h, and the culture supernatant was collected. The concentrations of the standard in the Bio-Tech NO Detection Kit (Cat. No. S0021S) were 0, 1, 2, 5, 10, 20, 40, 60, 100μM, and the standard and sample were added to the 96-well plate at 50μL / well. Then, 50μL of Griess Reagent I and Griess Reagent II, which were restored to room temperature, were added to each well in turn. After mixing evenly, the absorbance was measured at 540nm, and the concentration of nitric oxide in the sample was calculated according to the standard curve.

[0225] 4. Detection of the expression level of inflammatory factors by compounds

[0226] Raw 264.7 cells were cultured at 2×105 / mL, 1mL / well density was inoculated in 12-well plates. When the cell coverage rate was about 80%, positive control drug (dexamethasone, final concentration 1μM) and candidate compound (DMSO solvent control, candidate compound 10μM) were added. After 1 hour, LPS with a final concentration of 100ng / mL was added. LPS stimulation continued for 4 hours.

[0227] (1) RNA extraction and qPCR determination

[0228] After completely discarding the culture medium of the cultured cells, 0.5 mL of RA2 in the RNA extraction kit was added to each well for lysis; the cell lysate was collected and Total RNA was extracted according to the kit instructions (Vazyme, catalog number: RC112), and its concentration was measured using an ultra-micro UV-visible spectrophotometer;

[0229] (2) Reverse transcription reaction

[0230] A 20 μL reverse transcription system was established, including 4 μL 5× Buffer, X μL Total RNA (500 ng), and DEPC-H2O (16-X) μL in 8 tubes, which were marked with serial numbers and placed in a PCR instrument with the settings of 50°C, 15 min; 85°C, 5 s; and 16°C∝ for reverse transcription;

[0231] (3) Fluorescence quantification

[0232] Add 80 μL DEPC-H2O to the transcribed cDNA to make 100 μL, centrifuge and mix, and store at -20°C for later use; multiply (SYBR 10 μL + DEPC-H2O 10 μL + primer 0.5 μL) by the number of samples, take 18 μL / well to a 96-well plate, add 2 μL cDNA to form a 20 μL system, and centrifuge and mix at 1200 rpm for 1 min; place the sample plate in the CFX Connect Real-Time System (real-time fluorescence quantitative PCR instrument) for detection, and perform detection according to the program of (95°C, 2 min; 95°C, 20 s; 57°C, 20 s; 72°C, 20 s) for a total of 39 cycles, 95°C, 1 min; 55°C, 30 s; 95°C, 30 s;

[0233] (4) Analysis

[0234] Adoption 2 -ΔΔCt The experimental results were analyzed by the following calculation formula: △Ct target gene = Ct target gene - Ct internal reference gene, △△Ct target gene = △Ct experimental group target gene - △Ct control group target gene. -ΔΔCt Indicates the experimental group

[0235] The expression fold of the target gene relative to the control group.

[0236] The primers are shown in Table 1 below.

[0237] Table 1 Primer sequences.

[0238]

[0239] The results are as follows Figure 1 As shown, the synthesized cis-enamide-pyrazole derivatives, except A5, showed no obvious toxicity. The results of inflammation screening showed that most compounds could effectively inhibit the level of NO in Raw264.7 cells, and could also significantly inhibit the secretion levels of inflammatory factors IL-1β and IL-6, among which compound A2 had the strongest in vitro anti-inflammatory activity.

[0240] Activity Example 2 Study on the inhibition of inflammatory factors in Raw 264.7 cell model by cis-enamide-pyrazole derivatives (Compound A2).

[0241] The cis-enamide-pyrazole derivative (Compound A2) was subjected to an inhibition test on the expression level of cellular inflammatory factors using a conventional ELISA method.

[0242] 1. Cell Culture

[0243] Mouse mononuclear macrophage Raw 264.7 cells were cultured in DMEM high glucose medium (Gibco, Catalog No.: 11995065) supplemented with 10% (V / V) FBS (Gibco, Catalog No.: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, Catalog No.: 15140122). The culture conditions were 37°C, 5% CO2, and the cells were passaged when they reached 80% confluence.

[0244] 2. Cellular administration and induction of cellular inflammation

[0245] The experiment was conducted using cells in the logarithmic growth phase. The cells were cultured at a rate of 2×10 5 / well was inoculated in a 12-well plate, and cultured in an environment of 37°C and 5% CO2 until it grew to 70%, and then set aside. Set up groups: blank stimulation control group, LPS stimulation group and drug treatment group (compound A2). Carefully remove the culture medium, add fresh complete culture medium containing the compound to the positive control group and the drug treatment group, and add an equal volume of DMSO to the blank stimulation control group and the LPS stimulation group. After 1 hour, 1μg / mL LPS was added to all groups except the blank stimulation control group for 4 hours to induce cell inflammation. Take out the well plate and perform the ELISA experiment according to the instructions of the kit.

[0246] Collect the culture medium supernatant: collect the supernatant into a 1.5 mL EP tube, centrifuge at 1000 rpm for 10 minutes, and use the supernatant for ELISA detection of cytokines.

[0247] The secretion of mouse IL-6 and TNF-α in the culture medium supernatant was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Biyuntian, catalog number: IL-6: PI236; TNF-α: PT512). The specific steps are as follows:

[0248] (1) Reagent preparation

[0249] ① After taking it out of the refrigerator, place it at room temperature and equilibrate it for 20 minutes. ② Dilute the washing solution (20×) with double distilled water to 1× to prepare the required washing solution. ③ Add the standard diluent to 1 bottle of standard according to the volume marked on the standard label and incubate at room temperature for 15 minutes. ④ Take 5 clean 1.5 ml centrifuge tubes, add 250μL of standard diluent to each tube in advance, and dilute the standard in multiples to obtain six standard concentrations of 1000, 500, 250, 125, 62.5, and 31.25pg / mL. Finally, add the diluted standards to the pre-coated plate wells in turn, and add the standard diluent directly as 0pg / mL concentration, for a total of seven standard concentrations. ⑤ Add 300μL to each well, and proceed to the next time after about 15-30 seconds. Wash the plate five times in total and pat it dry on paper.

[0250] (2) Operation steps

[0251] ① Calculate the number of pre-coated strips required for one experiment, take out the required strips and place them in the 96-well frame.

[0252] ② Add samples or standards of different concentrations into the corresponding wells at 100 μL / well, seal the reaction wells with a sealing film (transparent), and incubate at room temperature for 120 minutes.

[0253] ③ Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0254] ④ Add 100 μL / well of biotinylated antibody, seal the reaction wells with a sealing film (transparent), and incubate at room temperature for 60 minutes.

[0255] ⑤ Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0256] ⑥Add horseradish peroxidase-labeled streptavidin 100 μL / well. Seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes.

[0257] ⑦ Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0258] ⑧Add 100 μL / well of TMB solution, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes.

[0259] ⑨Add 50 μL / well of stop solution, mix well, and immediately measure the expression levels of TNF-α and IL-6.

[0260] The results are as follows Figure 2 As shown in Figure 2, compared with the LPS group, compound A2 at 10 μM, 20 μM, 50 μM, and 100 μM concentrations significantly downregulated the IL-6 inflammatory factor level (IC 50 =4.816μM), and down-regulated the IL-1β inflammatory factor level at 50μM and 100μM concentrations (IC 50 =90.11 μM), and down-regulated in a dose-dependent manner.

[0261] Active Example 3 Anti-inflammatory effect of compound A2 on PM2.5-induced lung injury mice

[0262] 1. Purpose of the experiment

[0263] The therapeutic effect of compound A2 on the PM2.5-induced mouse lung injury model was investigated.

[0264] 2. Experimental Materials

[0265] Experimental animals: C57BL / 6 male mice, 36 mice, age: 6-8 weeks; weight: about 18-22 g, purchased from Zhuhai Baishitong Biological Co., Ltd.

[0266] Experimental instruments: heating stirrer, paraffin slicer, microscope, syringe, cell counter, balance, oven, refrigerated centrifuge, blood analyzer, etc.

[0267] Experimental reagents: HE staining related reagents, PBS solution, Masson staining kit (Solebo, product number G1340), immunohistochemistry kit (Zhongshan Jinqiao, product number PV9005), DAB color development kit (Zhongshan Jinqiao, product number ZLI9018), etc.

[0268] 3. Animal Experiments

[0269] 1. Establishment of PM2.5-induced lung injury mouse model: ① Modeling drug - PM2.5 particles; ② Modeling method - 50 μL of PM2.5 particle suspension was instilled into the trachea of ​​mice on the first and third days of modeling, with a dose of 3 mg / kg.

[0270] 2. Animal grouping: blank group, model group, positive drug group (dexamethasone), treatment group (low, medium and high doses of compound A2: 15 mg / kg, 30 mg / kg, 60 mg / kg), 6 animals in each group; body weight was measured every day. Figure 3 .

[0271] 3. Animal administration: Low (15 mg / kg), medium (30 mg / kg), and high (60 mg / kg) doses of compound A2 were administered by gavage. Dexamethasone was used as the positive control group. The control group and the model group were given an equal volume of normal saline by gavage once a day for 7 consecutive days. PM2.5 particles were instilled through the trachea on the 1st and 3rd days. Blood was collected from the eyeballs 24 hours later, and euthanasia was given by intraperitoneal injection of an overdose of 3% sodium pentobarbital.

[0272] 4. Animal sampling: After the mice were killed, the alveolar lavage fluid and lung tissue were collected.

[0273] 5. Observation indicators: total number of inflammatory cells, mouse lung tissue pathology, collagen formation, inflammatory protein expression, etc.

[0274] (1) Use a cell counter to count the total number of cells in the mouse bronchoalveolar lavage fluid; use a blood analyzer to count the total number of white blood cells in the mouse whole blood. The results are as follows: Figure 4 As shown, compared with the blank group, the total number of cells in the alveolar lavage fluid and the total number of white blood cells in the whole blood of mice in the model group were significantly increased; compared with the model group, dexamethasone and high-dose compound A2 reduced the total number of cells in the alveolar lavage fluid and the total number of white blood cells in the whole blood.

[0275] (2) Tissue fixation, dehydration and embedding

[0276] ① Place the mouse tissues of each group in 10% neutral formaldehyde solution and fix for 24 hours.

[0277] ② Place the trimmed tissue block in an embedding box and wash it with running water for 24 hours to completely remove residual formaldehyde.

[0278] ③ Dehydrate the tissue in an alcohol gradient, with the following specific steps: 70% alcohol for 12 hours, 80% alcohol for 1.5 hours, 95% alcohol I for 45 minutes, 95% alcohol II for 30 minutes, 100% alcohol I for 25 minutes, and 100% alcohol II for 20 minutes.

[0279] ④ After dehydration, place the tissue in alcohol / xylene (1:1, v / v) solution for 20 minutes.

[0280] ⑤ Soak the tissue in xylene I for 20 minutes and then in xylene II for 10 minutes.

[0281] ⑥ Place the tissue in preheated melted paraffin I and paraffin II in an oven at 60-65℃ for 1 hour each.

[0282] ⑦ Pour a small amount of embedding paraffin into the preheated metal embedding frame, place the skin tissue block in it, perpendicular to the embedding frame, with the bottom flat, pour paraffin again, embed the tissue, and cool it.

[0283] ⑧ Paraffin section: Fix the tissue paraffin block on the Leica slicer, slice the slices to a thickness of 5 μM, and slice them continuously. Use toothless forceps to place the slices in 40℃ water for spreading, pick up the slices with a slide, bake the slices at 60℃ for 2h, and store them in a slice box at room temperature for later use.

[0284] (3) HE staining

[0285] ① Dewaxing and hydration: Put the skin tissue paraffin sections into xylene I and xylene II for 15 minutes each, then put them into 100% ethanol I and ethanol II for 3 minutes each, 95% ethanol I and ethanol II for 3 minutes each, 80% ethanol for 3 minutes, and double distilled water for 1 minute;

[0286] ②Hematoxylin staining for 15 minutes, then wash off the excess stain on the slide;

[0287] ③1% hydrochloric acid ethanol (99mL 70% ethanol + 1mL concentrated hydrochloric acid) color separation for 3s, the cell nucleus and chromatin should be clear under the microscope;

[0288] ④ Rinse with running water for 15 minutes, and then with distilled water for 1 minute;

[0289] ⑤ Eosin for 2 minutes, rinse with running water for 1 minute;

[0290] ⑥ Dehydrate with 80% and 100% ethanol for 2s and 7min respectively;

[0291] ⑦ Xylene I and II for 5 min each;

[0292] ⑧ Sealing: Take the slide out of xylene II, add neutral gum to the tissue, gently cover with a coverslip, and let it dry naturally;

[0293] ⑨Observation: Observe pathological changes under a microscope, take photos and analyze.

[0294] like Figure 5 As shown, in the model group, alveoli collapsed, alveolar walls thickened, PM2.5 particles aggregated in the intercellular spaces, and there was obvious inflammatory cell infiltration. In the drug-treated group (compound A2), inflammatory cells were significantly reduced, and the degree of alveolar collapse and alveolar wall thickening was alleviated, indicating that compound A2 has an alleviating effect on PM2.5-induced lung damage.

[0295] (3) Masson staining

[0296] ① Paraffin sections were routinely dewaxed into distilled water.

[0297] ② According to the instructions of the Solebau Masson kit, mix reagents A1 and A2 in a ratio of 1:1 to prepare Weigert iron hematoxylin staining solution before use, add it to cover the slices and stain for 10 minutes.

[0298] ③ Wash away excess staining solution with distilled water, add acidic ethanol differentiation solution for 5-15 seconds, and wash with distilled water for 30 seconds.

[0299] ④Return to blue with Masson bluing solution for 3 minutes and wash with distilled water for 30 seconds.

[0300] ⑤Stain with Ponceau fuchsin staining solution for 10 min.

[0301] ⑥ During the above operation, prepare weak acid working solution in the ratio of distilled water: weak acid solution = 2:1, and add weak acid working solution dropwise to wash for 30 seconds.

[0302] ⑦ Pour off excess liquid, add phosphomolybdic acid solution for 1 minute, and add weak acid working solution for 30 seconds.

[0303] ⑧ Pour off excess liquid, add aniline blue dye solution and dye for 1 minute. Add weak acid working solution and wash for 30 seconds.

[0304] ⑨ Rapid dehydration with 95% ethanol for 2-3 seconds, and dehydration with anhydrous ethanol twice, each time for 5 seconds.

[0305] ⑩ Clear with xylene twice, 2 min each time, and seal with neutral gum.

[0306] Microscopic observation showed (see Figure 6 ), compared with the blank group, the proportion of blue collagen fibers in the model group increased; compared with the model group, the proportion of collagen fibers in the low, medium and high dose groups of compound A2 decreased to varying degrees, indicating that compound A2 has a therapeutic effect on alleviating pulmonary fibrosis caused by PM2.5-induced lung injury.

[0307] (4) Immunohistochemical staining

[0308] ① Place the paraffin sections of lung tissue in an oven and bake at 60°C for 1 hour.

[0309] ② Dewaxing: xylene 10 min three times → anhydrous ethanol I 5 min → anhydrous ethanol II 5 min → 95% ethanol 5 min → 85% ethanol 5 min → 75% ethanol 5 min → ddH2O 5 min.

[0310] ③ Antigen repair: Boil 400ml antigen repair solution (800mL dd H2O + 3g sodium citrate + 400mg citric acid and then dilute to 1000mL) in advance, put the slices into a beaker and boil for 20min. After cooling, rinse the slices with PBS 3 times, 3min each time.

[0311] ④ Add 3% hydrogen peroxide, incubate at room temperature for 5 minutes, and wash the sections 3 times with PBS, each time for 3 minutes.

[0312] ⑤ Remove PBS and add 10% goat serum blocking solution at room temperature for 1 hour.

[0313] ⑥ Remove the goat serum, dry and directly add the primary antibody, and incubate in a wet box at 4°C overnight (about 14 hours).

[0314] ⑦ Take out the slices, rewarm for 30 minutes, and rinse with PBS three times, 3 minutes each time.

[0315] ⑧Remove PBS, add secondary antibody for immunohistochemistry to each sample, incubate at room temperature for 15 minutes, and rinse with PBS three times, each time for 3 minutes.

[0316] ⑨ Dry the slices, place them on the microscope immediately, add DAB for staining, and rinse with PBS for 5 minutes after color development.

[0317] ⑩Re-stain with hematoxylin for 40 seconds → rinse with running water for 2 minutes → return to blue for 7 seconds → rinse with running water for 10-15 minutes → soak in ddH2O for 5 minutes.

[0318] 95% ethanol (1 min) → anhydrous ethanol I (1 min) → anhydrous ethanol II (1 min) → xylene (1 min) → xylene (1 min) → xylene (1 min) and seal the slides with neutral gum.

[0319] The results are as follows Figure 7 As shown in the data, compared with the blank control group, the expression levels of the macrophage M1 marker iNOS and the neutrophil marker LY6G in the model group were significantly increased, and the expression level of the M2 marker Arg-1 was lower; while in the drug-treated group (compound A2), the expression levels of iNOS and LY6G were significantly decreased, and the expression of Arg-1 was increased, indicating that compound A2 has an inhibitory effect on the inflammatory infiltration of M1 macrophages and neutrophils in lung tissue.

Claims

1. A cis-enamide-pyrazole derivative, characterized in that: Cis-enamide-pyrazole derivatives having the chemical formula of formula I or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof R1 is C6-14 aryl, five to fourteen membered heteroaryl, C6-10 cycloalkyl, five to fourteen membered heterocyclyl, C1-C6 alkyl, C1-C6 cycloalkyl, halogen, cyano; optionally, the C6-14 aryl, five to fourteen membered heteroaryl, C6-10 cycloalkyl, or five to fourteen membered heterocyclyl is substituted by one or more substituents selected from halogen, halogenated C1-C6 alkyl, carbonyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, carboxyl, cyano, hydroxyl, carbonyl, phenyl-SO2-, nitro, C1-C6 alkyloxycarbonyl; the five to fourteen membered heteroaryl or five to fourteen membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; preferably, the halogen is F, Cl, Br or I; R2 is H, C1-C6 alkyl, C1-C6 alkoxy, halogen, C1-C6 haloalkyl, C1-C6 haloalkoxy.

2. The cis-enamide-pyrazole derivative according to claim 1, characterized in that: When the amide group is located at the 5-position of the pyrazole, R1 is located at the 3-position of the pyrazole. When the amide group is located at the 3-position of the pyrazole, R1 is located at the 5-position of the pyrazole.

3. The cis-enamide-pyrazole derivative according to claim 1, characterized in that: The formula I is R1 is tert-butyl, methyl, or phenyl; R2 is hydrogen, fluorine, chlorine, bromine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

4. The cis-enamide-pyrazole derivative according to claim 1, characterized in that: The formula I is R1 is methyl, tert-butyl, or phenyl; R2 is hydrogen, fluorine, chlorine, bromine, methyl, methoxy, trifluoromethyl, or trifluoromethoxy.

5. The cis-enamide-pyrazole derivative according to claim 1, characterized in that: The formula I is R1 is 6. A cis-enamide-pyrazole derivative, characterized in that: Includes the following chemical formula, 7. A method for preparing the compound according to any one of claims 1 to 6, characterized in that: include (1) a) adding the compounds of formula I and formula II to a sodium ethoxide solution at -20°C-40°C, and reacting at 20°C-50°C for 8-12 hours; b) adding the product compound of formula III obtained in step a) to an anhydrous methanol solution of the compound of formula IV at 0°C, heating the mixture to room temperature and stirring for 1-2 hours, and then reacting at 60°C-90°C for 2-4 hours; c) dissolving the product compound of formula V obtained in step b) in an ammonia methanol solution and reacting at 20° C.-50° C. for 2-8 days; d) reacting the product compound of formula VI obtained in step c) with the compound of formula VII at 40° C.-80° C. for 0.5-3 hours; e) reacting the product compound of formula VIII obtained in step d) with sodium hydride at -20°C-5°C with stirring for 0.5-1 hour, then adding iodomethane at this temperature, and finally raising the temperature to 20°C-50°C with stirring for 2-6 hours to obtain a compound of formula IX; (2) f) adding the product compound of formula III obtained in step a) to an anhydrous ethanol solution of the compound of formula IV at 0°C, heating the mixture to room temperature and stirring for 1-2 hours, and then reacting at 60°C-90°C for 5-10 hours; g) dissolving the product compound of formula X obtained in step f) in an ammonia methanol solution and reacting at 20° C.-50° C. for 3-9 days; h) reacting the product compound of formula XI obtained in step g) with the compound of formula VII at 40° C.-80° C. for 1-4 hours; i) reacting the product compound of formula XII obtained in step h) with sodium hydride at -20°C-10°C with stirring for 0.5-1 hour, then adding iodomethane at this temperature, and finally raising the temperature to 20°C-50°C with stirring for 3-7 hours to obtain a compound of formula XIII; (3) j) stirring the compound of formula IV, the compound of formula XIV and methyl propiolate at 0°C for 5 minutes, then adding potassium carbonate, potassium bromide and potassium peroxymonosulfonate double salt (Oxone), and the reaction temperature was raised to 20°C-40°C and stirred for 9-12 hours; k) dissolving the product compound of formula XV obtained in step j) in an ammonia methanol solution and reacting at 20° C.-50° C. for 3-10 days; l) reacting the product compound of formula XVI obtained in step k) with the compound of formula VII at 40° C.-80° C. for 1-4 hours; m) reacting the product compound of formula XVII obtained in step l) with sodium hydride at -20°C-10°C with stirring for 0.5-1 hour, then adding iodomethane at this temperature, and finally raising the temperature to 20°C-50°C with stirring for 3-7 hours to obtain a compound of formula XVIII; Wherein, R1 and R2 are as described in any one of claims 1-5.

8. A pharmaceutical composition, characterized in that The composition comprises the compound according to any one of claims 1 to 6 and a pharmaceutically acceptable auxiliary material or excipient.

9. Use of the compound according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating inflammatory diseases and respiratory diseases.

10. Use of the compound according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating diseases mediated by IL-6 and / or TNF-α and / or IL-1β, an inhibitor of IL-6, an inhibitor of TNF-α, an inhibitor of IL-1β, or an inhibitor of IL-6, TNF-α and IL-1β.

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