阅读说明：本技术 桃金娘酮类化合物及其在制备抗流感病毒药物中的应用 (Myrtle ketone compound and application thereof in preparation of anti-influenza virus drugs ) 是由 邱声祥 赵丽云 于 2020-05-29 设计创作，主要内容包括：本发明公开了桃金娘酮类化合物及其在制备抗流感病毒药物中的应用。本发明的桃金娘酮类化合物对流感病毒具有明显的抑制作用,其作用机理包括(但不限于)抑制流感病毒进入细胞,阻止流感病毒在宿主细胞中的复制,适时调控受感染细胞的凋亡程序。本发明提供的桃金娘酮类化合物对甲型和乙型流感病毒均有抑制作用,活性优于阳性药利巴韦林(Ribavrin),因此可以用于预防或治疗流感。本发明有望为临床治疗流感提供新的活性先导化合物或者候选药物分子。(The invention discloses a myrtle ketone compound and application thereof in preparing anti-influenza virus drugs. The myrtle ketone compound has obvious inhibition effect on influenza virus, and the action mechanism of the myrtle ketone compound comprises (but is not limited to) inhibiting the influenza virus from entering cells, preventing the influenza virus from replicating in host cells and timely regulating and controlling the apoptosis program of infected cells. The myrtle ketone compound provided by the invention has an inhibitory effect on influenza A and B viruses, and the activity of the myrtle ketone compound is superior to that of a positive drug ribavirin (Ribavrin), so that the myrtle ketone compound can be used for preventing or treating influenza. The invention is expected to provide a new active lead compound or candidate drug molecule for clinical treatment of influenza.)

1. The structure of the myrtle ketone compound is shown as a formula I:

wherein the dotted line represents an optional C — C single bond, which forms a C — C single bond when the dotted line is absent and a C ═ X double bond when the dotted line is present;

when the dotted line is present, a C ═ X double bond, R, is formed₄、R₅And R₆Each independently selected from O, S or NR 1', R1' is hydrogen, or C1-C15 straight chain, branched chain naphthenic base or aromatic group containing benzene ring;

R₁is hydrogen, or C1-C15 straight chain, branched chain naphthenic base or aromatic group containing benzene ring;

R₂and R₃、R₇Each is independently selected from hydrogen, or a substituent group selected from the following;

wherein n is any number from 0 to 15, R₈And R₉Each independently selected from hydrogen, or C1-C15 straight chain, branched chain cycloalkyl or a cyclic alkyl group containingAromatic radicals of benzene rings, or R₈And R₉Form a cycloalkyl or heterocycle.

2. A myrtle ketone compound according to claim 1, wherein R is₁Is a straight chain, branched chain or cyclic alkyl of C3-C10.

3. A myrtle ketone compound according to claim 1, which is represented by formula II:

wherein R is₃Is H, R₁Is a linear or branched alkyl group of C3-C4, R₂Is hydrogen, or a substituent group from below;

wherein n is any number from 0 to 15, R₈And R₉Each independently selected from hydrogen, or C1-C15 straight chain, branched chain cycloalkyl or aromatic group containing benzene ring, or R₈And R₉Form a cycloalkyl or heterocycle.

4. A myrtle ketone compound according to claim 1, which is characterized in that any one of the following compounds is represented by:

5. a myrtle ketone compound according to claim 1, wherein n is 1 to 8.

6. A myrtle ketone compound, or a stereoisomer, an epimer, a configurational isomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or an extract of a part or whole plant (including but not limited to leaves, stems, flowers, fruits, roots, endophyte) of myrtle as claimed in claim 1, 2,3,4 or 5, wherein the extraction solvent can be water or (more effective) organic solvent including but not limited to ethanol, acetone, methanol, ethyl acetate, n-hexane, etc. or a mixture of the above solvents in different proportions) and CO₂Supercritical or subcritical extracting, or physically and chemically concentrating, distributing, enriching, and partially and purifying the above extract to obtain fraction or part, and its application in preparing medicine for preventing or treating influenza virus.

7. The use of claim 6, wherein the medicament for preventing or treating influenza virus is a medicament for preventing or treating influenza virus A/WSN/33(H1N1), A/PR/8/34(H1N1) or B/Massachusetts/2/2012.

8. A drug for preventing or treating influenza virus, which comprises the myrtle ketone compound according to claim 1, 2,3,4 or 5, or a stereoisomer, an epimer, a configurational isomer or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or an extract of a part or whole plant (including but not limited to leaves, stems, flowers, fruits, roots, endophytes) of myrtle, wherein the extraction solvent can be water or (more effective) an organic solvent including but not limited to ethanol, acetone, methanol, ethyl acetate, n-hexane and the like or a mixture of the above solvents in different proportions) and CO₂Supercritical or subcritical extracting, or physically and chemically concentrating, distributing, enriching, and partially and purifying the above extract to obtain fraction or fraction as active ingredient.

9. The pharmaceutical composition of claim 8, wherein the agent for preventing or treating influenza virus is an agent for preventing or treating influenza virus A/WSN/33(H1N1), A/PR/8/34(H1N1) or B/Massachusetts/2/2012.

Technical Field

The invention belongs to the technical field of medicine and pharmacy, and particularly relates to a myrtle ketone compound and application thereof in preparation of anti-influenza virus medicines.

Background art:

influenza virus (inflenzavirus) is an RNA virus causing influenza in humans and animals, and classified into a type a, a type b and a type c according to the difference in antigenicity of its internal Nucleoprotein (NP) and matrix protein (M), and belongs to the family Orthomyxoviridae in taxonomy. Among them, the Influenza A Virus (IAV) is susceptible to variation in antigenicity, and causes a pandemic worldwide many times, which may cause extremely high morbidity and mortality, and seriously threatens human health. For example, in 1918-1919 pandemics, at least 2000-4000 million people died from IAV worldwide, and in 2009 IAV infection was also outbreaked, which is rapidly spread and has attracted widespread attention. According to statistics, 30-50 million people still die of influenza every year in average all over the world at present, and IAV is always one of the main viruses seriously harming global health.

Although vaccines are an important preventive strategy, the lag time between virus identification and vaccine distribution impairs the preventive effect of the vaccine. Antiviral therapy is the best option to control influenza spread in a short time. To date, clinically approved anti-influenza virus drugs include only M2 ion channel blockers (amantadine and rimantadine), neuraminidase inhibitors (oseltamivir and peramivir), and RNA-dependent RNA polymerase (RdRp) inhibitors [ favipiravir (T705) ]. The rapid emergence of drug-resistant virus mutants limits the application of partial drugs, and in addition, few anti-influenza virus drugs with obvious curative effects exist at present, and the current situation that no drugs are available in the face of epidemic situations exists. Therefore, there is an urgent need to discover and develop new anti-influenza virus drugs.

The myrtle is a Rhodomyrtus myrtus (Ait.) plant belonging to Rhodomyrtus genus of Rhodomyrtus family (Mytaceae) of Rhodomyrtaceae family (Mytaceae), and is named as Duoney, Gongta, Melastoma dodecandrum, dolichos scandens, Dandelion root, Melastoma dodecandrum, Dodonium dodecandrum, Duoyang, Myrtacium alternifolia and Dankui mud, is mainly distributed in southern China and southeast China, especially is produced in southern Ling, and is a commonly used herb of Guangdong province. The myrtle is sweet and astringent in nature and taste, is taken as a medicine or a food by using roots, leaves, flowers and fruits, and has the effects of clearing heat, detoxifying, nourishing blood, stopping bleeding, astringing intestines, securing essence, warming the abdominal organs and benefiting muscles, so that the myrtle is an economic plant with potential development and utilization values. In addition, it has a long history of use, and it has been documented in Tang Liu 24642l Ling exterior recorded by strange records: "Backwan-zi … … is sweet and soft, warms abdomen and benefits muscles. "

At present, documents and patents report that a myrtle ketone compound and a derivative thereof in myrtle have an antibacterial effect (patent application number: CN 104761565A myrtle ketone compound and application thereof in preparing antibacterial drugs; CN 108752305A closed-loop myrtle ketone analogue and application thereof in antibacterial drugs; CN 105859537A open-loop myrtle ketone analogue and preparation method thereof and application thereof in antibacterial drugs), but no research report related to the antivirus of myrtle and active ingredients thereof is found before the patent.

The invention content is as follows:

the first purpose of the invention is to provide a myrtle ketone compound with anti-influenza virus activity.

The structure of the myrtle ketone compound is shown as a formula I:

wherein the dotted line represents an optional C — C single bond, which forms a C — C single bond when the dotted line is absent and a C ═ X double bond when the dotted line is present;

when the dotted line is present, a C ═ X double bond, R, is formed₄、R₅And R₆Each independently selected from O, S or NR 1', R1' is hydrogen, or C1-C15 straight chain, branched chain naphthenic base or aromatic group containing benzene ring;

R₁is hydrogen, or C1-C15 straight chain, branched chain naphthenic base or aromatic group containing benzene ring;

R₂and R₃、R₇Each is independently selected from hydrogen, or a substituent group selected from the following;

wherein n is any number from 0 to 15, R₈And R₉Each independently selected from hydrogen, or C1-C15 straight chain, branched chain cycloalkyl or aromatic group containing benzene ring, or R₈And R₉Form a cycloalkyl, heterocycle.

Preferably, n is 1 to 8.

Preferably, R is₁Is a straight chain, branched chain or cyclic alkyl of C3-C10.

Preferably, the myrtle ketone compound is represented by formula II:

wherein R is₃Is H, R₁Is a linear or branched alkyl group of C3-C4, R₂Is hydrogen, or a substituent group from below;

wherein n is any number from 0 to 15, R₈And R₉Each independently selected from hydrogen, or C1-C15 straight chain, branched chain cycloalkyl or aromatic group containing benzene ring, or R₈And R₉Form a cycloalkyl or heterocycle.

More preferably, the myrtle ketone compound is as follows:

the second object of the present invention is to provide an application of the aforementioned myrtle ketone compound, or a stereoisomer, an epimer, a configurational isomer, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or an ethanol extract, an n-hexane extract portion of the ethanol extract, and an ethyl acetate extract portion of the ethanol extract in the preparation of a medicament for preventing or treating influenza virus.

The medicine for preventing or treating influenza virus is a medicine for preventing or treating influenza virus A/WSN/33(H1N1), A/PR/8/34(H1N1) or B/Massachusetts/2/2012.

The myrtle ketone compound as a natural product of plant source can be extracted from any part of a whole plant of myrtle, i.e. a myrtaceae plant, by a physical and/or chemical method, can be extracted from other myrtaceae plants, or can be realized by a semisynthetic and fully synthetic chemical method, and is used for preparing a medicament for preventing or treating influenza virus.

The myrtle ketone compound has obvious inhibition effect on influenza virus, and the action mechanism of the myrtle ketone compound comprises (but is not limited to) inhibiting the influenza virus from entering cells, preventing the influenza virus from replicating in host cells and timely regulating and controlling the apoptosis program of infected cells. The myrtle ketone compound provided by the invention has an inhibitory effect on influenza A and B viruses, and the activity of the myrtle ketone compound is superior to that of a positive drug ribavirin (Ribavrin), so that the myrtle ketone compound can be used for preventing or treating influenza. The invention is expected to provide a new candidate drug molecule for clinical treatment of influenza.

Description of the drawings:

FIG. 1 is a time course analysis of the inhibition of influenza A virus strain A/WSN/33(H1N1) by compounds 2-H.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

the invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Example 1 isolation of Myrtaceae plants from Myrtaceae

1.1 plant Material

The plant Myrtus communis of Myrtaceae Myrtus is used as an experimental raw material, and the plant is widely distributed in the south of China, especially in the south of Lingnan. The plant material of the experiment is collected from Nankang county (region) of Ganzhou city in Jiangxi province, 20kg after being dried, and is identified as the myrtle (R.tominosa) of myrtle in Myrtaceae by the researchers of the Wangfang country in south China, academy of China. Plant specimens are currently available in the laboratories for natural products and chemical and biological research in the plantations of south China, academy of sciences.

1.2 laboratory instruments and reagents

The optical rotation data was measured using a Perkin-Elmer 341 polar imeter (Perkin-Elmer Co., U.S.A.). The UV spectrum was measured by a Perkin-Elmer Lambda 35 UV-vis spectrophotometer (Perkin-Elmer Co., U.S.A.) using methanol or chloroform as a solvent. The IR spectrum was measured by a Bruker Vertex 33 infrared spectrophotometer (Bruker, Germany) which requires tabletting before measurement. The NMR spectra were determined on a hydrogen, carbon, DEPT-135 and two-dimensional NMR spectrometer of the Bruker type Bruker AVIII500, TMS being the internal standard, δ being ppm and J being Hz. The preparative HPLC is L3000 type HPLC (Beijing Innovation technology Co., Ltd.), and the chromatographic column is C18 column (ALLTIMA C1810U, 250nm × 10nm, 3mL/min), and is equipped with single wavelength ultraviolet detector. High resolution mass spectra were determined by a Bruker Bio TOF IIIQ mass spectrometer from Bruker. 100-200, 200-300 and 300-400 mesh silica gel and thin-layer chromatography plates are produced by Qingdao spectral separation materials, Inc. MCI gel (CHP20P,75-150mm) was manufactured by Mitsubishi chemical corporation of Japan. Sephadex LH-20 gel was produced by Amersham biosciences, Sweden. The organic solvent is from Shanghai chemical materials, Inc. The thin-layer chromatography developer is 5% concentrated sulfuric acid-ethanol solution, and compounds with ultraviolet absorption need to be observed under an ultraviolet lamp. The proportion of the mixed solvent used in the experimental process is volume ratio.

1.3 obtaining the extract

Sufficiently crushing (20KG) the dried myrtle leaves, extracting 3 times (30L multiplied by 3) with a 95% ethanol aqueous solution with volume fraction, carrying out rotary evaporation on the combined solvents under reduced pressure to obtain a brown syrupy residue which is an ethanol part (2.5KG), suspending the brown syrupy residue in water (1:1, weight ratio), extracting (3L multiplied by 3) with n-hexane, and carrying out rotary drying on the extracted part by using the solvent to obtain the n-hexane part; extracting with ethyl acetate (3L × 3), and spin-drying the extracted part with solvent to obtain ethyl acetate part and the residual water part to obtain water part.

1.4 isolation to obtain monomeric Compounds

In the experiment, the n-hexane part and/or the ethyl acetate part of myrtle leaves are/is completely dissolved in a sample mixing pot by using chloroform as little as possible, then 500g of silica gel (80-100 meshes) is used for mixing the samples, the mixture is uniformly stirred, after the solvent is completely volatilized, the samples are loaded by a dry method, gradient elution is carried out by using an n-hexane-ethyl acetate system 10:1, 5:1, 2:1, 1:1 and 0:1v/v, finally, a column is flushed by using methanol, fractions with the same main point are combined after TLC thin-layer chromatography detection, and TLC detection (a developing solvent n-hexane: ethyl acetate is 5:1v/v) is collected to show blue fluorescence under an ultraviolet lamp; performing MCI column chromatography to remove pigment, performing SephadexLH-20 gel column chromatography, performing gradient elution with n-hexane-ethyl acetate system (8:1 → 1:1v/v), and collecting blue fluorescence under ultraviolet lamp by TLC detection (developing solvent n-hexane: ethyl acetate 5:1 v/v); c2, orange part under the action of sulfuric acid-ethanol color developing agent. And Fr, carrying out Sephadex LH-20 gel column chromatography on the C2, and eluting with chloroform-methanol (1:1v/v) to obtain compounds 2-h, 3 and 4. Rf for compounds 2-h, 3 and 4 were 0.4, 0.35, 0.42, respectively, as determined by TLC (developing solvent n-hexane: ethyl acetate: -4: 1 v/v).

The compound 2-h is a compound myrtle ketone, is a light yellow needle crystal and is easily dissolved in chloroform; nuclear magnetic data:¹H NMR(CDCl₃,500MHz):δ_H6.12(1H,s,H-5),4.27(1H,t,J＝5.6Hz,H-9),2.99(3H,m,H-1”,H-2”),2.28(1H,dp,J＝13.3,6.6Hz,H-3'),1.55,1.43,1.41,1.37(each3H,s,H-11,H-12,H-13,H-14),0.98(6H,d,J＝6.7,Hz,H-4',H-5'),0.87,0.83(each3H,d,J＝6.0Hz,H-3”,H-4”)；¹³CNMR(CDCl₃,125MHz):δC 212.2(C-3),206.7(C-1'),198.3(C-1),167.5(C-4a),162.8(C-8),158.7(C-6),155.7(C-10a),114.3(C-9a),107.7(C-7),106.4(C-8a),94.7(C-5),56.1(C-2),53.2(C-2'),46.4(C-4),45.8(C-1 "), 25.5(C-9),25.5 (C-2"), 25.2,25.1(C-13, C-14),24.7,24.6(C-11, C-12),24.2(C-3'),23.5,23.2 (C-3', C-4 "), 22.8(C-4', C-5'). The structural formula of compounds 2-h is shown below:

the compound 3 is myrtle isoflavone, yellow jelly and is easily dissolved in chloroform; nuclear magnetic data:¹H NMR(CDCl₃,500MHz):δ_H0.85(d,J＝6.3Hz,3H,4”-CH₃),0.86(d,J＝6.3Hz,3H,3”-CH₃),0.99(d,J＝6.6Hz,3H,5'-CH₃),1.01(d,J＝6.6Hz,3H,4'-CH₃),1.38(s,10-CH₃),1.41(s,11-CH3),1.46(s,13-CH₃),1.62(s,12-CH3),1.35(obscured,1H,2”-H),1.35(obscured,2H,1”-H),2.35(qqdd,J＝6.8,6.8,6.6,6.6Hz,1H,3'-H),2.93(dd,J＝17.2,6.1Hz,1H,2'-Ha),3.17(dd,J＝17.2,7.3Hz,1H,2'-Hb),4.29(t,J＝6.1Hz,1H,9-H),6.25(s,1H,7-H),8.08(s,1H,OH),13.49(s,1H,OH)；¹³C NMR(CDCl₃126 MHz. delta.C 211.8(C-3),203.9(C-1'),198.5(C-1),167.4(C-4a),159.8(C-6),159.7(C-8),153.1(C-4b),114.6(C-9a),106.0(C-5),105.5(C-8a),100.2(C-7),56.1(C-2),53.4(C-2'),47.3(C-4),46.9(C-1 "), 25.4(C-13),25.0(C-9),24.8 (C-2"), 24.7(C-12),24.5(C-3'),24.5(C-11),24.2(C-10),23.4(C-3 "), 23.1 (C-4"), 22.9(C-4'),22.6(C-5 '). The structural formula of compound 3 is shown below:

compound 4 is myrciaroneA, a yellow amorphous powder, readily soluble in chloroform; spectral data: UV λ max 299nm, "a" D +7.5 ° (c ═ 0.24, CHCl3), HR-MS; m/z429.2282[ M + H ]]⁺(calcd.forC₂5H₃₃O₆429.2268) nuclear magnetic data:¹H NMR(CDCl₃,500MHz):6.07(1H,s,H-5),4.24(1H,t,J＝5.9Hz,H-9),3.88(1H,m,J＝6.6,H-2’),1.41-1.44(2H,obscure,H-1”),1.36-1.46(2H,obscure,H-2”),1.36(3H,s,2-Me),1.39(3H,s,2-Me),1.42(3H,s,4-Me),1.54(3H,s,4-Me),1,20(3H,d,J＝5.5Hz,2’-Me),1.19(3H,d,J＝6,6Hz,2’-Me),0.83(3H,d,J＝6.0Hz,2”-Me),0.87(3H,d,J＝6.0Hz,2”-Me).¹³C NMR(CDCl₃197.5(C-1),56.1(C-2),212.1(C-3),47.1(C-4),166.8(C-4a),94.5(C-5),158.0(C-6),106.7(C-7),158.0(C-8),106.7(C-8a),25.2(C-9),114.2(C-9a),155.6(C-10a),210.9(C-1 '), 39.8 (C-2'), 45.9(C-1 "), 25.1 (C-2"), 24.2(2-Me),24.5(2-Me),24.6(4-Me),24.7(4-Me),19.1(2 '-Me), 19.2 (2' -Me),23.1(2 '-Me), 23.5 (2' -Me), compound 4, the formula is shown below:

example 2 Total Synthesis of Myrtle Ketone Compounds

2.1 materials and laboratory instruments for chemical moieties

All chemical reactions were carried out under anhydrous conditions, in dry solvents and under nitrogen unless otherwise indicated. Reagents were purchased in high commercial quality and used without further purification. All solvents were of analytical grade (Shanghai chemical plant, Shanghai, China). Thin Layer Chromatography (TLC) was performed with a 0.25mm Qingdao silica gel plate (60F-254) and developed by exposure to UV light (254nm) or staining with potassium permanganate. Silica gel (ZCX-II, 200-300 mesh) for flash column chromatography was purchased from Qingdao ocean chemical industries, Inc. of China. The NMR spectra were determined on a hydrogen, carbon, DEPT-135 and two-dimensional NMR spectrometer of the Bruker type Bruker AVIII500, TMS being the internal standard, δ being ppm and J being Hz. The preparative HPLC is L3000 type HPLC (Beijing Innovation technology Co., Ltd.), and the chromatographic column is C18 column (ALLTIMA C1810U, 250nm × 10nm, 3mL/min), and is equipped with single wavelength ultraviolet detector. High resolution mass spectra were measured using a Bruker Bio TOF IIIQ mass spectrometer from Bruker. The synthesis process is as follows:

2.2 Synthesis of Acylphuloroglucinol (6)

30.0g of the compound phloroglucinol (5) (0.238mol) was added to a suspension stirrer containing a mixture of 240mL of methylene chloride and 240mL of nitromethane, and 127.0g of aluminum trichloride (0.952mol, 4-fold equivalent) was added to the mixture, followed by stirring at room temperature for thirty minutes. Then 17mL of acetyl chloride (0.238mol,1 eq.) was added to the black reaction flask using a syringe. After the addition was complete, the reaction was refluxed for three hours while the progress of the reaction was checked by a thin layer. Subsequently, the reaction mixture was cooled to room temperature, evaporated under reduced pressure, added 400mL of ice water, and extracted three times with 500mL of ethyl acetate. The mixture solution was washed once with saturated sodium chloride solution, taken up with anhydrous sodium sulfate, filtered and concentrated in vacuo. Petroleum ether: flash column chromatography with ethyl acetate (2:1) gave compound 6, as pale yellow crystals which could be recrystallized from toluene. R_f＝0.26(petroleum ether/ethyl acetate,2:1)；¹H NMR(400MHz,DMSO-d₆):d 2.53(s,3H,CH₃),5.78(s,2H,Ar),10.39(br s,1H,OH),12.23(br s,2H,OH)；¹³C NMR(100MHz,DMSO-d₆):d 32.5(CH₃),94.6(C-3,C-5),104.1(C-1),164.4(C-2,C-6),164.8(C-4),202.6(C＝O).

2.3 Synthesis of 4-Acetyl-5-hydroxy-2,2,6, 6-tetramethylcyclohexox-4-ene-1, 3-dione (acylsyncarpic a cid) (7)

Preparation of sodium methoxide: 30.6g of sodium (1.33mol,8.16equiv) was added in several portions to 1L of an anhydrous methanol solution. Stir at room temperature until completely dissolved. After addition of 142mL of methyl iodide (2.28mol,14equiv), 27.46g of Compound 6(0.163mol) were added and the mixture was refluxed for 6h (during which time the plates were examined on a thin plate, petroleum/ether ethyl acetate 2:1v/v development) and cooled to room temperature. Concentrated under reduced pressure, the remaining part was dissolved in water, extracted four times with 300Ml of diethyl ether and acidified with 1M hydrochloric acid. The organic phase was acidified with 400mL of saturated sodium sulfite solution and 3M hydrochloric acid was added until a white color appeared. Extracted three times with 100mL of diethyl ether. Combine all compounds, add magnesium sulfate, filter, concentrate, and dry in vacuo. A yellow color is obtainedThe solid, recrystallized from petroleum ether to give compound 7(30.6g, 84%).¹H NMR(400MHz,CDCl₃):d1.33(s,6H,2CH₃),1.42(s,6H,2CH₃),2.57(s,3H,COCH₃)；¹³CNMR(100MHz,CDCl₃):d23.8(2CH₃),24.3(2CH₃),27.4(COCH₃),52.0(C-6),56.7(C-2),109.4(C-4),196.7(C-5),199.1(C-3),201.7(CH₃ C＝O),210.0(C-1).

2.4 Synthesis of 5-Hydroxy-2,2,6, 6-tetramethylcyclohexox-4-ene-1, 3-dione (syndrome acid) (8)

Dissolve compound 7 with about 1.4L of 3M hydrochloric acid and stir at reflux for 6h until all is dissolved (monitored by thin layer). Subsequently, the reaction solution was cooled to room temperature and extracted four times with 400mL of ethyl acetate. Washing the extractive solution with water twice, adding sodium sulfate to absorb water, filtering, and vacuum drying. The crude product was recrystallized from toluene. 18.3g of pale yellow compound 8 are obtained. R f1/40.37(petroleum ether/ethyl acetate,1: 1);¹H NMR(400MHz,CDCl₃),mixture of keto and enol tautomers in a ratio of 2:1；keto tautomer:d 1.31(s,12H,4CH 3),3.61(s,2H,CH 2)；enol tautomer:d 1.40(s,12H,4CH3),5.74(brd,J1/42.3Hz,1H,CH),8.00(br s,1H,OH)；¹³C NMR(100MHz,CDCl₃):keto tautomer:d 21.8(4CH 3),50.2(CH2),59.1(2C(CH₃)2),204.3(2CO),208.9(CO)；enol tautomer:d 24.6(4CH₃),51.2(C-6),59.1(CH₃),101.7(C-4),191.9(C-5),204.3(C-3),212.6(C-1).

2.5 Synthesis of 2,2,4, 4-tetramethylol-6- (3-methylisobriene) cyclohexane-1,3,5-trione (9)

To 110mL of dichloromethane to which 6.76g of Compound 8(0.037mol) was added 7.4mL of piperidine (0.075mol,2equiv) followed by 6mL of isovaleraldehyde (0.056mol,1.5 equiv). Ten minutes before starting stirring, the reaction was quenched with 50mL of 1M hydrochloric acid, saturated ammonium chloride solution was added, and stirring was vigorously carried out for one hour. The reaction solution was extracted three times with 150mL of dichloromethane, taken up with water over sodium sulfate, filtered and dried in vacuo. A crude product of compound 9 was prepared using a 5 cm thin silica gel plate in dichloromethane as solvent. Easy isomerization and needs to be used at present.

2.6 Synthesis of 6,8-Dihydroxy-9-isobutyl-2,2,4,4-tetramethyl-4, 9-Dihydroxy-1H-xanthene-1, 3(2H) -dione (10)

To a 15mL THF mixture containing compound 9(5.94mmol,1.5equiv) was added phloroglucinol (0.5g,3.97mmol) and p-TsOH.H₂O (2.263g,11.9mmol,3equiv), refluxed for two hours (TLC). Phloroglucinol is consumed in a few minutes at room temperature and the intermediate product slowly converts to compound 10. The reaction mixture was slowly cooled to room temperature and quenched with water. Extracted four times with 50mL of diethyl ether. After drying over magnesium sulfate, filtration and vacuum drying. Silica gel column chromatography (petroleum ether: ethyl acetate 2:1) followed by recrystallization from toluene gave compound 10.

2.7 Synthesis of Myrtle Ketone Compound 2-R₁

Compound 10(400mg,1.116mmol) was suspended and dissolved in 25mL of dichloromethane, and titanium tetrachloride was added and stirred at room temperature for ten minutes. Then adding R₁ClO (0.145mL,1.19mmol,1.07equiv), stirred at room temperature for 26 hours. The reaction was quenched with 40mL of water until the main product was formed. It is then extracted four times with 50mL of dichloromethane, washed with saturated sodium chloride solution and dried over sodium sulfate. Filtered and concentrated in vacuo. Brown oil, column chromatographed with ethyl ether acetate 4: 1. R1 ═ h (b), CH₃(c),C₂H₅(d),n-C₃H₇(e),i-C₃H₇(f),n-C₄H₉(g),i-C₄H₉(h),n-C₅H₁₁(i),n-C₆H₁₃(j),n-C₇H₁₅(k),n-C₈H₁₇(l),n-C₉H₁₉(m),n-C₁₀H₂₁(n),n-C₁₁H₂₃(o),n-C₁₃H₂₇(p),n-C₁₅H₃₁(q),c-C₄H₇(r),c-C₅H₉(s),c-C₆H₁₁(t),CH₂Ph(u,C₂H₄Ph(v).

When R is₁＝i-C₄H₉(h) The structural formula is as follows:

the compound 2-h is a compound rhodogyrthone, a light yellow needle crystal and is easily soluble in chloroform; nuclear magnetic data:¹H NMR(CDCl₃,500MHz):δ_H6.12(1H,s,H-5),4.27(1H,t,J＝5.6Hz,H-9),2.99(3H,m,H-1”,H-2”),2.28(1H,dp,J＝13.3,6.6Hz,H-3'),1.55,1.43,1.41,1.37(each3H,s,H-11,H-12,H-13,H-14),0.98(6H,d,J＝6.7,Hz,H-4',H-5'),0.87,0.83(each3H,d,J＝6.0Hz,H-3”,H-4”)；¹³C NMR(CDCl₃125 MHz. delta.C 212.2(C-3),206.7(C-1'),198.3(C-1),167.5(C-4a),162.8(C-8),158.7(C-6),155.7(C-10a),114.3(C-9a),107.7(C-7),106.4(C-8a),94.7(C-5),56.1(C-2),53.2(C-2'),46.4(C-4),45.8(C-1 "), 25.5(C-9),25.5 (C-2"), 25.2,25.1(C-13, C-14),24.7,24.6(C-11, C-12),24.2(C-3'),23.5,23.2(C-3 ', C-4', 22.8, 22.8(C-4', C-5 '). The structural formula of compounds 2-h is shown below:

example 3 further Synthesis of related derivatives on the basis of the Myrtle Compounds obtained in example 1 or example 2

The synthesis process is as follows:

the materials of the chemical part and the laboratory instruments,

same as example 2.1 part

Synthesis of 3.28-hydroxy-9-isobutyl-6-methoxy-2, 2,4, 4-tetramethylyl-7- (3-methylbutanoyl) -4,9-dihydro-1H-xanthene-1,3(2H) -dione (11)

To myrtle ketone (compound 2-h) (22.7mg, 0.0513mmol) and K in N, N-Dimethylformamide (DMF)₂CO₃To a stirred solution of (12mg, 0.1026mmol) was added methyl iodide (1 mL). The reaction mixture was stirred at room temperature until the conversion was acceptable. The mixture was stirred with distilled water (50ml) and extracted with hydrogen chloride (3X 50 ml). By distillationThe combined organic layers were washed with water (100mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The crude product was purified by column chromatography using 100:0 to 84:16 n-hexane-dichloromethane to give yellow amorphous compound 11(5.5mg, 23%).¹H NMR(CDCl₃):δ0.80(d,J＝5.8Hz,3H,CH₃-3″),0.84(d,J＝5.9Hz,3H,CH₃-4″),0.95(d,J＝6.6Hz,6H,CH₃-4′and CH₃-5′),1.34,1.37(each s,2×3H,C(CH₃)₂),1.42,1.54(each s,2×3H,C(CH₃)₂),2.19(m,1H,H-3′),2.83(dd,J＝6.7,15.4Hz,1H,H-2a′),2.90(dd,J＝6.6,15.4Hz,1H,H-2b′),3.88(s,3H,OCH₃),4.25(t,J＝5.5Hz,1H,H-9),6.09(s,1H,H-5),14.1(br s,1H,8-OH)；MS(ES):m/z:455([M-H]^-,100％).

Synthesis of 3.39-isobutyl-2, 2,4,4-tetramethyl-7- (3-methylbutanyl) -1,3-dioxo-2,3,4, 9-tetrahedron-1H-xanthene-6, 8-diyl diacetate (12)

To a stirred solution of myrtle ketone (compound 2-h) (21.1mg, 0.0477mmol) in pyridine (2mL) was added dropwise acetic anhydride (0.5mL) and 4-dimethylaminopyridine (10mg, 0.0818 mmol). The reaction was monitored by thin layer chromatography until the conversion was acceptable. Distilled water (50ml) was added and the mixture was extracted with ethyl acetate (3X 50 ml). The combined ethyl acetate extracts were washed with distilled water (100mL), dried over anhydrous sodium sulfate, and evaporated to dryness. Purification by column chromatography using n-hexane acetone (98:2) as an eluting solvent gave compound 12(5.2mg, 21%) as a colorless amorphous form.¹H NMR(CDCl₃):δ0.79(d,J＝6.2Hz,3H,H-3″),0.89(d,J＝6.1Hz,3H,H-4″),0.93(d,J＝6.6Hz,6H,H-4′and H-5′),1.33,1.36(each s,2×3H,C(CH₃)₂),1.42,1.52(each s,2×3H,C(CH₃)₂),2.18(m,1H,H-3′),2.26,2.90(each s,6H,2×OAc),2.56(dd,J＝6.9,17.6Hz,1H,H-2a′),2.63(dd,J＝6.8,17.6Hz,1H,H-2b′),3.99(t,J＝6.0Hz,1H,H-9),6.91(s,1H,H-5)；MS(ES):m/z:483([M-H]^-,100％).

Synthesis of 46, 8-dihydroxy-9-isobutyl-2,2,4, 4-tetramethylyl-7- (3-methylbutanoyl) -4, 9-dihydroxy-1H-xanthene-1, 3(2H) -dione (13)

To a stirred solution of compound 2-h (20.1mg, 0.0454mmol) in dichloromethane/methanol (2mL) was added acetic acid (0.5mL) dropwise. Then phenylhydrazine (0.1mL) was added at room temperature. The progress of the reaction was monitored by thin layer chromatography. Distilled water (50ml) was added to the solution, and the mixture was extracted with ethyl acetate (3X 50ml) to give a reaction product. The organic phase was washed with distilled water (100ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude mixture was purified by column chromatography using dichloromethane as an eluting solvent to give 13(2.8mg, 11%) as a pale yellow amorphous compound.¹H NMR(CDCl₃):δ0.84(d,J＝6.1Hz,3H,H-3″),0.88(d,J＝6.1Hz,3H,H-4″),1.00(d,J＝6.6Hz,3H,CH₃-4′),1.03(d,J＝6.6Hz,3H,CH₃-5′),1.36,1.41(each s,2×3H,C(CH₃)₂),1.43,1.56(each s,2×3H,C(CH₃)₂),2.20(m,1H,H-3′),2.95(dd,J＝7.5,13.8Hz,1H,H-2a′),3.03(dd,J＝6.9,13.8Hz,1H,H-2b′),4.35(t,J＝6.3Hz,1H,H-9),5.77(br s,1H,6-OH),6.99(s,1H,H-5),7.31(t,J＝7.4Hz,1H,Ar-H),7.51(t,J＝7.9Hz,2H,Ar-H),7.66(d,J＝7.4Hz,2H,Ar-H)；MS(ES):m/z:533([M-H]^-,60％).

Synthesis (14) of 3.56, 8-dihydroxy-7- (1-hydroxy-3-methylbutylyl) -9-isobutyl-2,2,4, 4-tetramethylyl-4, 9-dihydroxy-1H-xanthene-1, 3(2H) -dione

To a stirred solution of myrtle ketone (compound 2-h) (19.8mg, 0.0447mmol) in methanol (2ml) was added dropwise acetic acid (1ml) and sodium borohydride (4mg, 0.1056 mmol). The reaction mixture was stirred at room temperature. The reaction mixture was diluted with distilled water (50ml), which was then usedExtraction was carried out with ethyl acetate (3X 50 ml). The resulting ethyl acetate extract was washed with distilled water (100mL), dried over anhydrous sodium sulfate, and evaporated to dryness. The crude product was purified by column chromatography using dichloromethane-methanol (99:1) as the eluting solvent to give orange amorphous compound 14(7.9mg, 40%).¹H NMR(CDCl₃):δ0.77(d,J＝6.1Hz,3H,H-3″),0.83(d,J＝6.1Hz,3H,H-4″),0.95(d,J＝6.6Hz,6H,CH₃-4′and CH₃-5′),1.34,1.37(each s,2×3H,C(CH₃)₂),1.41,1.52(each s,2×3H,C(CH₃)₂),1.62(m,1H,H-3′),2.57(m,2H,H-2′),4.15(t,J＝5.8Hz,1H,H-9),4.82(m,1H,H-1′),4.95(br s,1H,OH),6.19(s,1H,H-5)；MS(ES):m/z:455.3([M-H]^-,100％).

3.6 Synthesis of Compounds 15 and 16

Hydroxylamine hydrochloride (6mg, 0.086mmol) was added to a stirred solution of myrtle ketone (compound 2-h) (18.9mg, 0.0427mmol) in dichloromethane/methanol, followed by N, N-diisopropylethylamine (0.2 mL). The reaction mixture was stirred at room temperature until the conversion was acceptable. Distilled water (50ml) was added to prepare a mixture, which was extracted with ethyl glycolate (3X 50 ml). The organic phase was washed with distilled water (100ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography using dichloromethane/methanol as the eluting solvent to give yellow amorphous compound 15(0.9mg, 4.6%) and compound 16(11.9mg, 60.9%) with increasing methanol concentration.

Compound 15.¹H NMR(CDCl₃):δ0.76(d,J＝6.1Hz,3H,H-3″),0.82(d,J＝6.1Hz,3H,H-4″),0.85(d,J＝6.6Hz,3H,CH₃-4′)0.86(d,J＝6.6Hz,3H,CH₃-5′),1.35,1.38(each s,2×3H,C(CH₃)₂),1.41,1.52(each s,2×3H,C(CH₃)₂),1.89(m,1H,H-3′),2.92(dd,J＝7.5,13.8Hz,1H,H-2a′),2.98(dd,J＝6.9,13.8Hz,1H,H-2b′),4.22(t,J＝5.2Hz,1H,H-9),6.23(s,1H,H-5)；MS(ES):m/z:915([2M-H]^-,100％).

Compound 16.¹H NMR(CDCl₃):δ0.75-0.85(m,12H,H-3″,H-4″,CH₃-4′,CH₃-5′),1.35,1.38(each s,2×3H,C(CH₃)₂),1.41,1.53(each s,2×3H,C(CH₃)₂),1.61(m,1H,H-3′),2.62(dd,J＝7.5,13.8Hz,1H,H-2a′),2.72(dd,J＝6.9,13.8Hz,1H,H-2b′),4.29(t,J＝5.3Hz,1H,H-9),6.33(s,1H,H-5)；MS(ES):m/z:915([2M-H]^-,100％).

Synthesis of 76- (3-bromopropoxy) -8-hydroxy-9-isobutyl-2,2,4, 4-tetramethy-7- (3-methyl butanoyl) -4,9-dihydro-1H-xanthene-1,3(2H) -dione (17)

To a stirred solution of myrtle ketone (compound 2-h) (42mg, 0.0949mmol) in DMF (2mL) was added potassium carbonate (14mg, 0.1013mmol) and 1.3-dibromopropane (0.1 mL). The mixture was stirred at room temperature until acceptable conversion. The reaction mixture was reacted with distilled water (50mL), followed by extraction with ethyl acetate (3X 50 mL). The combined organic layers were washed with distilled water (50ml), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Column chromatography purification using 100:0 to 95:5 n-hexane-ethyl acetate as eluent gave colorless amorphous compound 17(27.7mg, 52%).¹H NMR(CDCl₃):δ0.80(d,J＝5.7Hz,3H,H-3″),0.83(d,J＝5.8Hz,3H,H-4″),0.94(d,J＝6.6Hz,6H,H-4′and H-5′),1.33,1.36(each s,2×3H,C(CH₃)₂),1.41,1.53(each s,2×3H,C(CH₃)₂),2.22(m,1H,H-3′),2.40(m,2H,H-2″′),2.86(dd,J＝7.1,16.4Hz,1H,H-2a′),2.92(dd,J＝7.0,16.4Hz,1H,H-2b′),3.59(t,J＝6.3Hz,2H,H-3″′),4.19(t,J＝5.3Hz,2H,H-1″′),4.24(t,J＝5.4Hz,1H,H-9),6.12(s,1H,H-5),14.1(br s,1H,8-OH)；MS(ES):m/z:563([M-H]^-,80％),565([M-H]^-,100％).

Synthesis of 3.88-hydroxy-9-isobutyl-2, 2,4, 4-tetramethylyl-7- (3-methylbutanoyl) -6- (3-mor pholinopropoxy) -4, 9-dihydo-1H-xanthene-1, 3(2H) -dione (18)

Morpholine (0.2ml) was added to a stirred solution of compound 17(6.4mg, 0.0011mmol) in DMF (0.5 ml). The reaction was stirred at room temperature to an acceptable conversion and was carried out by adding distilled water (25 ml). The mixture was extracted with ethyl acetate (3X 50 mL). The organic phase was washed with distilled water (50ml), dried over anhydrous sodium sulfate and evaporated to dryness. Purification by column chromatography using dichloromethane/methanol (98:2) as the eluting solvent gave compound 18(4.1mg, 63%) as a colorless amorphous form.¹H NMR(CDCl₃):δ0.80(d,J＝5.5Hz,3H,H-3″),0.84(d,J＝5.7Hz,3H,H-4″),0.95(d,J＝6.6Hz,6H,H-4′and H-5′),1.33,1.37(each s,2×3H,C(CH₃)₂),1.42,1.53(each s,2×3H,C(CH₃)₂),2.07(m,2H,H-2″′),2.24(m,1H,H-3′),2.49(m,4H,H-1″″),2.57(m,J＝6.3Hz,2H,H-3″′),2.88(dd,J＝6.8,16.1Hz,1H,H-2a′),2.95(dd,J＝6.8,16.1Hz,1H,H-2b′),3.74(m,4H,H-2″″),4.10(t,J＝5.2Hz,2H,H-1″′),4.25(t,J＝5.2Hz,1H,H-9),6.10(s,1H,H-5),14.1(br s,1H,8-OH)；MS(ES):m/z:570([M-H]^-,100％).

Synthesis of 98-hydroxy-9-isobutyl-2, 2,4,4-tetramethyl-7- (3-methylbutanoyl) -6- (3- (piperidin-1-yl) propoxy) -4,9-dihydro-1H-xanthene-1,3(2H) -dione (19)

To a stirred solution of compound 17(3.5mg, 0.0062mmol) in DMF (0.5mL) was added piperidine (0.2mL) dropwise. The mixture was stirred at room temperature. The reaction was monitored by thin layer chromatography until the conversion was acceptable. Distilled water (25mL) was added to the reaction mixture, which was then extracted with ethyl glycolate (3X 50 mL). The organic phase was washed with distilled water (50ml), dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. With methylene chloride/methanol(98:2)) as an eluting solvent, the crude product was purified by column chromatography to give colorless amorphous compound 19(2.6mg, 74%).¹H NMR(CDCl₃):δ0.80(d,J＝5.5Hz,3H,H-3″),0.84(d,J＝5.6Hz,3H,H-4″),0.95(d,J＝6.5Hz,6H,H-4′and H-5′),1.33,1.37(each s,2×3H,C(CH₃)₂),1.41,1.53(each s,2×3H,C(CH₃)₂),1.46(m,2H,H-4″″),1.63(m,4H,H-3″″and H-5″″),2.09(m,2H,H-2″′),2.24(m,1H,H-3′),2.47(m,4H,H-2″″and H-6″″),2.56(t,J＝6.7Hz,2H,H-3″′),2.89(dd,J＝6.8,16.1Hz,1H,H-2a′),2.95(dd,J＝6.8,16.1Hz,1H,H-2b′),4.09(m,2H,H-1″′),4.25(t,J＝5.4Hz,1H,H-9),6.10(s,1H,H-5),14.1(br s,1H,8-OH)；MS(ES):m/z:568([M-H]^-,100％).

Synthesis of 3.108-hydroxy-9-isobutyl-2,2,4,4-tetramethyl-7- (3-methylbutanoyl) -6- (3- (pipe razin-1-yl) propoxy) -4,9-dihydro-1H-xanthene-1,3(2H) -dione (20)

Piperazine (1mg, 0.0116mmol) was added to a stirred solution of compound 17(3.8mg, 0.0067mmol) in DMF (0.5mL stirring the reaction at room temperature until acceptable conversion distilled water (25mL) was added to the reaction mixture, the combined ethyl acetate extracts were extracted with ethyl glycolate (3 × 50mL), washed with distilled water (50mL), dried over anhydrous sodium sulfate and evaporated to dryness, the starting mixture was purified with 100:0 to 87:13 column chromatography alcohol with dichloromethane/methanol to give compound 20 as a colorless amorphous form (1.4mg, 37%).¹H NMR(CDCl₃):δ0.80(d,J＝5.7Hz,3H,H-3″),0.83(d,J＝5.8Hz,3H,H-4″),0.94(d,J＝6.6Hz,6H,H-4′and H-5′),1.33,1.37(each s,2×3H,C(CH₃)₂),1.41,1.53(each s,2×3H,C(CH₃)₂),2.04(m,2H,H-2″′),2.23(m,1H,H-3′),2.56(m,6H,H-1″″and H-3″′),2.87(dd,J＝6.8,16.0Hz,1H,H-2a′),2.94(dd,J＝6.8,16.0Hz,1H,H-2b′),3.02(m,4H,H-2″″),4.08(t,J＝5.3Hz,2H,H-1″′),4.25(t,J＝5.4Hz,1H,H-9),6.08(s,1H,H-5),14.1(br s,1H,8-OH)；MS(ES):m/z:569([M-H]^-,100％).

Synthesis of 3.116- (3- (dimethylamino) proxy) -8-hydroxy-9-isobutyl-2,2-dimethyl-7- (3-methyl butanoyl) -4,9-dihydro-1H-xanthene-1,3(2H) -dione (21)

0.2mL of diethylamine was added to a stirred solution of Compound 17(3.8mg, 0.0067mmol) in DMF (0.5mL, stirred at room temperature until acceptable conversion. distilled water (25mL) was added to the reaction mixture, the combined ethyl acetate extracts were extracted with ethyl glycolate (3X 50mL), washed with distilled water (50mL), dried over anhydrous sodium sulfate, and evaporated to dryness. the starting mixture was purified with 100:0 to 87:13 column chromatography alcohol with dichloromethane/methanol to give compound 21 as a colorless amorphous form (2.6mg, 72%).¹H NMR(CDCl₃):δ0.80(d,J＝5.7Hz,3H,H-3″),0.83(d,J＝5.8Hz,3H,H-4″),0.94(d,J＝6.6Hz,6H,H-4′and H-5′),1.33,1.37(each s,2×3H,C(CH₃)₂),1.41,1.53(each s,2×3H,C(CH₃)₂),2.04(m,2H,H-2″′),2.23(m,1H,H-3′),2.56(m,6H,H-1″″and H-3″′),2.87(dd,J＝6.8,16.0Hz,1H,H-2a′),2.94(dd,J＝6.8,16.0Hz,1H,H-2b′),3.02(m,4H,H-2″″),4.08(t,J＝5.3Hz,2H,H-1″′),4.25(t,J＝5.4Hz,1H,H-9),6.08(s,1H,H-5),14.1(br s,1H,8-OH)；MS(ES):m/z:527([M-H]^-,100％).

Example 4 assay of anti-influenza Virus Activity of Myrtle Compounds

4.1 preparation of cells and viruses

The dog kidney cell MDCK is subcultured according to the conventional method, and the cell density is adjusted to 2 multiplied by 10⁴one/mL was seeded into 96-well plates and the cells in the wells were grown to a complete monolayer ready for use. The cell culture solution is DEME culture solution containing 10% FBS and 1% streptomycin mixed solution, and the cell maintenance solution and the virus maintenance solution are DEME culture solution containing 3% FBS and 1% streptomycin mixed solution.

Influenza A virus strain A/WSN/33(H1N1),Respectively inoculating A/PR/8/34(H1N1) and influenza B virus B/Massachusetts/2/2012 to MDCK monolayer cells, incubating at 37 deg.C for 1H, removing supernatant, replacing DMEM maintenance solution containing pancreatin, freezing and thawing for three times when cells have 100% pathological changes, collecting supernatant as seed virus, TCID₅₀The titer of the virus was determined.

TCID₅₀The determination of (1): MDCK monolayer cells cultured in 96-well plates, supernatant was removed and then washed twice with PBS, plus 100 μ L of diluted virus in multiple dilutions, 8 replicates per dilution, incubated at 37 ℃ for 2h, supernatant was removed, washed twice with PBS, and 150 μ L of virus maintenance solution containing TPCK pancreatin was added. After three days, the lesion count lesion wells were observed under a microscope, and the virus titer TCID was calculated by the Reed-Muench method₅₀。

4.2 determination of the cytotoxicity of Compounds

After the compound or ribavirin solution was diluted 2-fold in a cell maintenance medium, the diluted solution was added to a 96-well culture plate in which cells were grown in a monolayer at 100. mu.L/well and 3 wells were repeated for each dilution, a control group of cells without the compound was cultured in a 5% CO2 incubator at 37 ℃ for 72 hours, and cytopathic effect (CPE) was observed and recorded every day. And after the culture is carried out for 72 hours, abandoning the supernatant, washing the supernatant for 2 times by using PBS buffer solution, adding 20 mu L of MTT into each hole, continuing to culture the supernatant for 4 hours, abandoning the MTT supernatant, adding 150 mu L of DMSO into each hole, shaking the mixture for 5 to 10min, and measuring OD570 by using a microplate reader after the crystals are completely dissolved. Calculating the pathogenic rate of drug induced cells, CC, based on the degree of CPE and the measured OD₅₀Is the drug concentration that causes 50% of the cells to develop lesions.

4.3 inhibition of IAV Virus replication by Compounds

After MDCK cells in 96-well plates were grown into monolayers, the culture was aspirated and 100 × TCID was used₅₀Adsorbing 100 μ L of virus to cells, incubating at 37 deg.C for 1h, washing off free virus, adding 100 μ L of compound diluted by multiple times of cell maintenance solution, repeating 3 wells for each dilution, and keeping the virus and compound freeCell control group, virus control group without compound and virus, 37 deg.C, 5% CO₂And (3) continuously culturing, observing cytopathic effect, determining whether the cytopathic effect is a specific effect caused by the IAV, ending the test when the CPE of the virus control group reaches 80% -90%, recording the CPE of each hole, measuring the OD570 value of each hole according to 4.2 operation, and calculating the inhibition rate of the sample on the virus by using the following formula. The concentration of the sample that inhibits half of the cells from infecting the virus (IC) was determined as a result of CPE and inhibition rate₅₀). Finally, the selection index SI ═ CC is calculated₅₀/IC₅₀

TABLE 1 Myrtus communis extract fraction, fraction anti-A/WSN/33 (H1N1) virus activity and cytotoxicity

The screening of anti-IAV activity is carried out on the ethanol extract, the n-hexane part, the ethyl acetate part and the water part of the myrtle respectively, and the results show that the ethanol extract, the n-hexane part and the ethyl acetate part have good anti-AIV activity, particularly the n-hexane part has stronger antiviral activity and relatively better selectivity (larger SI value), so that main antiviral active ingredients in the myrtle are considered to be easier to enrich in the n-hexane part. And (3) further performing silica gel column chromatography, MCI column chromatography and Sephadex LH-20 gel column chromatography on the n-hexane part, gradually separating to obtain a Fr.C fraction and a Fr.C2 fraction, and enhancing the antiviral activity, which indicates that the antiviral active ingredients are further enriched.

TABLE 2 Myrtle Compounds inhibit the Activity of influenza A Virus strain A/WSN/33(H1N1)

Antiviral Effect of 3 Compounds such as Table 32-h on different strains of influenza Virus

The results in Table 2 show that the myrtle ketone compounds 2-H, 3 and 4 obtained by separating from Fr.C2 fraction and the series of myrtle ketone compounds obtained by synthesis have inhibitory activity to influenza A virus strain A/WSN/33(H1N1) with different degrees, in particular to the compounds 2-H, 18 and 21 and IC thereof₅₀Is about 0.01 times of ribavirin, and the selectivity coefficient SI is more than 10, which shows that the anti-influenza A virus effect is obvious and the cytotoxicity is relatively small. The antiviral effects of 3 compounds such as 2-h on different strains of influenza viruses are further verified, and the results in table 3 show that the 2-h rhodomyrnone compounds have obvious inhibitory activity on influenza A viruses and influenza B viruses.

4.4 time course analysis of IAV inhibition by Compound 2-h

Group 1: adding 0.5 μ M compound 100 μ L into each well after MDCK cells in 96-well culture plate grow into monolayer, adsorbing for 2 hr, washing with PBS (pH7.2) for 2 times, adding 100TCID₅₀The virus cells were maintained at 100. mu.L/well for a time of-2. Group 2: will contain 100TCID₅₀The virus cell maintenance solution and 0.5. mu.M compound were mixed for 2-h and immediately added to the monolayer of cells, 100. mu.L per well, for a time of 0. Group 3, addition of 100 TCID-containing solution to cells grown in monolayer₅₀The virus cell maintenance solution is 100 mu L/well, the supernatant is discarded after 2h, and 100 mu L of cell maintenance solution containing 0.5 mu M compound is added into each well after PBS washes for 2 times. Additional virus controls and cell controls were provided. And (3) culturing in a 5% CO2 incubator at 37 ℃, observing CPE every day until 80% -90% of the virus control group is reached, recording the CPE of each hole, and determining the virus titer of each group by a Reed-Muench method.

The results are shown in FIG. 1, and the addition of the compound 2h before the infection of the cells and the simultaneous addition and the addition 2h after the infection can greatly reduce the virus titer 4An order of magnitude (i.e., 99.99% inhibition of virus) but the effect on viral titer was not very different between these three time points, indicating thatCompound pairThe proliferation and/or release of the influenza A virus strain A/WSN/33(H1N1) has obvious inhibition effect, and can be used as a candidate drug molecule for clinical prevention and treatment of influenza.

In summary, the present invention provides compounds having structural formula I and their use against influenza virus.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.