A class of isopentenyl flavonoid derivatives and their application

Abstract

The present invention relates to a class of isopentenyl flavonoid derivatives and their applications, and their structural formulas are shown in (I) and (2). The present invention has outstanding beneficial effects: 1. So far, there is no report in the prior art of a pharmaceutical composition with such isopentenyl flavonoid derivatives (1-2 shown in formula (I)) as an effective ingredient, and there is no report on the application of such isopentenyl flavonoid derivatives and their pharmaceutical compositions in the preparation of drugs for treating Zika virus (ZIKV) inhibitors. 2. The two isopentenyl flavonoid derivatives obtained by the present invention can significantly inhibit the expression of ZIKV E protein; and have no obvious toxicity to Vero cells, and their CC 50 All were greater than 100μM.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology and relates to the preparation and application of a class of isopentenyl flavonoid derivatives as a pharmaceutical composition for treating Zika virus (ZIKV) disease as an inhibitor. Background Art

[0002] Zika virus (ZIKV) belongs to the genus Flavivirus of the family Flaviviridae. It is mainly transmitted by Aedes mosquitoes and is a single-stranded RNA virus wrapped in polyprotein. In addition to infection mainly through the bites of Aedes mosquitoes, it can also be transmitted through blood, sexual transmission, and mother-to-child transmission. The disease caused by ZIKV infection is called Zika virus disease, also known as Zika fever. The clinical symptoms caused by ZIKV infection are generally mild, and its early symptoms are often confused with those of dengue virus infection. The main symptoms of most patients are fatigue, muscle aches, conjunctivitis, etc., but these symptoms generally disappear on their own after 2-7 days. In recent years, more and more studies have shown that ZIKV infection can cause serious damage to the patient's nervous and immune systems, leading to serious adverse consequences. For example, ZIKV infection in pregnant women can cause microcephaly in newborns. In addition, adult infection can also lead to Guillain-Barre syndrome, and male infected patients will suffer from testicular damage, which has attracted great attention from health organizations around the world. Currently, there are no approved drugs or preventive vaccines for the treatment of ZIKV infection, and most clinical treatment strategies use analgesics to relieve symptoms. Natural products have complex and diverse structures and rich biological activities. It is of great significance to find and discover safe and effective anti-ZIKV drugs from natural products to treat diseases caused by ZIKV.

[0003] Isopentenyl flavonoids are active ingredients of various Chinese herbal medicines characterized by isopentenyl side chains attached to the flavonoid skeleton. They are a branch of flavonoid compounds. Due to the diversity of their structural types and substituents, isopentenyl flavonoids have a wide range of biological activities, such as promoting stem cell differentiation, anti-oxidation, anti-inflammatory and immunomodulatory, anti-tumor and anti-viral effects, and have great development prospects. Studies have reported that five natural flavonoid compounds, myricetin, quercetin, luteolin, isorhamnetin and apigenin, have inhibitory activity against the NS2B-NS3 protease of ZIKV, EC 50The range is 1.3-56.3 μM. The presence of isopentenyl side chains can significantly enhance the antiviral activity of flavonoids. Therefore, isopentenyl flavonoids can be one of the effective ways to develop highly effective and low-toxic natural anti-ZIKV drugs, and should be further studied and developed. Summary of the invention

[0004] The present invention is to solve the above problems and defects and provides a class of isopentenyl flavonoid derivatives and applications thereof.

[0005] The present invention is implemented by the following technical solution.

[0006] A class of isopentenyl flavonoid derivatives, the chemical structure is:

[0007]

[0008] The present invention protects the use of the isopentenyl flavonoid derivative in inhibiting Zika virus.

[0009] A Zika virus inhibitor. The Zika virus inhibitor of the present invention comprises the component of the chemical structural formula (1) or the component of the chemical structural formula (2) of the isopentenyl flavonoid derivative.

[0010] A Zika virus inhibitor. The Zika virus inhibitor of the present invention comprises the above-mentioned isopentenyl flavonoid derivative chemical formula (1) component and chemical formula (2) component.

[0011] The present invention protects the medicine prepared from the above-mentioned Zika virus inhibitor.

[0012] The beneficial effects of the present invention are as follows: 1. So far, there is no report in the prior art of a pharmaceutical composition containing such isopentenyl flavonoid derivatives (1-2 shown in formula (I)) as an active ingredient, nor is there a report on the use of such isopentenyl flavonoid derivatives and their pharmaceutical compositions in the preparation of drugs for treating Zika virus (ZIKV) inhibitors. 2. The two isopentenyl flavonoid derivatives obtained by the present invention can significantly inhibit the expression of ZIKV E protein; and have no obvious toxicity to Vero cells, and their CC 50 All were greater than 100μM.

[0013] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Compound 1 and compound 2 inhibit RNA replication in ZIKV and their cytotoxicity to Vero cells. A is the inhibitory effect of compound 1 and 2 on RNA replication in ZIKV detected by qRT-PCR; B is the EC of compound 1 and compound 2 50; C is the CCK-8 method to detect the cytotoxicity of compounds 1 and 2 on Vero cells.

[0015] Figure 2 Western Blotting detection of the inhibitory effect of compound 1 and compound 2 on ZIKV E protein expression. D is the inhibition of ZIKV E protein expression and grayscale analysis by compound 1; E is the inhibition of ZIKV E protein expression and grayscale analysis by compound 2.

[0016] Figure 3 Immunofluorescence detection of the inhibitory effect of compound 1 and compound 2 on ZIKV E protein expression. DETAILED DESCRIPTION

[0017] In order to better understand the essence of the present invention, the following embodiments of the present invention are used to further illustrate the essential content of the present invention, but the present invention is not limited thereto.

[0018] Example 1 Preparation of isopentenyl flavonoid derivatives (1-2)

[0019] 1. Instruments and Materials

[0020] Instrument / Material Model Manufacturer

[0021] RE-2000E rotary evaporator (Buchi, Switzerland); low-temperature coolant circulation pump (DLSB-5L, Gongyi Yuhua Instrument); SK2200HP ultrasonic cleaner (Shanghai Kedao Ultrasonic Instrument Co., Ltd.); Agilent 1260 high-performance liquid chromatograph (Agilent Technology Co., Ltd., USA); Agilent Zorbax SB-C18 semi-preparative chromatographic column (9.4mm×250mm, 5μm) (Agilent Technology Co., Ltd., USA); Agilent Zorbax SB-C18 analytical chromatographic column (4.6mm×250mm, 5μm) (Agilent Technology Co., Ltd., USA); Bruker AV 400 nuclear magnetic resonance instrument (Bruker, Switzerland); Bruker AV III 600 nuclear magnetic resonance instrument (Bruker, Switzerland); Agilent 1100 HPLC / TOF high-resolution mass spectrometer (Agilent Technology Co., Ltd., USA); Applied Photophysics Chirascan V100 circular dichroism spectrometer (Applied Photophysics, UK); Thermo Scientific Nicolet IS10 infrared spectrometer (Thermo Fisher Scientific, USA); Shimadzu UV-2401PC UV-visible spectrophotometer (Shimadzu, Japan); Rudolp Autopol VI polarimeter (Rudolph, USA); ZF-1 dark box three-purpose UV analyzer (Qilin Bell Instrument Manufacturing Co., Ltd.); GF 254 Silica gel plates (Qingdao PUCO Separation Materials Co., Ltd.); silica gel for column chromatography (80-100, 100-200, 200-300 mesh) (Qingdao Ocean Chemical Plant); D-101 macroporous adsorption resin (Tianjin Haoju Resin Technology Co., Ltd.); reverse phase filling material RP-18 (40-60 μm) (Merck, USA); Sephadex LH-20 hydroxypropyl dextran gel (Pharmacia, USA); the solvent for column chromatography is a redistilled industrial grade solvent.

[0022] 2 Plant Sources

[0023] The stems and leaves of Macaranga denticulata Bl. were collected from Medog County, Tibet Autonomous Region, China in June 2009. The plant was identified by Yang Yongping, a researcher at the Kunming Institute of Botany, Chinese Academy of Sciences. The plant specimens are preserved in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (specimen number: LXL2009001).

[0024] 3 Extraction and separation

[0025] The dried stems and leaves of the crushed Zhongping tree (5 kg) were soaked in an 85% methanol (MeOH) aqueous solution (3×24 h) at room temperature, and the methanol extracts were combined and concentrated to obtain an extract. The extract was added to water to make a suspension, and extracted with ethyl acetate (EtOAc) for 3 times to obtain an ethyl acetate extract (512 g). MeOH was used as a solvent, and the sample was mixed with a macroporous resin. After the sample was completely dried, it was loaded into a column by dry method, and gradient eluted with MeOH / H2O (5:5, 7:3, 8:2, 9:1, 1:0, v / v) respectively. The eluted solution of each component was concentrated under reduced pressure and weighed separately to obtain 5 fractions (A-E).

[0026] D fraction (35 g) was fractionated on a silica gel column with PE / EtOAc (20:1-0:1, v / v) as the eluent to obtain 6 fractions: D1-D6.

[0027] D5 was purified by RP-18 column, and gradient elution was performed with MeOH / H2O (6:4-1:0, v / v) as eluent to obtain 6 subfractions from D5-1 to D5-6. D5-6 was purified by silica gel column, eluted with PE / EtOAc (3:2, v / v), and then separated by semi-preparative HPLC (CH3CN / H2O, 80:20, v / v, 3.0 mL / min) to obtain compound 1 (1.7 mg, t R =16.79min).

[0028] D6 was purified and separated using LH-20 gel column (eluent: MeOH) to obtain four subcomponents, D6-1 to D6-4. D6-4 was purified by silica gel column, eluted with PE / EtOAc (5:1-3:2, v / v), and then separated by semi-preparative HPLC (CH3CN / H2O, 85:15, v / v, 3.0 mL / min) to obtain compound 2 (1.9 mg, t R =18.20min).

[0029] 4 Results

[0030] The structure of the obtained isopentenyl flavonoid derivative (1-2) was confirmed by NMR, HRMS, IR, and UV data. The data of the isopentenyl flavonoid derivative (1-2) are as follows:

[0031] Table 1 Compound 1 1 H NMR and 13 C NMR data (δ in ppm, J in Hz)

[0032]

[0033] Note: Hmeasured at 600MHz in Methanol-d4.δ C measured at 150MHz inMethanol-d4.

[0034] Table 2 Compound 2 1 H NMR and 13 C NMR data (δ in ppm, J in Hz)

[0035]

[0036] Note: H measured at 600MHz in Methanol-d4.δ C measured at 150MHz inMethanol-d4.

[0037] Denticulain P(1)

[0038] Yellow solid; (c 0.06, MeOH); UV(MeOH)λ max (logε)250(2.73),284(2.65),332(2.50),400(2.12)nm; IR(KBr)ν max 3423,2925,1630,1511,1457,1384,1224,1165,1113,802cm -1 ; 1 H NMR and 13 C NMR data are shown in Table 1; HR-ESI-MS [M+Na] + m / z 529.2195 (calcd for C 30 H 34 O7Na,529.2191).

[0039] Denticulain V (2)

[0040] Yellow solid; (c 0.07, MeOH); UV(MeOH)λ max (logε)203(4.51),252(4.28),282(3.97),357(3.55),423(3.28)nm; CD(MeOH,nm)λ max (Δε)290(-0.43),340(+0.03); IR(KBr)ν max3419,2925,2854,1647,1515,1460,1391,1267,1145,1088,816cm -1 ; 1 HNMR and 13 C NMR data are shown in Table 2; HR-ESI-MS [M+H] + m / z313.0710(calcd for C 17 H 13 O6,313.0707).

[0041] Example 2 Inhibitory Effect of Prenylated Flavonoid Derivatives (1-2) of Formula (I) on Zika Virus 1. Instruments and Materials

[0042] Zika virus (ZIKV) (kindly provided by Dr. Zhang Bo, Wuhan Institute of Virology, Chinese Academy of Sciences); African green monkey kidney cells (Vero) (Wuhan Pronosai Biotechnology Co., Ltd.); culture medium and fetal bovine serum (Thermo Fisher); ZIKV E protein antibody (Beijing Sino Biological Technology Co., Ltd.); BCA protein concentration assay kit (Beyotime Biotechnology Co., Ltd.); Western cell lysis buffer (Beyotime Biotechnology Co., Ltd.); primers and TaqMan probes (Beijing Qingke Biotechnology Co., Ltd.); one-step quantitative kit RNA-direct TM (Hangzhou Aikerui Biotechnology Co., Ltd.); viral RNA extraction kit (Hangzhou Aikerui Biotechnology Co., Ltd.); CCK-8 kit (Proteintech Co., Ltd.).

[0043] 2 Methods

[0044] 2.1 Detection of the effects of compounds 1 and 2 on RNA replication in ZIKV

[0045] 2.1.1 Collection of progeny virus supernatant

[0046] Vero cells were cultured at 3 × 10 5 / mL, 1mL per well was inoculated in a 12-well plate and cultured overnight in a 37°C, 5% CO2 incubator. After the cells grew into a monolayer, the culture supernatant was discarded and ZIKV solution (MOI=1) was added for adsorption for 2h. The virus solution was discarded, the residual unadsorbed virus was washed off with PBS, and 1mL of compound 1 and compound 2 with a final concentration of 50μM (diluted with DMEM medium containing 2% FBS) were added. At the same time, a negative control group without drug, a positive control group with only virus, and a 50μM ribavirin (RBV) positive drug control group were set up. After the cells were cultured in a 37°C, 5% CO2 incubator for 72h, the virus supernatant was collected and stored in a -80°C refrigerator for later use.

[0047] 2.1.2 Extraction of RNA from viral supernatant

[0048] Use the SteadyPure Viral DNA / RNA Extraction Kit to extract viral RNA, and follow the instructions of the kit for experimental operations. For specific experimental steps, see the kit instruction manual.

[0049] 2.1.3 Viral load detection

[0050] qRT-PCR assay was used to detect the ZIKV viral load in the cell supernatant after treatment with compound 1 and compound 2. The experiment was performed using a one-step quantitative PCR kit and TaqMan probe.

[0051] ZIKV primer and probe sequences:

[0052]

[0053] 2.1.4 EC of Compound 1 and Compound 2 50 calculate

[0054] Compound 1 and compound 2 were diluted to 40, 20, 10, 5, and 2.5 μM in 2% FBS DMEM medium and added to the cultured cells. The supernatant was collected after 72 h of culture, and the viral RNA in the supernatant was extracted and quantified using a qRT-PCR quantitative kit to calculate the EC values ​​of compound 1 and compound 2. 50 (50% effective concentration).

[0055] 2.2 CCK-8 cytotoxicity assay

[0056] Dilute the cells to a density of 1 × 10 5 / mL, use a spray gun to spread 100μL per well in a 96-well plate, and culture overnight in a 37℃, 5% CO2 incubator. Compound 1 and compound 2 were gradiently diluted to 100μM, 50μM, 25μM, 12.5μM, 6.25μM, and 3.125μM using 2% FBS DMEM maintenance medium for later use. Different concentrations of compound 1 and compound 2 were added to a 96-well plate, and 2 replicates were made for each concentration. At the same time, the group with only culture medium was set as the negative control group and the group without drug was set as the positive control group, and the cells were cultured in a 37℃, 5% CO2 incubator for 3d. After 3d, new culture medium was replaced, 20μL CCK-8 reagent was added to each well, and the OD value at a wavelength of 450nm was measured with an enzyme marker after incubation at 37℃ for 2h. The OD values ​​under different concentration gradients were recorded, and the inhibition rate of the compound on cell viability was calculated by comparing with the blank control and positive control groups, and the CC of the compound was calculated based on this. 50 .

[0057] 2.3 Detection of the effects of compound 1 and compound 2 on ZIKV E protein expression

[0058] 2.3.1 Collection of cell proteins

[0059] Vero cells were cultured at 3 × 10 5 / mL, 2mL per well was inoculated in a 6-well plate, the cells were gently shaken and placed in an incubator for overnight culture. After the cells grew into a monolayer, the culture supernatant was discarded, and ZIKV solution (MOI=1) was added for adsorption for 2h. Compound 1 and compound 2 were gradiently diluted to 40μM, 20μM, 10μM, 5μM, and 2.5μM using 2% FBS DMEM maintenance medium for standby use. The virus-infected cells were removed from the incubator and gently washed 3 times with PBS to remove the unadsorbed virus. The diluted compound 1 and compound 2 were added to a 6-well culture plate, 2mL per well, and placed in an incubator for continued culture for 72h. At the same time, a negative control group without drug and a positive control group with only virus were set up. After 72h of culture, the cell state was observed and the total intracellular protein was extracted.

[0060] 2.3.2 Total intracellular protein extraction

[0061] After culturing the cells with compound 1 and compound 2 for 72 hours, discard the cell culture supernatant and wash gently with PBS twice. Aspirate the PBS, add 100 μL of cell lysis solution and place on ice for 15 minutes. After the cells are fully lysed, scrape the lysis solution with a cell scraper and aspirate it into an EP tube. After complete lysis, centrifuge at 12000g in a 4°C centrifuge for 15 minutes, collect 60 μL of protein supernatant and transfer it to a new EP tube. The obtained protein supernatant is the total intracellular protein.

[0062] 2.3.3 Protein concentration determination

[0063] The protein concentration was determined using the Bio-Time protein concentration assay kit according to the kit instructions. The specific experimental steps are shown in the kit instructions.

[0064] 2.4 Western Blotting assay to detect the inhibitory effects of compound 1 and compound 2 on ZIKV E protein expression

[0065] Prepare SDS-PAGE gel with a concentration of 10%. Add an appropriate amount of concentrated 4×SDS-PAGE protein loading buffer to the protein sample in proportion. Denature the protein by heating at 95℃ for 5-10min. After the protein sample cools to room temperature, add 20-30μg of protein sample to the SDS-PAGE gel loading well for electrophoresis. The electrophoresis conditions of the concentrated gel are: low voltage constant voltage electrophoresis (80v, 30min). When the bromophenol blue dye enters the separation gel, the electrophoresis conditions of the separation gel are: high voltage constant voltage electrophoresis (120v, 1-1.5h). Use a wet transfer device to transfer the PVDF membrane. Under ice bath conditions, adjust the current so that the voltage is about 100V, and the transfer time is 30-60min. After transfer, place the membrane in 2% BSA and block it at room temperature for 1h. Dilute ZIKV E and β-actin antibodies using Western primary antibody diluent according to an appropriate ratio (1:1000). Place the target band in the diluted primary antibody and incubate overnight on a shaker at 4°C. After the primary antibody incubation is completed, wash with 1×TBST solution for 10 minutes, discard the solution, and repeat 3 times. Dilute the horseradish peroxidase (HRP) labeled secondary antibody with 1×TBST solution at a ratio of 1:5000. Place the membrane in the diluted secondary antibody and incubate at room temperature for 1 hour. Wash with 1×TBST solution for 20 minutes, discard the solution, and repeat 3 times. Use ECL developer to detect the expression of the protein, and use it in the dark for color development to detect the expression of ZIKV E protein.

[0066] 2.5 Immunofluorescence assay to detect the inhibitory effects of compounds 1 and 2 on ZIKV E protein expression

[0067] Place a poly-lysine-treated 12-well plate in a 12-well plate and inoculate Vero cells at a density of 3 × 10 5 / mL, 1mL per well was inoculated in a 12-well plate, placed in a 37°C, 5% CO2 incubator overnight, and the culture supernatant was discarded after the cells grew into a monolayer on the slide. After adding ZIKV solution (MOI=1) for adsorption for 2h, the virus solution was discarded, and the residual virus solution was washed with PBS, and 2mL of 2% FBS DMEM medium was added and placed in a 37°C, 5% CO2 incubator for culture. After 72h of infection, the culture supernatant was discarded, leaving the cell slide, and it was washed with PBS 3 times, each time for 3min. The slide was fixed with 4% paraformaldehyde for 15min, and the slide was washed with PBS 3 times, each time for 3min. 0.5% Triton X-100 (prepared with PBS) was permeabilized at room temperature for 20min, and the slide was washed with PBS 3 times, each time for 3min. Sheep serum was added to the slide and blocked at room temperature for 30min. Remove the blocking solution with filter paper, do not wash, add a sufficient amount of diluted primary antibody to each slide: ZIKV E protein (1:500 dilution), and incubate in a wet box at 4°C overnight. Wash the slides with PBST 3 times, 3 minutes each time, remove excess liquid, add diluted fluorescent secondary antibody, incubate in a wet box at 37°C for 1 hour, and wash the slides with PBST 3 times, 3 minutes each time. Add DAPI and incubate in the dark for 5 minutes to stain the cells, wash the slides with PBST 4 times, 5 minutes each time, and wash away excess DAPI. Remove the liquid with filter paper, seal the slides with sealing solution containing anti-fluorescence quencher, and observe the expression of ZIKV E protein under a fluorescence microscope.

[0068] 2.6 Calculation formula and data analysis

[0069] Western blotting results were analyzed using Image J. The EC values ​​of compound 1 and compound 2 were 50 、CC 50 All calculations will be performed according to the Reed & Muench method. All experimental data and graphs were processed using GraphPad Prism 8 software.

[0070] (1) Cell survival rate (%) = (1-((OD value of experimental well-OD value of blank well) / (OD value of control well-OD value of blank well)) × 100%

[0071] (2)EC 50 =10EXP[lg(>50% concentration)-(>50% inhibition rate-50) / ((>50% inhibition rate)-(<50% inhibition rate))×lg(dilution multiple)]

[0072] (3)CC 50 =10EXP[lg(>50% concentration)-(>50% cell survival rate-50) / ((>50% cell survival rate)-(<50% cell survival rate))×lg(dilution multiple)]

[0073] 3 Results

[0074] See Figure 1 , Figure 2 , Figure 3 shown.

[0075] The experimental results show that the virus copy number mainly represents the activity of the virus. The higher the copy number value, the higher the activity of the virus, indicating that the virus has a stronger ability to replicate. The virus copy number was measured, and the copy number of the ZIKV group increased significantly. The positive drug ribavirin group can significantly inhibit the replication of RNA in ZIKV, and compound 1 and compound 2 can significantly inhibit the replication of RNA in ZIKV. The EC values ​​of compound 1 and compound 2 are 50 The toxicity of compound 1 and compound 2 to Vero cells was determined by CCK-8 method, and it was found that compound 1 and compound 2 had no obvious toxicity to Vero cells. 50 All greater than 100μM ( Figure 1 ).

[0076] ZIKV E protein is an important component of ZIKV capsid protein and the main mediator for virus entry into host cells. It is involved in virus assembly and release and plays a variety of key roles in virus replication and transmission. Western Blotting experiments revealed that compounds 1 and 2 inhibited the expression of ZIKV E protein in a dose-dependent manner ( Figure 2 Immunofluorescence experiments further demonstrated that compounds 1 and 2 could significantly inhibit the expression of ZIKV E protein ( Figure 3 ).

[0077] In summary, the present invention demonstrates that Compound 1 and Compound 2 inhibit the expression of ZIKV E protein in a dose-dependent manner, thereby exerting an anti-ZIKV replication effect and having anti-ZIKV activity.

[0078] The above are only some specific embodiments of the present invention (since the present invention includes a numerical range, the embodiments cannot be exhaustive, and the protection scope recorded in the present invention includes the numerical range and other technical key points of the present invention). The specific content or common sense known in the scheme is not described in detail here (including but not limited to abbreviations, abbreviations, and units commonly used in the art). It should be pointed out that the above embodiments do not limit the present invention in any way. For those skilled in the art, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention. The scope of protection claimed in this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A flavonoid derivative, characterized in that: The chemical structural formula of the flavonoid derivative is: 。 2. A flavonoid derivative, characterized in that: The chemical structural formula of the flavonoid derivative is: 。 3. Use of the flavonoid derivative according to claim 1 or claim 2 in the preparation of a drug for inhibiting Zika virus.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the flavonoid derivative according to claim 1 or 2 as an active ingredient.

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