Benzyl alcohol derivative with anti-influenza virus and anti-inflammatory activity

Abstract

The invention belongs to the technical field of medicines, and relates to a benzyl alcohol derivative with anti-influenza virus and anti-inflammatory activity. According to the invention, on the basis of 2, 5-dihydroxybenzyl alcohol, 14 benzyl alcohol derivatives are synthesized by combining 2, 5-dihydroxybenzyl alcohol with cysteine, amantadine or sesquiterpene. All the derivatives show excellent anti-influenza virus activity and also have a relatively good anti-inflammatory effect at the same time. The compounds can inhibit virus replication by acting with influenza virus hemagglutinase or M2 ion channels, can also reduce excessive inflammatory response by activating a host Nrf2 / HO-1 signal axis, can inhibit virus replication driven by NF-kappa B, can reduce over-high expression of inflammatory factors such as host cells IL-6, IL-1beta and TNF-alpha, and can reduce inflammatory factor storm. The action mechanism not only reduces the generation of drug resistance, but also reduces pathological effects caused by oxidative damage and inflammatory factor storm, and lays a foundation for developing novel anti-influenza virus drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to benzyl alcohol derivatives with anti-influenza virus and anti-inflammatory activities. BACKGROUND

[0002] Influenza virus is the main pathogen causing influenza. Human influenza virus is divided into four types, i.e., type A (A type), type B (B type), type C (C type) and type D (D type). Among them, the antigenicity of type A influenza virus is prone to variation, which poses a serious threat to human health and life, and has caused global pandemics many times in history. At present, the drugs approved by the US Food and Drug Administration (FDA) for clinical treatment of influenza virus mainly include neuraminidase inhibitors (oseltamivir and zanamivir), M2 ion channel inhibitors (amantadine) and cap-dependent endonuclease inhibitors (baloxavir). However, since almost all influenza virus strains have developed resistance to amantadine, M2 ion channel inhibitors are no longer used in clinical practice. In addition, it has been reported that the 2008-2009 North American seasonal H1N1 influenza virus strain has almost completely developed resistance to oseltamivir. Therefore, with the continuous emergence of drug-resistant influenza viruses, it is particularly urgent to develop new anti-influenza drugs.

[0003] To explore new anti-influenza, anti-inflammatory and antioxidant active substances, the research group focuses on traditional Chinese medicine Red Kallophycus. In the previous study, a phenolic molecule 2,5-dihydroxybenzyl alcohol was successfully isolated and identified from the fermentation broth of the endophytic fungus Penicillium coprophilum Mzz9. Biological activity tests confirmed that the molecule has anti-influenza virus, anti-inflammatory and antioxidant activities. Based on this finding, to further tap the potential medicinal value, a series of benzyl alcohol derivatives were innovatively prepared by chemical synthesis. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the present application provides a series of benzyl alcohol derivatives with anti-influenza virus and anti-inflammatory activities, which provides a new drug selection for anti-influenza virus and anti-inflammatory.

[0005] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0006] The present application provides the use of benzyl alcohol derivatives in the preparation of anti-influenza virus drugs and / or anti-inflammatory drugs, wherein the benzyl alcohol derivatives include at least one of SG1-14:

[0007]

[0008]

[0009] Preferably, the benzyl alcohol derivatives include at least one of SG-7, SG-9 and SG-14.

[0010]

[0011] Preferably, the influenza virus includes Influenza A / Aichi / 2 / 68 (H3N2), A / WSN / 1933 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), A / Fort Monmouth / 1 / 1947 (H1N1) mouse-adapted strains and A / Puerto Rico / 8 / 34 (H1N1) with NA-H274Y mutation.

[0012] Preferably, the benzyl alcohol derivatives exert anti-influenza virus effect by inhibiting NF-κB-driven viral replication, and the benzyl alcohol derivatives exert anti-inflammatory effect by reducing excessive inflammation through activating Nrf2 / HO-1 axis.

[0013] The second aspect of the present application provides an anti-influenza virus and / or anti-inflammatory drug, which uses the benzyl alcohol derivatives of the first aspect as the main active ingredient.

[0014] Preferably, to enrich the application form of the drug and make it suitable for different ranges, the drug further includes pharmaceutically acceptable excipients.

[0015] More preferably, the excipients include at least one of excipients, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavorings, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, antioxidants, adsorbents, filter aids, release retardants.

[0016] More preferably, to improve the use form of the drug, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations or suppositories. The drug preparation can be administered orally or parenterally (e.g. intravenously, subcutaneously, intraperitoneally or topically), and if certain drugs are not stable under stomach conditions, they can be prepared into enteric-coated tablets.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The application is based on 2,5-dihydroxybenzyl alcohol, and 14 benzyl alcohol derivatives are designed and synthesized by combining with cysteine, amantadine or sesquiterpene. Research shows that the 14 benzyl alcohol derivatives such as SG-7, SG-9 and SG-14 all exhibit excellent antiviral activity in in vitro experiments (for example, the antiviral activities of SG-7, SG-9 and SG-14 are 2.45±0.41, 0.60±0.19 and 2.20±0.43 μM respectively), and also have certain anti-inflammatory effect. Further research shows that these derivatives can not only inhibit the replication of viruses by acting on the influenza virus hemagglutinin enzyme or M2 ion channel, but also reduce excessive inflammation by activating the host Nrf2 / HO-1 signal axis, and inhibit the virus replication driven by NF-κB, thereby reducing the overexpression of inflammatory factors such as IL-6, IL-1β and TNF-α in host cells, and reducing the inflammatory factor storm. This multiple action mechanism not only reduces the emergence of traditional drug resistance, but also solves the pathological effects of oxidative damage and cytokine storm, and has multiple therapeutic advantages. The above research results show that the 14 benzyl alcohol derivatives SG-7, SG-9 and SG-14 designed by the application exhibit the value of further development as potential candidates in the aspects of anti-influenza and anti-inflammatory, and provide new insights for host-oriented anti-influenza virus and anti-inflammatory strategies. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The compound structure (A) and the synthesis route (B) of SG1-8.

[0020] Figure 2 The synthesis route and the structural formula of SG 9-13.

[0021] Figure 3 The synthesis route and the structural formula of the Vilsmeier ketone 1 and the sesquiterpene 7.

[0022] Figure 4 The synthesis route and the structural formula of the azide compound N-2 and SG-14.

[0023] Figure 5 The effect of SG-5, SG-7, SG-9 and SG-14 on the viral NP protein level detected by indirect immunofluorescence technology (100x).

[0024] Figure 6To evaluate the upregulation of antioxidant factors, inhibition of NF-κΒ activation, and suppression of inflammatory cytokine overexpression by SG-7, SG-9, and SG-14. (A) RAW264.7 cells were treated with SG-7, SG-9, SG-14, and amantadine or curcumin for 1 hour, followed by viral infection. After 24 hours, total RNA was extracted from the cells, and the mRNA expression levels of IL-6, IL-Ιβ, and TNF-α were measured by qRT-PCR. (B) The above procedure was repeated, and the expression of Nrf2, HO-1, and NQO1 was measured by qRT-PCR. (C-D) The above procedure was repeated, and mL385 or Nrf2 siRNA was added, and the expression of Nrf2, HO-1, NQO1, IL-6, and IL-Ιβ and TNF-α was measured by qRT-PCR. (E) The ability of SG-7, SG-9, and SG-14 to inhibit PR8- or LPS-induced NF-κΒ expression was evaluated by luciferase reporter gene assay.

[0025] Figure 7 To evaluate the protective effects of SG-7, SG-9, and SG-14 in IAV-infected mice. (A) Mice were dissected on day 5 after IAV infection and SG-7 administration, and lung tissues were collected for observation and weighed to calculate the lung index. (B) The body weight changes of the mice in each group were recorded. (C) Lung tissues were ground into lung suspensions, and the virus titers in the suspensions were measured, and (D) the death of the mice was recorded for 15 days. (E) Mice were dissected on day 5 after IAV infection and SG-9 administration, and lung tissues were collected for observation and weighed to calculate the lung index. (F) The body weight changes of the mice in each group were recorded. (G) Lung tissues were ground into lung suspensions, and the virus titers in the suspensions were measured, and (H) the death of the mice was recorded for 15 days. (J) Mice were dissected on day 5 after IAV infection and SG-14 administration, and lung tissues were collected for observation and weighed to calculate the lung index. (I) The body weight changes of the mice in each group were recorded. (L) Lung tissues were ground into lung suspensions, and the virus titers in the suspensions were measured, and (K) the death of the mice was recorded for 15 days. DETAILED DESCRIPTION

[0026] Further description will be given to the specific embodiments of the present application. It should be noted that the description of the embodiments is provided to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each of the embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0028] Example 1: Structures and synthesis of benzyl alcohol derivatives

[0029] 1. Synthesis of benzyl alcohol derivatives SG1-8 (as shown in Figure 1

[0030] 1.1. Synthesis of SG 1-2:

[0031] Into a 10 mL round-bottom flask, 100 mg (0.725 mmol) of 2,3-dihydroxybenzaldehyde or 2,5-dihydroxybenzaldehyde was added, and 2 mL of anhydrous ethanol was added. The round-bottom flask was stirred at 0°C for 10 min, and then 8.7 mg (0.363 mmol) of sodium borohydride was added. The reaction progress was monitored by TLC. After the reaction was completed, 100 μL of glacial acetic acid was added to quench the reaction. After the solvent was rotary evaporated, 5 mL of saturated sodium bicarbonate was added, and the mixture was extracted with 5 mL of ethyl acetate three times. The organic layers were combined and dried over anhydrous sodium sulfate. After filtration, concentration, and column chromatography purification (V dichloromethane:V methanol = 8:1) elution, the white compound SG1-2 was obtained.

[0032] SG-1: 1 H NMR (400 MHz, CD3OD): 6.77 (s, 1H), 6.64 (s, 1H), 6.65 (s, 1H), 4.61 (s, 2H).

[0033] SG-2: 1 H NMR (400 MHz, CDCl3): 6.90-6.83 (m, 1H), 6.79-6.73 (m, 1H), 6.65-6.56 (m, 1H), 4.9-4.74 (d, 2H).

[0034] 1.2. Synthesis of SG-3:

[0035] Into a 10 mL round-bottom flask, 100 mg (0.725 mmol) of 2,5-dihydroxybenzaldehyde, 2 mL of DMF, 472.4 mg (1.45 mmol) of cesium carbonate, and 108 μL (1.74 mmol) of iodomethane were sequentially added. The round-bottom flask was placed in an 80°C water bath for 2 h, and the reaction progress was monitored by TLC. After the reaction was completed, 2 mL of saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with 3 mL of ethyl acetate three times, and the organic layers were combined. The organic layers were washed with 5 mL of water and saturated physiological saline two times each, and the combined organic layers were dried over anhydrous sodium sulfate. After filtration, concentration, and column chromatography purification (V petroleum ether:V ethyl acetate = 2:1) elution, the yellow solid SG-3 was obtained.

[0036] SG-3: 1 ​H NMR (400MHz, CDCl3): 10.44(s,1H),7.33(d,1H),7.14-7.12(m,1H),6.94(d,1H),3.89(s,3H),3.80(s,3H).

[0037] 1.3 Synthesis of SG-4:

[0038] To a 10 mL round-bottom flask, add 20 mg (0.12 mmol) of SG-3 and 1 mL of anhydrous ethanol. Stir the flask at 0°C for 10 minutes, then add 1.44 mg (0.06 mmol) of sodium borohydride. Monitor the reaction progress by TLC. After completion, quench the reaction by dropwise addition of 20 μL of glacial acetic acid. The solvent is evaporated, and 1 mL of saturated sodium bicarbonate is added. Extract the mixture three times with 2 mL of ethyl acetate. The organic layers are combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (V petroleum ether:V ethyl acetate = 1:2) to obtain SG-4, a pale yellow viscous liquid.

[0039] SG-4: 1 H NMR (400MHz, CDCl3): 6.88 (d, 1H), 6.80-6.78 (m, 2H), 4.65 (s, 2H), 3.82 (s, 3H), 3.77 (s, 3H).

[0040] 1.4. Synthesis of SG 5-8:

[0041] 100 mg (0.724 mmol) of the starting material (2,3-dihydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, or 2,5-dimethoxybenzaldehyde) and 105.3 mg (0.869 mM) of L-cysteine ​​were weighed separately and poured into a 25 mL round-bottom flask. 5 mL of anhydrous ethanol was then added and the mixture was stirred at room temperature for 24-36 hours. The reaction solution was filtered and the filter residue was washed with 50 mL of methanol / dichloromethane solution (methanol:dichloromethane = 1:1, v:v). The filter residue was collected and dried in vacuo to obtain a white solid SG 5-8. Each compound was detected as a single peak by HPLC, and the H NMR spectrum ( 1 HNMR) identified it as a mixture of isomers.

[0042] SG-5: 1 H NMR (400MHz, CD3OD): 6.80-6.57(m,3H), 5.85-5.65(d,1H), 4.22-4.83(m,1H), 3.23-2.94(m,2H).

[0043] SG-6: 1H NMR (400 MHz, DMSO-d6): 7.09 (m, 1H), 6.32-6.16 (m, 2H), 5.74-5.57 (d, 1H), 3.75 (s, 1H), 3.10-2.90 (m, 2H).

[0044] SG-7: 1H NMR (400 MHz, DMSO-d6): 6.77-6.45 (m, 3H), 5.78-5.58 (d, 1H), 4.18-3.79 (m, 1H), 3.20 (m, 1H), 2.98 (m, 1H).

[0045] SG-8: 1 H NMR (400 MHz, DMSO-d6): 7.15-6.78 (m, 3H), 5.82-5.67 (d, 1H), 4.19 (m, 1H), 3.76-3.71 (m, 6H), 3.22-3.17 (m, 1H), 3.01-2.97 (m, 1H).

[0046] 2. Synthesis of benzyl alcohol derivatives SG9-13 (as shown in Figure 2 )

[0047] 2.1. Synthesis of SG-9:

[0048] A mixture of 302 mg (2 mmol) of adamantylamine and 276 mg (2 mmol) of 2,5-dihydroxybenzaldehyde in 4 mL of toluene was added with one drop of triethylamine. The reaction mixture was refluxed at room temperature in a Dean-Stark apparatus for 6 hours to ensure efficient reaction. Subsequently, the solvent toluene was removed by a rotary evaporator. The reaction mixture was then dissolved in 5 mL of absolute ethanol and 3.0 equivalents of sodium borohydride were added. The resulting mixture was stirred at room temperature under nitrogen protection overnight. After quenching the reaction mixture with water, the product was concentrated under reduced pressure. The resulting crude product was purified by a silica gel column (300-400 mesh) and a mixture of ethyl acetate and MeOH (2: 1, v:v) to obtain a yellow solid product. The purity of the resulting compound was more than 95% as determined by a Shimadzu 10A high-performance liquid chromatograph, and its nuclear magnetic resonance spectroscopy data was obtained using a Bruker DRX-400 spectrometer.

[0049] SG-9: light yellow solid, yield 82.8%. 1 H NMR (400 MHz, CD3OD): 6.76-6.68 (m, 3H), 4.04 (s, 2H), 2.21 (s, 3H), 1.99 (s, 6H), 1.82-1.73 (m, 6H). 13C NMR (100 MHz, CD3OD): δ 151.43, 150.44, 120.64, 118.52, 118.23, 117.18, 58.15, 41.05, 39.73, 36.72, 30.66.C 17 H 24 NO2,(ESI + ), m / z: [M+H] + = 274.36.

[0050] 2.2, Synthesis of SG-10:

[0051] A mixture of 302 mg (2 mmol) of adamantylamine and 276 mg (2 mmol) of 2,4-dihydroxybenzaldehyde in 4 mL of toluene was mixed with a drop of triethylamine. The reaction mixture was refluxed in a Dean-Stark apparatus for 6 hours to ensure efficient reaction. Subsequently, the solvent toluene was removed by a rotary evaporator. The reaction mixture was then dissolved in 5 mL of absolute ethanol and 3.0 equivalents of sodium borohydride was added. The resulting mixture was stirred at room temperature under nitrogen protection overnight. After the reaction mixture was quenched with water, the product was concentrated under reduced pressure. The resulting crude product was purified by a silica gel column (300-400 mesh) with a mixture of ethyl acetate and MeOH (2: 1, v:v) to obtain a yellow solid product. The purity of the resulting compound was more than 95% as determined by a Shimadzu 10A high-performance liquid chromatograph, and its nuclear magnetic resonance spectral data was obtained using a Bruker DRX-400 spectrometer.

[0052] SG-10: yellow solid, yield 75.3%. 1 H NMR (400 MHz, CD3OD): δ 7.08 (d, J = 8 Hz, 1H), 6.39 (s, 1H), 6.31 (d, J = 8 Hz, 1H), 4.03 (s, 2H), 2.21 (s, 3H), 2.00-1.99 (m, 6H), 1.83-1.73 (m, 6H). 13 C NMR (100 MHz, CD3OD): δ 161.19, 158.94, 133.08, 110.84, 107.90, 103.59, 58.15, 40.55, 39.59, 36.69, 30.61.C 17 H 24 NO2,(ESI + ), m / z: [M+H] + = 274.12.

[0053] 2.3, Synthesis of SG-11:

[0054] A mixture of 302 mg (2 mmol) of adamantylamine and 276 mg (2 mmol) of 2,3-dihydroxybenzaldehyde in 4 mL of toluene was added with one drop of triethylamine. The reaction mixture was refluxed in a Dean-Stark apparatus for 6 hours to ensure the reaction was carried out efficiently. Subsequently, the solvent toluene was removed by a rotary evaporator. The reaction mixture was then dissolved in 5 mL of absolute ethanol and 3.0 equivalents of sodium borohydride was added. The resulting mixture was stirred at room temperature under nitrogen protection overnight. After the reaction mixture was quenched with water, the product was concentrated under reduced pressure. The resulting crude product was purified by a silica gel column (300-400 mesh) with a mixture of ethyl acetate and MeOH (2: 1, v:v) to obtain a yellow solid product. The purity of the resulting compound was more than 95% as determined by a Shimadzu 10A high-performance liquid chromatograph, and its nuclear magnetic resonance spectroscopy data was obtained using a Bruker DRX-400 spectrometer.

[0055] SG-11: light yellow solid, yield 72.8%. 1 HNMR (400 MHz, CD3OD): δ 6.68 (s, 1H), 6.53-6.50 (m, 2H), 3.94 (s, 2H), 2.11 (s, 3H), 1.82 (s, 6H), 1.77-1.67 (m, 6H). 13 CNMR (100 MHz, CD3OD): δ 148.95, 147.49, 124.09, 120.53, 118.68, 115.05, 53.66, 43.70, 41.92, 37.35, 30.82.C 17 H 24 NO2, (ESI + ), m / z: [M+H] + = 273.88.

[0056] 2.4, Synthesis of SG-12:

[0057] A mixture of 302 mg (2 mmol) of adamantylamine and 276 mg (2 mmol) of 3,4-dihydroxybenzaldehyde in 4 mL of toluene was added with one drop of triethylamine. The reaction mixture was refluxed in a Dean-Stark apparatus for 6 hours to ensure efficient reaction. Subsequently, the solvent toluene was removed by a rotary evaporator. The reaction mixture was then dissolved in 5 mL of absolute ethanol and 3.0 equivalents of sodium borohydride was added. The resulting mixture was stirred at room temperature under nitrogen overnight. After quenching the reaction mixture with water, the product was concentrated under reduced pressure. The resulting crude product was purified by a silica gel column (300-400 mesh) with a mixture of ethyl acetate and MeOH (2: 1, v:v) to obtain a yellow solid product. The purity of the resulting compound was more than 95% as determined by a Shimadzu 10A high-performance liquid chromatograph, and its nuclear magnetic resonance spectroscopy data was obtained using a Bruker DRX-400 spectrometer.

[0058] SG-12: yellow solid, yield 81.1%. 1 H NMR (400 MHz, CD3OD): δ 6.89 (s, 1H), 6.83-6.77 (m, 2H), 3.99 (s, 2H), 2.23 (s, 3H), 1.99 (s, 6H), 1.84-1.73 (m, 6H). 13 CNMR (100 MHz, CD3OD): δ 147.34, 147.06, 126.84, 121.97, 117.64, 116.54, 56.50, 44.94, 40.52, 36.97, 30.75.C 17 H 24 NO2, (ESI + ), m / z: [M+H] + = 274.29.

[0059] 2.5, Synthesis of SG-13:

[0060] 302 mg (2 mmol) of adamantane amine and 276 mg (2 mmol) of 3,5-dihydroxybenzaldehyde were mixed in 4 mL of toluene, and one drop of triethylamine was added. The reaction mixture was refluxed in a Dean-Stark apparatus for 6 hours to ensure efficient reaction. The toluene solvent was then removed by rotary evaporation. The reaction mixture was then dissolved in 5 mL of anhydrous ethanol, and 3.0 equivalents of sodium borohydride were added. The resulting mixture was stirred at room temperature overnight under nitrogen. The reaction mixture was quenched with water, and the product was concentrated under reduced pressure. The crude product was purified by silica gel column (300-400 mesh) using a mixture of ethyl acetate and MeOH (2:1, v:v) to obtain a yellow solid. The purity of the obtained compound exceeded 95% using a Shimadzu 10A high-performance liquid chromatograph, and its nuclear magnetic resonance spectroscopy data were obtained using a Bruker DRX-400 spectrometer.

[0061] SG-13: yellow solid, yield 71.1%. 1 H NMR (400MHz, CD3OD): δ6.39-6.38(m,1H),6.32(m,1H),3.95(s,2H),2.22(s,3H),1.99-1.98(m,6H),1.83-1.73(m,6H). 13 C NMR (100MHz, CD3OD): δ160.46,135.02,109.06,104.23,58.66,44.89,39.44,36.61,30.65.C 17 H 24 NO2,(ESI + ),m / z:[M+H] + =274.09.

[0062] 3. Synthesis of SG-14 (Synthesis route see Figure 3 、 4 )

[0063] (1) 4-Hydroxy-2-butanone (30 g, 1 eq) was dissolved in 200 mL of dichloromethane. After cooling to 0°C, 4 drops of DMF were added, followed by the slow addition of SOCl2 (27 mL, 1.1 eq). After stirring for 30 min, the mixture was allowed to warm to room temperature and continued to react with stirring for 4 h. After the reaction, the reaction flask was placed in an ice bath and cooled to 0°C. Then, a saturated NH4Cl solution was slowly added dropwise to quench the unreacted SOCl2. The mixture was then extracted three times with dichloromethane. The organic layers were combined, dried over anhydrous Na2SO4, filtered through filter paper, and the dichloromethane was evaporated under reduced pressure. The mixture was then purified by silica gel column chromatography (V petroleum ether: V ethyl acetate = 15:1) to obtain 34.9 g of 4-chloro-2-butanone with a yield of 96.6%.

[0064] Dissolve 2-methyl-1,3-cyclohexanedione (10 g, 1 eq) in 150 mL of dry ethyl acetate, add Et3N (33 mL, 3 eq) followed by 4-chloro-2-butanone (9.5 mL, 1.2 eq) and heat to reflux at 80 °C for 16 h, monitor the progress of the reaction by TLC. After completion of the reaction, cool to room temperature, filter the reaction mixture to remove triethylamine hydrochloride on filter paper and wash the residue with 50 mL of ethyl acetate. Collect the filtrate and wash the filtrate with 60 mL of 10% dilute hydrochloric acid three times, 20 mL each time. Continue washing the organic layer with saturated NaHCO3solution and saturated NaCl solution three times, 20 mL each time. Dry the organic layer over anhydrous Na2SO4, filter and concentrate under reduced pressure to obtain the crude product. No further purification is required, use directly for the next reaction.

[0065] Dissolve the crude product from the previous step (1 eq) in 120 mL of dichloromethane, cool to 0 °C, then add L-proline (9.1 g, 1 eq) and AcOH (4.5 mL, 1 eq) and continue the reaction for 10 h, monitor the progress of the reaction by TLC. After completion of the reaction, evaporate the solvent under reduced pressure, dissolve the residue in 100 mL of ethyl acetate and wash with 50 mL of saturated NaHCO3solution, 50 mL of distilled water and 50 mL of saturated NaCl solution. Collect the organic layer, dry over anhydrous Na2SO4, filter and concentrate and purify by silica gel column chromatography (V petroleum ether:V ethyl acetate = 4:1) to obtain the Wieland-Michel ketone 1 Figure 3 ) in 85% yield as a yellow-brown oily liquid. 1 HNMR (400 MHz, Chloroform-d) δ 5.87 (s, 1H), 2.74 (t, J = 12.8 Hz, 2H), 2.57 - 2.40 (m, 4H), 2.15 (d, J = 10.7 Hz, 3H), 1.80 - 1.68 (m, 1H), 1.47 (s, 3H). 13 CNMR (101 MHz, Chloroform-d) δ 211.03, 198.31, 165.77, 125.82, 50.56, 37.63, 33.57, 31.72, 29.63, 23.24, 22.87. MS (ESI) m / z: calcd for C 11 H 14 O2 (M+H) + : 179.11; found: 178.97.

[0066] (2) Take 50 mg of raw material Vilsmeier ketone 1 in a 10 mL round-bottom flask, add 600 mL of anhydrous ethanol and stir to dissolve. Place the round-bottom flask in an ice bath for 10 minutes until the temperature drops to 0°C. Weigh 3.3 mg of sodium borohydride and add it to the round-bottom flask. During the reaction, TLC is used to detect the disappearance of the raw material and the formation of the product, both of which can be observed as dark spots under UV 254 nm. After adding sodium borohydride at 0°C for 15-20 minutes, the raw material disappears. Add 30 mL of ice acetic acid to quench the remaining sodium borohydride and neutralize the alkaline environment. After stirring at 0°C for 15 minutes, the solution in the round-bottom flask is rotary evaporated to dryness. Neutralize the ice acetic acid with 3 mL of saturated sodium bicarbonate solution, and extract with 3-5 mL of ethyl acetate until there are no dark spots on the plate. Combine the organic layer solutions, reduce pressure and distill, then purify the crude product by column chromatography on a 300-400 mesh normal silica gel column with DCM:EA = 10:1 as the eluent. The product 2 is a colorless oil, 49.5 mg, yield 98%. 1 H NMR (400 MHz, CDCl3) δ 5.78 (s, 1H), 3.42 (dd, J = 11.6, 4.3 Hz, 1H), 2.49-2.28 (m, 3H), 2.19 (m, 2H), 1.84-1.41 (m, 5H), 1.19 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 199.9, 168.8, 125.6, 78.5, 41.8, 34.4, 33.9, 32.2, 30.5, 23.4, 15.5.

[0067] (3) Take 49.5 mg of compound 2 in a 10 mL round-bottom flask, add 3.4 mg of DMAP (0.027 mmol, 0.1 eq). Dissolve 200 mL of pyridine in the above material. Slowly add 40 mL of acetic anhydride (0.42 mmol, 1.5 eq) to the reaction system. The reaction is complete after 1 h. Add 2 mL of saturated ammonium chloride solution to quench the reaction, and extract with 2 mL of ethyl acetate for 3 times. Combine the organic layers and rotary evaporate to dryness to obtain 60.9 mg of product 3. No further purification is needed. Yield 99.8%. 1 H NMR (400 MHz, CDCl3) δ 5.81 (s, 1H), 4.65 (dd, J = 11.8, 4.1 Hz, 1H), 2.45-2.31 (m, 3H), 2.26 (d, J = 12.4 Hz, 1H), 2.08 (s, 3H), 2.00-1.62 (m, 6H), 1.28 (s, 3H).

[0068] (4) Take 21 mg of compound 3 in a 25 mL round-bottom flask, add 200 mL of ethylene glycol, 0.6 mg of p-toluenesulfonic acid, and dissolve the above raw materials and catalyst in 5 mL of benzene. Heat the reaction to reflux at 100°C for 15 hours. After completion, remove the remaining solvent in the system by rotary evaporation, add saturated aqueous sodium bicarbonate solution, and extract with ethyl acetate. Combine the ethyl acetate layers and rotary evaporate, then purify the crude product by column chromatography with PE:EA = 10:1 as the eluent to obtain 12 mg of pure product 4. Compound 4 is a colorless oil with a yield of 87%. 1 H NMR (400 MHz, CDC13) δH: 5.33 (s, 1H), 4.81 (dd, J = 11.2, 4.5 Hz, 1H), 4.11 - 3.76 (m, 4H), 2.53 (d, J = 14.0 Hz, 1H), 2.18 (d, J = 14.2 Hz, 2H), 2.05 (d, J = 5.8 Hz, 3H), 1.82 - 1.63 (m, 5H), 1.49 (dd, J = 13.3, 4.2 Hz, 2H), 1.15 (s, 3H). 13 C NMR (100 MHz, CDC13) δC: 171.0, 138.7, 109.3, 64.6, 64.5, 41.2, 38.3, 30.8, 24.6, 24.0.

[0069] (5) Take 42.5 mg of compound 4 in a round-bottom flask and dissolve in 425 mL of methanol. Weigh 17 mg of sodium hydroxide solid and slowly add it to the solution. Stir the reaction for 4 hours and check by TLC that there is no starting material. Dry the methanol in the flask, add 3 mL of distilled water, and extract with 3 mL of ethyl acetate three times. Combine the organic layers and dry to obtain colorless oil product 5 without further purification. The yield is 99%. 1 H NMR (400 MHz, CDC13) δH: 5.30 (d, J = 2.6 Hz, 1H), 3.95 (s, 4H), 3.59 (t, J = 7.9 Hz, 1H), 2.52 (d, J = 14.1 Hz, 1H), 2.17 (d, J = 14.2 Hz, 2H), 2.08 - 1.91 (m, 2H), 1.85 - 1.67 (m, 5H), 1.45 (td, J = 13.5, 4.4 Hz, 1H), 1.08 (s, 3H). 13 C NMR (100 MHz, CDC13) δC: 139.0, 122.0, 77.9, 64.7, 64.5, 41.4, 39.3, 35.4, 31.0, 27.4, 25.0, 16.9.

[0070] (6) The reaction requires anhydrous and oxygen-free conditions. The stir bar, rubber stopper and syringe are dried before use. After drying, they are immediately taken out and placed in a desiccator for cooling. Compound 5 is taken in a suitable amount and added to a 10 mL round-bottom flask, which is dried and weighed (42 mg). The flask is tightly capped with a rubber stopper, and a silicone tube connected to an oil pump and a syringe needle is used to insert the rubber stopper to remove the oxygen in the flask. A nitrogen balloon connected to a syringe is used to replenish the inert environment in the flask. The oxygen is removed and the nitrogen is replenished three times in this way. The flask is injected with 500 mL of dichloromethane dried overnight with 4A molecular sieves using a syringe. The raw material is completely dissolved by stirring, and the flask is placed in an ice bath for cooling. After cooling, 800 mL of diethylzinc (1 mol / L in hexane) is added dropwise to the flask using a syringe, and the reaction solution is stirred at 0°C for 15 minutes. 50 mL of diiodomethane is taken with another dry syringe and slowly added dropwise to the reaction system. The solution gradually turns white. The reaction is stirred at 0°C for 10 hours, and then transferred to room temperature for 2.5 hours. After TLC detection shows that there is no raw material, 200 mL of triethylamine is added using a syringe and stirred for 2 minutes. After the end, saturated ammonium chloride solution is added to quench the reaction, and the same volume of dichloromethane is used for repeated extraction. The dichloromethane after extraction is filtered using diatomite. The combined organic layer is dried to obtain the crude product, which is purified by silica gel column chromatography to obtain the product 6, 39 mg in total, with a yield of 91.8%. 6 is a colorless oily compound. 1 HNMR (400 MHz, CDC13) δH: 3.98 - 3.78 (m, 4H), 3.39 (d, J = 6.3 Hz, 1H), 2.12 (d, J = 14.2 Hz, 1H), 1.99 - 1.89 (m, 1H), 1.86 - 1.67 (m, 5H), 1.45 - 1.13 (m, 3H), 1.09 (d, J = 1.7 Hz, 3H), 0.88 (q, J = 8.1 Hz, 1H), 0.63 (d, J = 14.1 Hz, 1H), 0.58 (t, J = 4.7 Hz, 1H), 0.07 (dd, J = 9.1, 4.2 Hz, 1H). 13 C NMR (100 MHz, CDC13) δC: 109.6, 75.0, 64.4, 64.0, 43.8, 34.8, 33.3, 31.2, 24.9, 23.2, 20.3, 19.9, 19.8, 15.9.

[0071] (7) Take 1.8 mg of compound 6 dissolved in 180 mL of dichloromethane, and cool to 0°C in an ice bath. Slowly add 10 mL of 70% perchloric acid to the round-bottom flask. Stir the reaction at 0°C for 1 hour, then transfer to room temperature and stir for 2-3 hours. Stop the reaction when the starting material is consumed according to TLC. After the reaction is complete, add 2 mL of saturated aqueous sodium bicarbonate solution to quench, and extract with 2 mL of ethyl acetate. Combine the organic layers, and purify the crude product obtained after rotary evaporation by normal-phase silica gel column chromatography with PE:EA = 2:1 as the eluent to obtain 1.2 mg of sesquiterpene 7 with a yield of 82.2%. Sesquiterpene 7 Figure 3 ) is a colorless oil with dark spots under UV 254 nm wavelength and black color with sulfuric acid vanillin heating. Rf = 0.33 (PE:EA = 2:1). 1 HNMR (400 MHz, CDC13) δH: 5.81 (s, 1H), 3.43 (dd, J = 11.4, 4.6 Hz, 1H), 2.40 (dd, J = 11.3, 6.1 Hz, 3H), 2.15 (dt, J = 13.6, 4.7 Hz, 1H), 1.95 - 1.79 (m, 6H), 1.19 (s, 3H), 1.06 (d, J = 6.5 Hz, 3H). 13 C NMR (100 MHz, CDC13) δC: 200.2, 171.7, 123.2, 78.3, 41.9, 34.6, 33.7, 33.6, 32.2, 30.4, 17.9, 16.5.

[0072] (8) Add 2,5-bis(tert-butyldimethylsilyloxy)benzaldehyde (1 eq) to anhydrous ethanol to completely dissolve the compound, then cool to 0°C, and slowly add sodium borohydride (1 eq) to react at 0°C for 1 h, monitoring the reaction progress by TLC. After the reaction is complete, carefully add acetic acid to quench the reaction, then transfer to room temperature to continue the reaction for 30 min. Remove the solvent under reduced pressure, dissolve the residue (completely dissolved) in ethyl acetate, and extract with saturated NaHCO3 solution three times, 20 mL each time. Dry the organic layer with anhydrous sodium sulfate, then filter and concentrate under reduced pressure to obtain (2,5-bis((tert-butyldimethylsilyl)oxy)phenyl)methanol.

[0073] (9) Add (2,5-bis((tert-butyldimethylsilyl)oxy)phenyl)methanol (1 eq) to anhydrous tetrahydrofuran to completely dissolve the compound, add DPPA (1.5 eq) and DBU (2 eq), and react at room temperature for 10 h. After the reaction is complete, add pure water to quench the reaction, and extract with ethyl acetate three times. Combine the organic layers, dry with anhydrous sodium sulfate, then filter, concentrate under reduced pressure, and purify by column chromatography with guaiac to obtain azide compound N-1.

[0074] (10) The azide compound N-1 (1 eq) was dissolved in anhydrous tetrahydrofuran, cooled to 0 °C, and then acetic acid (2.5 eq) and TABF (2.5 eq) were added. The reaction was continued for 30 min. After the reaction was completed, 10 mL of water was added, and then the reaction solution was extracted with ethyl acetate three times, 10 mL each time. The ethyl acetate layer was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography (V petroleum ether:V ethyl acetate = 4:1) to obtain azide compound N-2 Figure 4 ), with a product yield of 76.7%. 1 H NMR (400 MHz, Chloroform-d): δ 6.87 (s, 3H), 4.36 (s, 2H), 3.82 (d, J = 14.3 Hz, 6H). 13 C NMR (100 MHz, Chloroform-d): δ 153.44, 151.67, 124.77, 115.97, 113.99, 111.48, 55.86, 55.72, 50.03.

[0075] (11) Sesquiterpene 7 (100 mg, 51.5 mmol) was dissolved in 5 mL of dichloromethane treated with anhydrous Na2SO4, cooled to 0 °C under nitrogen protection, and then diisobutylaluminum hydride (1 mL, 1 M / L) was slowly added. The reaction was continued at 0 °C for 3 h. After the reaction was completed, 40 μL of water was slowly added, followed by 40 μL of 15% NaOH solution and 100 μL of water. The temperature was raised to room temperature and stirred for 15 min. 80 mg of anhydrous Na2SO4 was added, stirred for 15 min, filtered with diatomite, and then the filter cake was washed with dichloromethane. The dichloromethane washings were combined, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain intermediate 8, which was a light yellow oily liquid with a product yield of 73%. 1 H NMR (400 MHz, Chloroform-d): δ 6.87 (s, 3H), 4.36 (s, 2H), 3.82 (d, J = 14.3 Hz, 6H). 13CNMR (101 MHz, Chloroform-d) δ 149.81, 121.17, 77.67, 65.17, 40.76, 33.28, 31.94, 30.97, 30.69, 30.36, 18.11, 17.60. MS (ESI) m / z: calcd for C 12 H 20 O2(M+H) + : 197.15; found: 197.13.

[0076] (12) Intermediate 8 (50 mg, 0.255 mmol) was dissolved in 3 mL of dry tetrahydrofuran, cooled to 0 °C, and 1 mL of 60% sodium hydride in dry tetrahydrofuran (20.4 mg, 0.51 mmol) was added. After 15 min, the reaction was transferred to an oil bath at 100 °C and heated to reflux for 1 h. Then 26 μL of 20% 3-bromopropyne in toluene was added, and the reaction was continued at 105 °C for 10 h, monitoring by TLC. After the reaction was completed, the reaction was cooled to 0 °C, and water was added carefully to quench the reaction. The aqueous layer was extracted with ethyl acetate three times, and the ethyl acetate layers were combined, washed with dilute hydrochloric acid and saturated NaHC03solution, dried over anhydrous Na2S04, and filtered, and finally concentrated under reduced pressure to give intermediate 9 in 78% yield. The product was a light yellow oily liquid. 1 HNMR (400 MHz, Chloroform-d) δ 5.42 (s, 1H), 4.23 (dd, J = 6.5, 2.4 Hz, 2H), 4.11 (ddd, J = 8.3, 5.1, 2.6 Hz, 1H), 3.26 (dd, J = 11.5, 4.4 Hz, 1H), 2.42 (t, J = 2.4 Hz, 1H), 2.18 (dddd, J = 12.9, 6.4, 4.3, 2.2 Hz, 1H), 1.99 (dt, J = 9.9, 5.0 Hz, 1H), 1.91 - 1.84 (m, 1H), 1.76 (m, 2H), 1.65 (dd, J = 12.9, 3.6 Hz, 1H), 1.53 - 1.40 (m, 3H), 1.07 (s, 3H), 1.00 (d, J = 6.4 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 148.85, 120.34, 80.35, 78.93, 74.29, 73.89, 55.20, 40.79, 33.52, 33.20, 32.07, 30.46, 24.30, 18.14, 17.54. MS (ESI) m / z: calcd for C 15 H 21 O2(M+Na)+ : 257.15; found: 257.13.

[0077] (13) Intermediate 9 (15 mg, 0.064 mmol) was dissolved in 2 mL of dichloromethane, CuI (1.22 mg, 0.006 mmol) was added, followed by azide compound N-2 and 1 drop of DIEA, and the reaction was allowed to proceed at room temperature for 3 h, with TLC monitoring. After the reaction was completed, 2 mL of water was added, and the aqueous layer was extracted with dichloromethane 3 times. The dichloromethane extracts were combined, dried over anhydrous Na2S04, filtered with filter paper, and concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the target compound SG-14 Figure 4 ).

[0078] SG-14: yield 85.3%; 1 H NMR (400 MHz, Methanol-d4) δ 7.91 (s, 1H), 6.71 (d, J = 8.8 Hz, 1H), 6.67 - 6.58 (m, 2H), 5.50 (s, 2H), 5.39 (s, 1H), 4.67 (q, J = 12.3 Hz, 2H), 4.04 (s, 1H), 3.18 (d, J = 8.6 Hz, 1H), 2.25 - 2.12 (m, 1H), 2.02 - 1.93 (m, 1H), 1.90 - 1.82 (m, 1H), 1.82 - 1.73 (m, 2H), 1.52 - 1.39 (m, 2H), 1.38 - 1.28 (m, 2H), 1.06 (s, 3H), 0.97 (d, J = 6.9 Hz, 3H). 13 CNMR (101 MHz, Methanol-d4) δ 149.94, 148.98, 148.04, 145.03, 123.61, 121.84, 119.95, 116.21, 116.00, 115.77, 78.08, 75.06, 60.59, 40.65, 33.33, 33.22, 31.90, 29.81, 29.29, 24.17, 17.15, 16.87. MS (ESI) m / z: calcd for C 22 H 29 N3O4 (M+H) + : 400.22; found: 400.12.

[0079] Example 2: Anti-influenza virus, anti-inflammatory activity verification of benzyl alcohol derivatives

[0080] 1. Materials and methods

[0081] 1.1. Materials

[0082] L-cysteine, 3,4-dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, 2,3- dihydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde and 2,5-dihydroxybenzaldehyde were purchased from Shanghai Biotechmed Pharmaceutical Technology Co., Ltd. (Shanghai, China). Sodium borohydride and iodomethane were purchased from Guangzhou Chemical Reagent Factory. Abiraterone hydrochloride was purchased from Aladdin (China). Amantadine and triethylamine were purchased from Shanghai Biotechmed Pharmaceutical Technology Co., Ltd. (Shanghai, China). Sodium borohydride and toluene were purchased from Guangzhou Chemical Reagent Factory. Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (USA). Dimethyl sulfoxide (DMSO) and N-dimethylformamide (DMF) were purchased from Tianjin Zindong Tianzheng Fine Chemical Reagent Factory (Tianjin, China). MTT, medium (DMEM) and trypsin TPCK were purchased from Thermo Fisher Scientific (USA). Trizol and lipopolysaccharide (LPS) were purchased from Beijing Solabio Technology Co., Ltd. (Beijing, China). Luciferase reagent was purchased from Suzhou Youyilan Di Biological Technology Co., Ltd. mL385 was purchased from InVivo Chemical Technology (Guangzhou) Co., Ltd. Positive controls were amantadine (Aladdin, China) and curcumin (AnNaiJi, China).

[0083] 1.2, Cells and viruses

[0084] Madin-Darby canine kidney (MDCK), adenocarcinoma human alveolar basal epithelial cells (A549) and mouse macrophage RAW264.7 were cultured in medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin / streptomycin. Influenza A / Aichi / 2 / 68 (H3N2), A / WSN / 1933 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), A / Fort Monmouth / 1 / 1947 (H1N1) mouse-adapted strains and A / Puerto Rico / 8 / 34 (H1N1) with NA-H274Y mutation virus strains were stored in the Polypeptide and Natural Products Research Group of the School of Pharmaceutical Sciences, Southern Medical University, and were cultured in 9-day-old fertilized chicken embryos at 37°C, respectively. Clinical isolates 699 (H3 subtype) and 690 (H3 subtype) were provided by the Guangdong Center for Disease Control and Prevention. These two strains were amplified in MDCK cells to obtain progeny virus particles. The virus titers were determined by analyzing the 50% tissue culture infective dose (TCID 50 ) in MDCK cells, and the viruses were stored at -80°C until use.

[0085] 1.3, Cytopathic effect (CPE) and cytotoxicity test

[0086] MDCK cells were seeded at a density of 2 x 105cells per well in 96-well plates and allowed to adhere overnight. After washing with PBS, the antiviral activity was evaluated using a full-dose administration protocol: the test compounds were diluted in DMEM containing 0.6% TPCK-treated trypsin and an equal volume of 100 TCID50of A / PR / 8 / 34 virus was added to the cells. The cells were incubated for 48 hours. After 48 hours of infection, 0.5 μg / mL of MTT solution was added and incubated for 4 hours; then 150 μL of DMSO was added to dissolve the crystals and the absorbance was measured at 570 nm and the half-inhibitory concentration value was calculated. 4 MDCK cells were seeded at a density of 2 x 105cells per well in 96-well plates and allowed to adhere overnight. After washing with PBS, the antiviral activity was evaluated using a full-dose administration protocol: the test compounds were diluted in DMEM containing 0.6% TPCK-treated trypsin and an equal volume of 100 TCID50of A / PR / 8 / 34 virus was added to the cells. The cells were incubated for 48 hours. After 48 hours of infection, 0.5 μg / mL of MTT solution was added and incubated for 4 hours; then 150 μL of DMSO was added to dissolve the crystals and the absorbance was measured at 570 nm and the half-inhibitory concentration value was calculated. 50 A / Puerto Rico / 8 / 34 virus solution was mixed and then added to the cells, and incubated for 48 hours. After 48 hours of infection, 0.5 μg / mL of MTT solution was added and incubated for 4 hours; then 150 μL of DMSO was added to dissolve the crystals and the absorbance was measured at 570 nm and the half-inhibitory concentration value was calculated.

[0087] MDCK cells were seeded in 96-well plates and incubated overnight. Subsequently, the compounds were diluted in DMEM to different concentrations and added to the cells, and incubated for 48 hours. After 48 hours, 0.5 mg / mL of MTT solution was added and incubated for 4 hours; then 150 μL of DMSO solution was added to dissolve the crystals and the absorbance was measured at 570 nm to evaluate the toxicity of the compounds to the cells.

[0088] 1.4. Immunofluorescence assay

[0089] MDCK cells were seeded in 24-well plates with cell slides and treated as described above (1.3). After 24 hours of co-incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde (Beijing Reagent Biotech Co., Ltd.) for 20 minutes. Subsequently, 0.25% Triton X-100 was added to the cells and treated at room temperature for 5 to 10 min. After washing with PBS, the cells were incubated with 500 μL of 4% bovine serum albumin (BSA) (Sigma-Aldrich) solution at 37°C for 4 hours. Next, the primary antibody for influenza virus nucleoprotein (NP) (diluted 1:200 in 4% BSA) was added and incubated at 4°C. After 18 hours, the primary antibody was removed, the cells were washed with PBS three times, and then the secondary antibody (fluorescein isothiocyanate (FITC)-labeled) (Sigma-Aldrich) was added, which was diluted 1:200 in 4% BSA. After incubation at room temperature for 2 hours, 4', 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) was added to label the cell nuclei for 10 min. Finally, the cells were observed under a confocal microscope (Nikon, Japan) and quantitative analysis was performed using Image J software.

[0090] 1.5. Quantitative real-time RT-PCR

[0091] (1) Cell treatment: A549 cells or RAW264.7 cells were respectively plated in 6-well plates and cultured overnight. The drugs were diluted in DMEM to a series of concentrations at a 2-fold ratio, and then added to the cells at 100 μL / well. After incubation for half an hour, the medium containing the drugs was discarded, and the cells were washed twice with PBS, and then 100 TCID 50 A / Puerto Rico / 8 / 34 virus liquid medium was added, and after 1 h of virus adsorption, the medium containing unabsorbed virus was discarded, and the cells were washed twice with PBS, and then DMEM medium was added for continuous culture for 24 h.

[0092] (2) Total RNA extraction

[0093] After 24 h, the supernatant was discarded, and the cells were washed with PBS, and then total RNA was extracted by the Trizol method. The specific operation was as follows: 1 mL of Trizol was added to each well, and lysis was performed at room temperature for 5 min. After blowing, it was transferred to a 1.5 mL EP tube. 0.2 mL of chloroform was added to each tube, and it was mixed uniformly after shaking for 30 s, and then it was allowed to stand for 2-3 min. After low-temperature centrifugation at 12000 rpm for 10 min, the supernatant was transferred to a new EP tube. 400 μL of the supernatant was taken from each tube, 400 μL of pre-cooled isopropanol was added, and it was mixed uniformly by inverting up and down several times. It was allowed to stand at room temperature for 10 min, and then low-temperature centrifugation was continued at 12000 rpm for 10 min. After centrifugation, a white precipitate was observed at the bottom of the EP tube. The supernatant was removed, 1 mL of 75% ethanol solution was added, and low-temperature centrifugation was performed at 12000 rpm for 2 min. The ethanol solution was discarded, and low-temperature centrifugation was performed again for 2 min to remove the residual ethanol solution. The precipitate was dried at room temperature for 5-10 min, and 30-60 μL of DEPC water was added for dissolution. The concentration and purity of the RNA were measured by a micro ultraviolet spectrophotometer, and the OD 260 / OD 280 It was within the range of 1.8-2.0 and could be used for subsequent experiments.

[0094] (3) Reverse transcription experiment: The experimental operation was performed according to the instructions of the Takara reverse transcription kit.

[0095] ①: Genomic DNA removal reaction: The preparation process was always kept at low temperature, and after preparation was completed, it was placed in a 42℃ water bath for 2 min. The reaction was prepared according to the components in Table 1.

[0096] Table 1 Reagent system for genomic DNA removal reaction

[0097]

[0098] *The amount of Total RNA was determined according to the specific requirements of the experiment.

[0099] ②: Reverse transcription reaction: The preparation of the reaction system was performed on ice, and the reaction solution was prepared according to Table 2:

[0100] Table 2 Reverse transcription reaction liquid system

[0101]

[0102] After the reaction system is prepared, set a: 37°C, 15 min; b: 85°C, 5 sec; c: 4°C. Perform reverse transcription reaction according to the procedure.

[0103] (4) Real-time fluorescent quantitative PCR reaction

[0104] 1. Preparation of PCR reaction liquid: according to Table 3, the preparation process should be performed on ice.

[0105] Table 3 PCR reaction liquid system

[0106]

[0107] 2. PCR amplification reaction: add the reaction liquid to a 96-well PCR reaction plate, centrifuge at 2500 rpm for 5 min, and use LC480 PCR instrument for amplification. Perform three-step PCR amplification reaction, and the setting program is as follows: a denaturation: 95°C for 30 s, one cycle; b amplification: 95°C for 5 s, 60°C for 30 s, 45 cycles; c melting: 95°C for 5 s, 60°C for 1 min, 95°C, one cycle; d cooling: 50°C for 30 s, one cycle.

[0108] After the reaction is completed, use LC480 analysis software to calculate the CT value of the sample, use GAPDH gene as the internal reference gene, and calculate all results using 2 -ΔΔCT formula, and the experiment is repeated more than 3 times.

[0109] Among them, the primers used in real-time fluorescent quantitative PCR are shown in Table 4.

[0110] Table 4 qRT-PCR primers

[0111]

[0112] 1.6, Luciferase assay

[0113] Determine the inhibitory activity of the compound on NF-κB by luciferase reporter gene detection method. 5×10 4 Stable transfection pNF-κB-Luc RAW 264.7 cells are inoculated into a 96-well plate and cultured overnight. After washing the cells twice with PBS, incubate with the compound for 1 hour, then add 200 microliters of 100 TCID 50A / Puerto Rico / 8 / 34 or 100 ng / mL LPS treatment. After 24 hours or 6 hours, cells were washed with PBS, and 100 μL of cell lysis solution was added and shaken for 20 min. After cell lysis was completed, the supernatant was transferred to a white opaque 96-well plate, and 100 μL of luciferase reagent was added to each well under light-protected conditions. The luminescence of firefly was detected immediately afterwards using a microplate reader.

[0114] 1.7. Animal Experiments

[0115] Male Kunming mice (4-6 weeks old, weighing 18-22 g) were provided by the Experimental Animal Center of Southern Medical University (License No. SCXK (Yue) 2021-0041, Guangzhou, China) for antiviral research. Animals were allowed free access to food, with 6 mice per cage and corn cobs as bedding. They were isolated for 48 hours before use and monitored according to the standard operating procedures and animal welfare law of the institution. In the experiment, mice were randomly divided into six groups of six, including: (I) virus control group, (II) blank control group, (III) non-infected virus + compound group, (IV) infected virus + compound group, (V) infected virus + compound group, and (VI) infected virus + positive drug group. At the beginning of the experiment, the mice were first lightly anesthetized with ether, and then infected with A / Puerto Rico / 8 / 34 (H1N1) virus twice through the intranasal route, with a virus titer of 5 x 10 6 TCID 50 , with a virus volume of 35 μL per drop. After infection, 300 μL of test compound was immediately administered by gavage according to the group. At the same time, the mice in the virus control and blank control groups were given normal saline by gavage. The mice received the test compound twice a day for 5 consecutive days. In the following 15 days, the body weight, mortality, and behavioral changes of the mice were recorded daily.

[0116] 1.8. Mouse Lung Index and Lung Virus Titer Determination

[0117] Using the same virus infection protocol as 1.7, the test compound was administered to the mice by gavage for 3 days after infection (dose given in Figure 7 ). By the 5th day, the lung tissue was collected and washed with normal saline, dried on gauze, and weighed to calculate the lung index (lung weight / body weight) of each group. The mouse lung tissue was mixed with normal saline at a ratio of 1:9 (weight / volume) to prepare a lung suspension using a tissue homogenizer. Then, the lung homogenate was centrifuged at 4°C (2500 r / min) for 20 minutes to obtain the supernatant. 200 μL of the supernatant was serially diluted (10 1 , 10 2 , 10 3 , 10 4 , 105 ,10 6 Supernatant (10-fold) was added to 96-well plates seeded with MDCK cells, and after 48 hours of incubation, the TCID50 50 (five-day infectious dose) of virus was measured.

[0118] 1.9. Statistical analysis

[0119] Data analysis and graph plotting were performed using Graph Prism 8.0. Each data point was expressed as the mean ± standard deviation (SD), and each experiment was repeated more than three times. The fluorescence intensity of images was quantified using ImageJ software. All data were analyzed by SPSS 25.0 software for non-paired t-test or one-way ANOVA. The significance level of statistical analysis was defined as: ns, no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0120] 2. Experimental results

[0121] 2.1. Benzyl alcohol derivatives exhibit inhibitory effect on viral infection

[0122] The anti-viral activity of derivatives SG-1 to SG-14 was tested by viral-induced cytopathic effect (CPE) assay and quantified by MTT assay. As shown in Table 5, all compounds exhibited relatively good anti-influenza A virus (IAV) activity with half inhibitory concentration (IC 50 ) values ranging from 0.60 ± 0.19 μΜ to 15.72 ± 0.04 μΜ. Further determination of the inhibitory activity of the compounds against influenza virus subtypes, including A / Fort Monmouth / 1 / 1947, A / Aichi / 2 / 68 (H3N2), A / Puerto Rico / 8 / 34 (H1N1) mutated with NA-H274Y, A / WSN / 1933 (H1N1), clinical isolates of 699 (H3 subtype) and 690 (H3 subtype), was performed. As shown in Table 6, the half inhibitory concentration values of the derivatives ranged from 0.32 ± 0.06 μΜ to 3.64 ± 0.69 μΜ, which further confirmed the broad-spectrum anti-IAV activity of these compounds.

[0123] The anti-IAV activity of SG-5, SG-7, SG-9 and SG-14 was confirmed by immunofluorescence assay, which characterized the level of presence of influenza virus nucleoprotein (NP) on MDCK cells by green fluorescence observed under a fluorescence microscope. As shown in Figure 5 , there was a significant decrease in green fluorescence compared to the virus group.

[0124] Table 5 In vitro anti-influenza A virus (A / PR / 8 / 34 / H1N1) activity and cytotoxicity of benzyl alcohol derivatives

[0125]

[0126]

[0127] a Compound's half inhibitory concentration (IC) 50 Measured by cytopathic effect assay using influenza A virus (A / PR / 8 / 34 / H1N1) and MDCK cells. b Cytotoxicity of all compounds (half inhibitory concentration, CC 50 ) was determined by MTT assay in MDCK cells. c Selectivity index (SI) was calculated by the formula CC 50 / IC 50 All data represent the mean of three independent experiments.

[0128] Table 6 Antiviral activity of benzyl alcohol derivatives against various influenza virus strains

[0129]

[0130] PR8,Influenza A / Puerto Rico / 8 / 34; FM-1, Mouse-adapted viral strain A / Fort Monmouth / 1 / 1947; 699(H3)and 690(H3),699(H3)and 690(H3)were clinicalisolate; H3N2,Influenza A / Aichi / 2 / 68; 274,Influenza A / Puerto Rico / 8 / 34NA-H274Ymutant; WSN, Influenza A / WSN / 1933; NT: Not tested.

[0131] 2.2 Benzyl alcohol derivatives SG-7, SG-9, and SG-14 block IAV-induced proinflammatory cytokine overexpression by inhibiting NF-κB and inducing Nrf2

[0132] The expression of virus-induced proinflammatory cytokines (such as IL-6, IL-1β, and TNF-α) was significantly inhibited after treatment with benzyl alcohol derivatives ( Figure 6 A). At the same time, SG-7, SG-9, and SG-14 significantly increased the mRNA levels of antioxidant factors Nrf2, HO-1, and NQO1, and were superior to curcumin, while the prototype drug amantadine had no regulatory effect ( Figure 6B). To further determine whether the inhibitory effect of SG-7, SG-9 and SG-14 on IAV was due to the induction of Nrf2 expression, further addition of Nrf2 inhibitor mL385 or transfection of Nrf2 siRNA (sense strand: CGAAUUACAGUGUCUUAAUTT, antisense strand: AUUAAGACACUGUAAUUCGTT) as a control. The results showed that the mRNA expression of Nrf2 and its downstream factors HO-1 and NQO1 induced by SG-7, SG-9 and SG-14 was inhibited compared with the group without inhibitor Figure 6 C). It was thus speculated that the ability of SG-7, SG-9 and SG-14 to inhibit the expression of pro-inflammatory cytokines might be closely related to the expression of Nrf2. At the same time, the mRNA levels of IL-6, IL-1β and TNF-α in the mL385 inhibitor group or the Nrf2 siRNA transfection group were significantly higher than those in the group without using mL385 inhibitor or transfecting Nrf2 siRNA after treatment with SG-7 and SG-9 Figure 6 D). These results indicated that the induction of Nrf2 was one of the main mechanisms by which SG-7, SG-9 and SG-14 exerted their anti-inflammatory effects. Subsequently, the relationship between SG-7, SG-9 and SG-14 and nuclear factor (NF-κΒ) was further verified by luciferase reporter gene detection. The results showed that at a concentration of 20 μΜ, SG-7, SG-9 and SG-14 significantly inhibited the activation of NF-κΒ Figure 6 E). These data indicated that SG-7, SG-9 and SG-14 could control the inflammatory response triggered by IAV infection by inhibiting NF-κΒ and inducing Nrf2.

[0133] 2.3, SG-7, SG-9 and SG-14 inhibit the replication of influenza virus in the lungs of mice

[0134] This experiment studied the effect of SG-7 on viral replication in infected mouse lung cells. In the experiment, the mice were first lightly anesthetized with ether, and then infected with A / Puerto Rico / 8 / 34 (H1N1) twice by intranasal route, with a titer of 5 x 10 6 TCID 50 , and the volume of each drop was 38 μΐ, and the mice received drug treatment twice a day for three consecutive days. It was found that the IAV-infected mice showed persistent griping and shaking symptoms on the 3rd day, and the lung tissue was dissected from the lungs of the mice on the 5th day, and the lung index (lung weight / body weight) Figure 7 A) and the virus titer in the lung suspension were determined. In the groups of mice treated with SG-7 (45 mg / kg) and oseltamivir (15 mg / kg), the virus titer was significantly lower than that in the control group without drug Figure 7 C).

[0135] The drug was administered for five consecutive days according to the above viral infection protocol. The protective effect of SG-7 on infected mice was evaluated according to the average survival days, survival rate and body weight change within 15 days. The infected mice began to show weight loss and flu symptoms from the third day, and approximately on the sixth day, the mice began to die. By the tenth day, all mice in the virus control group had died. However, from the eighth to the fifteenth day, the mice in the drug administration group gradually recovered their body weight Figure 7 B), and the survival rate of the oseltamivir (15 mg / kg) control group was 60% ( Figure 7 D).

[0136] Meanwhile, the effect of SG-9 on viral replication in the lung cells of infected mice was also studied. In the experiment, the mice were first lightly anesthetized with ether, and then infected with A / Puerto Rico / 8 / 34 (H1N1) twice through the intranasal route, with a titer of 5 x 10 6 TCID50per drop of 38 μL, and the mice received drug treatment twice a day for three consecutive days. It was found that the IAV-infected mice showed persistent scratching and shaking symptoms on the third day, and the lung tissue was dissected from the lungs of the mice on the fifth day to determine the lung index (lung weight / body weight) Figure 7 E) and the viral titer in the lung suspension. In the SG-9 (20 mg / kg) and amantadine (20 mg / kg) treatment groups, the viral titer was significantly lower than that in the untreated virus group Figure 7 G). Again, the drug was administered according to the above viral infection protocol, and the administration was continued for five days, and the protective effect of SG-9 on infected mice was evaluated according to the average survival days, survival rate and body weight change within 15 days. The infected mice began to show weight loss and flu symptoms on the third day Figure 7 F). On the ninth day, all mice in the virus control group had died. The survival rate of the virus + SG-9 (10 mg / kg) treatment group was 33%, and the survival rate of the virus + SG-9 (20 mg / kg) treatment group was 50%; the survival rate of the virus + amantadine (10 mg / kg) treatment group was also 33%. Until the fifteenth day, all mice in the virus infection group had died except for the virus + SG-9 (20 mg / kg) treatment group, and the survival rate of this group was 33%. In addition, from the eighth to the fifteenth day, the body weight of the mice in the virus infection + SG-9 (20 mg / kg) group slowly increased Figure 7 H).

[0137] In addition, in order to explore whether SG-14 also has a similar effect in vivo, a KM mouse survival experiment was further conducted. The ether-anesthetized mice were infected with A / Puerto Rico / 8 / 34 virus (5 x 10 6TCID50), 45 μL each time, was administered for 5 consecutive days after infection, and the general behavior, body weight changes, mortality, etc. of the mice were observed and recorded for 14 consecutive days.

[0138] like Figure 7 As shown in Figure 1J, during 14 consecutive days of observation, mice infected with the virus exhibited symptoms such as huddling, coarse fur, weight loss, and tremors. The weight of infected KM mice began to decrease on the third day, and deaths began on the fourth day, until all mice in the virus control group died on the ninth day. Starting on the eighth day, the weight of mice in the drug-treated group began to increase. The survival rate of infected mice in the SG-14 (40 mg / kg) and oseltamivir (15 mg / kg) groups was 28.57% (P<0.05), while the survival rate of the SG-14 (10 mg / kg) group was 14.29%. These results indicate that SG-14 can prolong the survival of mice after influenza virus infection.

[0139] The mouse survival rate experiment showed that the compound SG-14 can prolong the survival time of mice. The effect of SG-14 on the viral replication in the lungs of infected mice was further examined. KM mice were randomly divided into 5 groups. Except for the blank group, the mice in other groups were anesthetized with ether and infected with A / Puerto Rico / 8 / 34 virus. The drug was administered for 3 consecutive days. On the fifth day, the mice were weighed and recorded and killed by dislocation of the neck. The lung tissues of the killed mice were dissected and the lung tissues were cleaned with physiological saline and weighed. Part of the lung tissue was ground into lung suspension. After centrifugation of the lung suspension, the supernatant was taken to measure the virus titer. The experimental results showed that ( Figure 7 KL), compared with the virus control group, the lung index of the SG-14-treated group and the oseltamivir-treated group had no statistical difference (P>0.05), while the virus titer in the lung tissue suspension decreased.

[0140] In summary, 14 benzyl alcohol derivatives, including SG-7, SG-9 and SG-14, not only have strong intracellular antiviral activity, but also can inhibit the replication of influenza virus in the lungs of mice and reduce the mortality rate of mice.

[0141] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. Use of a benzyl alcohol derivative in the preparation of an anti-influenza virus drug and / or an anti-inflammatory drug, characterized in that: The benzyl alcohol derivative includes at least one of SG1-14:

2. The use according to claim 1, characterized in that The benzyl alcohol derivative includes at least one of SG-7, SG-9 and SG-14:

3. The use according to claim 1 or 2, characterized in that The influenza viruses include various current influenza A virus strains and influenza B virus strains, such as Influenza A / Aichi / 2 / 68 (H3N2), A / WSN / 1933 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), A / Fort Monmouth / 1 / 1947 (H1N1) mouse-adapted strains and A / Puerto Rico / 8 / 34 (H1N1) with NA-H274Y mutation, etc.

4. The use according to claim 1 or 2, characterized in that The benzyl alcohol derivatives exert anti-influenza virus effects by inhibiting NF-κB-driven viral replication, and the benzyl alcohol derivatives exert anti-inflammatory effects by activating the Nrf2 / HO-1 axis to reduce excessive inflammation.

5. An anti-influenza virus and / or anti-inflammatory drug, characterized in that: The medicine uses the benzyl alcohol derivative according to claim 1 or 2 as a main active ingredient.

6. The anti-influenza virus and / or anti-inflammatory drug according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The anti-influenza virus and / or anti-inflammatory drug according to claim 6, characterized in that: The excipients include at least one of excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, antioxidants, adsorbents, filter aids, and release retardants.

8. The anti-influenza virus and / or anti-inflammatory drug according to claim 6, characterized in that: The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations or suppositories.

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