WO2021179513A1 - Influenza virus neuraminidase inhibitor, preparation method therefor and application thereof - Google Patents
Influenza virus neuraminidase inhibitor, preparation method therefor and application thereof Download PDFInfo
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- WO2021179513A1 WO2021179513A1 PCT/CN2020/105256 CN2020105256W WO2021179513A1 WO 2021179513 A1 WO2021179513 A1 WO 2021179513A1 CN 2020105256 W CN2020105256 W CN 2020105256W WO 2021179513 A1 WO2021179513 A1 WO 2021179513A1
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- 0 C*NC(C(*)NC(CC(C)(C)C)=O)=O Chemical compound C*NC(C(*)NC(CC(C)(C)C)=O)=O 0.000 description 12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
- C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
- C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
Definitions
- the invention belongs to the technical field of medicinal chemistry, and relates to an influenza virus neuraminidase inhibitor and a preparation method and application thereof.
- Influenza the full name of influenza, is an acute respiratory infectious disease caused by influenza virus.
- seasonal influenza and highly pathogenic influenza viruses that have emerged from time to time have always warned of the potential threat of a new round of influenza outbreaks in humans.
- the prevention and control of influenza is important and urgent.
- oseltamivir (Tamiflu) and zanamivir (Relexa) represented by influenza virus neuraminidase (NA) inhibitors are still the main methods for preventing and treating influenza.
- NA neuraminidase
- the purpose of the present invention is to provide a class of influenza virus neuraminidase inhibitors and preparation methods and applications thereof. Compared with the existing zanamivir, the influenza virus neuraminidase inhibitor provided by the present invention has more efficient anti-influenza virus activity, and the water solubility is obviously improved, and it is expected to be made into an oral medicine.
- influenza virus neuraminidase inhibitor characterized in that the general structural formula of the influenza virus neuraminidase inhibitor is as follows:
- n is selected from any natural number in 3-12, and X is selected from OCOO or O;
- Y is selected from or,
- Said R in is selected from:
- R' is H or a fluorescent labeling group
- Z is selected from any natural number of 3-6.
- R ⁇ is a fluorescent group
- its main function is to label the virus, but the compound where the R ⁇ group is located It also has antiviral activity.
- the R' is selected from: biotin, CY3, CY5, or FITC.
- influenza virus neuraminidase inhibitor is selected from compounds I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14 or I-15;
- I-12’s m is 5, n is 6, X is O, Y is Among them, the R in is Said Z is 6, R ⁇ is biotin;
- m is 5
- n is 6
- X is O
- Y is in
- the R in is Said Z is 3 and R ⁇ is biotin.
- influenza virus neuraminidase inhibitor characterized in that the influenza virus neuraminidase inhibitor is prepared by compound VIII or compound XIV under reaction conditions g;
- m is any natural number from 2-11
- n is any natural number from 3-12
- X is OCOO or O
- Y 3 is
- the structural formula of the compound XIV is Wherein, m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, Y 5 is H or R; said R means Wherein R ⁇ is biotin, CY3 or CY5;
- the reaction condition g refers to: dissolving compound VIII or XIV in a solvent, adding sodium hydroxide aqueous solution dropwise, stirring at room temperature for 3 hours, neutralizing the solution with Dowex-50 (H+) ion exchange resin to a pH of 7, and filtering After the solution was concentrated, the concentrated residue was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid with a volume ratio of 1/1, reacted for 1 hour and then concentrated, and the concentrated residue was separated and purified by Sephadex G-15 gel column ;
- the effect of dropping sodium hydroxide aqueous solution is to remove the methyl ester group under alkaline conditions
- Dowex-50(H+) ion exchange resin is used to neutralize and at the same time adsorb Na ions on the resin to remove metal ions in the solution;
- Concentration is to evaporate the solvent, because the solvent system is not used in subsequent reactions, and the specific operation is to concentrate under reduced pressure on a rotary evaporator;
- the concentrated residue is dissolved in a mixed solution of dichloromethane and trifluoroacetic acid with a volume ratio of 1/1 to remove Boc and isopropylidene groups;
- concentration is required for the following "Sephadex G-15 gel column separation and purification".
- the sample concentration must not be too dilute during the gel column sample loading process, otherwise the separation effect will not be good.
- the specific operation is to concentrate under reduced pressure on a rotary evaporator;
- the concentrated residue is separated and purified by Sephadex G-15 gel column.
- the function of this step is to purify the target compound and separate impurities with a large difference in molecular weight from the target compound.
- the compound VIII is prepared by the following steps:
- the compound II is Where R 1 is ClCO or Ts;
- n is any natural number from 3-12, Y 1 is N 3 or COOMe;
- n is any natural number from 3-12, X is OCOO or O, and Y 2 is NH 2 or COOH;
- the compound V is Where m is any natural number from 2-11, and R 2 is
- the compound VI is Where m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, and Y 3 is
- the reaction condition a refers to dissolving the compound II and the compound III in a non-polar solvent, refluxing the reaction overnight at 100-150° C., the solution is concentrated and subjected to silica gel chromatography Column separation to obtain the precursor of compound IV;
- the temperature condition of reflux reaction overnight can reach the boiling point of 1,4-dioxane at 100-150°C, and the solvent can boil and reflux, which is the usual saying in chemical reactions;
- the function of concentration is for the separation and purification needs of the silica gel chromatography column.
- the sample concentration of the silica gel chromatography column separation and loading process requires that the sample concentration is not too dilute, otherwise the separation effect will not be good.
- the specific operation is to concentrate under reduced pressure on a rotary evaporator;
- the reaction condition a means that the compound II and the compound III are dissolved in pyridine, and the reaction is stirred overnight at room temperature; methanol is added to the reaction solution, which is dissolved in the solvent after concentration, and Washing with HCl solution and NaHCO 3 solution, the organic phase is concentrated and then separated by silica gel chromatography column to obtain the precursor of compound IV;
- the reaction condition b refers to: dissolving the precursor of compound IV in tetrahydrofuran, adding deionized water and triphenylphosphorus, and heating the reaction solution to 40-60°C, preferably 45°C , The reaction is stirred for 1-12h, preferably 3h; the reaction solution is concentrated and separated by silica gel chromatography column to obtain compound IV;
- the effect of adding deionized water is to make the reaction solution a homogeneous system, speed up the reaction process, and increase the yield; adding triphenylphosphorus is to reduce the azide group to an amino group;
- the reaction condition b refers to: dissolving the precursor of compound IV in a solvent, adding sodium hydroxide aqueous solution dropwise, stirring at room temperature for 1-6 hours, preferably 3 hours, and then using Dowex-50 The (H+) ion exchange resin is neutralized to the pH of the solution to 7, after filtration, the solution is concentrated and separated by a silica gel chromatography column to obtain compound IV;
- Adding sodium hydroxide aqueous solution dropwise has an alkaline effect and removes methyl ester;
- Dowex-50 (H+) ion exchange resin is used to neutralize and adsorb Na ions;
- the reaction condition c refers to dissolving compound IV and compound V in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature. After concentrating the reaction solution, it is separated by a silica gel chromatography column to obtain compound VI;
- N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are both amidation reagents and promote the forward progress of the reaction;
- the reaction condition f refers to dissolving compound VII in pyridine under nitrogen protection, stirring uniformly, adding compound VI and DMAP, and reacting at room temperature for 2-10h, preferably 5h; the reaction solution is concentrated and separated by silica gel chromatography column , Compound VIII is obtained.
- the compound XIV was prepared by the following steps:
- the compound IX is Where Y 4 is H or CH 2 SH;
- the compound X 1 is Wherein R ⁇ is biotin, CY3 or CY5;
- the compound XII is Where Y 5 is H or R, and the R means Wherein R ⁇ is biotin, CY3 or CY5;
- the compound XIII is Where Y 5 is H or R, and the R means Wherein R ⁇ is biotin, CY3 or CY5;
- n is any natural number from 3-12, X is OCOO or O, and Y 2 is NH 2 or COOH;
- the compound V is Where m is any natural number from 2-11, and R 2 is
- reaction condition h refers to dissolving compound IX and compound X 1 in a solvent under nitrogen protection, reacting at room temperature overnight, and then concentrating the solution and separating it on a silica gel column to obtain compound XI;
- the dosage ratio of the compound IX, X 1 and absolute ethanol is 1mmol:1mmol:2-50ml, preferably 1mmol:1mmol:20ml;
- the solvent is selected from the group consisting of absolute ethanol, water and methanol, preferably ethanol;
- the reaction condition e refers to dissolving compound IV and compound XI in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature.
- the reaction solution was concentrated and separated by silica gel chromatography column to obtain compound XII;
- the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.2 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
- the reaction condition i refers to dissolving compound XII in a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 1/1, reacting for 1-3h, preferably 1 hour, and then concentrating, and the residue is condensed by Sephadex G-15 After separation and purification by a gel column, the treated compound XII is obtained;
- the volume ratio of the mixed solution of compound XII, dichloromethane and trifluoroacetic acid with a volume ratio of 1/1 is 1mmol:1-50mL, preferably 1mmol:10mL;
- the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.1 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
- the reaction condition f refers to: under nitrogen protection, dissolve compound VII in pyridine, stir evenly, add compound XIII and DMAP, react at room temperature for 1-12h, preferably 5h, then concentrate the reaction solution and pass it through a silica gel chromatography column After separation, compound XIV was obtained.
- the solvent is selected from the group consisting of methanol, ethanol, and water;
- the dosage ratio of the compound VIII or XIV, methanol, aqueous sodium hydroxide solution, and a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 1/1 is 1mmol:1-100mL:0.5-2M:1-100mL.
- the dosage ratio of the compound II, the compound III, and the non-polar solvent is 1 mol: 1-1.5 mol: 10-200 ml; preferably 1 mol: 1.2 mol: 100ml; the non-polar solvent is 1,4-dioxane; the temperature condition for the reflux reaction overnight is 125°C;
- Non-polar solvents can be common non-polar solvents in the field, preferably 1,4-dioxane has the advantage that it can maximize the yield of the final product;
- the solvent is selected from dichloromethane or chloroform; the compound II, compound III, pyridine, methanol, dichloromethane, HCl solution, NaHCO 3
- the dosage ratio of the solution is 1mol:1.2mol:100mL:2mL:100mL:1M:1M;
- volume and concentration dosage is only an approximate dosage, and the increase or decrease of dosage does not affect the final result
- the dosage ratio of compound IV, tetrahydrofuran, deionized water, and triphenylphosphorus is 10mmol: 10-100ml: 10-100ml: 10-50mmol , Preferably 10mmol: 100ml: 20ml: 20mmol;
- the dosage ratio of the compound IV precursor, methanol, and aqueous sodium hydroxide solution is 10 mmol: 10-200 mL: 0.5-2M, preferably 10 mmol: 100 mL: 1M;
- the solvent is selected from the group consisting of methanol, ethanol, and water, preferably methanol;
- the dosage ratio of the compound IV, compound V, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-3mmol:1-50ml:1 -6mmol:0.1-2mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
- the dosage ratio of the compound VII, pyridine, compound VI, and DMAP is 1:1-50:1-3:0.1-1 by volume, preferably 1:10:1.5:0.2.
- influenza virus neuraminidase inhibitor and/or the influenza virus neuraminidase inhibitor prepared by the preparation method in the preparation of anti-influenza drugs.
- the dosage form of the medicine is selected from the group consisting of oral agents, nasal drops, injections, and nasal sprays.
- R' is biotin, CY3, CY5, FITC and other functional groups or fluorescent molecules.
- the present invention also provides two preparation methods of neuraminidase inhibitors, and the reaction equation is as follows:
- R 1 of the compound II is Ts
- the precursor of compound IV is obtained through the following reaction conditions a: a.
- R 1 of the compound II is ClCO
- the reaction condition b refers to dissolving the carboxymethyl ester compound (COOMe, 10 mmol) in methanol (100 mL), adding 1M aqueous sodium hydroxide solution (5 mL) dropwise, and stirring at room temperature for 3 After hours, use Dowex-50(H+) ion exchange resin to neutralize the solution to a pH of 7, after filtering, the solution is concentrated, and the organic phase is concentrated and separated by silica gel column to obtain the target compound. This step is the COOMe reaction to obtain COOH. Conditions, only compound IV is produced.
- Reaction condition c refers to: under the protection of nitrogen, a carboxyl compound, such as compound IV (COOH, 1 mmol) and an amino compound, such as compound V (NH 2 , 1 mmol) are dissolved in DMF (10 mL), and N,N-di Cyclohexylcarbodiimide (DCC, 2 mmol) and 4-dimethylaminopyridine (DMAP, 1 mmol) were reacted overnight at room temperature. The reaction solution is concentrated and separated by silica gel chromatography column to obtain the target compound (this step is the conditions for the amidation reaction of NH 2 and COOH to obtain CONH, and all such reactions are under this condition.
- a carboxyl compound such as compound IV (COOH, 1 mmol)
- an amino compound such as compound V (NH 2 , 1 mmol)
- DCC N,N-di Cyclohexylcarbodiimide
- DMAP 4-dimethylaminopyridine
- IV+V is used as the raw material
- the precursor of IV (N 3 ) is formed. If IV+XI is used under this condition, XII is formed. If the product treated with condition i of XII is reacted with V, XIII is formed.
- Reaction condition f Under nitrogen protection, dissolve compound VII (1mmol) in pyridine (10mmol), stir well and add amino compound, such as compound VI (NH 2 , 1.5mmol) and DMAP (0.2mmol), and react at room temperature 5h. The reaction solution is concentrated and separated by a silica gel chromatography column to obtain the target compound. (VI+VII produces VIII; VII+XIV precursor produces XIV).
- Reaction condition g Under nitrogen protection, the protective group precursor of compound I (compound VIII or XIV) (1 mmol) was dissolved in methanol (10 mL), and 1M aqueous sodium hydroxide solution (1 mL) was added dropwise, and stirred at room temperature for 3 After hours, neutralize the solution with Dowex-50(H+) ion exchange resin to the pH value of 7, and concentrate the solution after filtration. The remainder is dissolved in a mixed solution (10mL) with a volume ratio of dichloromethane and trifluoroacetic acid of 1/1. After reacting for 1 hour, it was concentrated, and the residue was separated and purified by Sephadex G-15 gel column to obtain compound I as a white solid.
- the reaction condition e refers to dissolving compound IV and compound XI in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature.
- the reaction solution was concentrated and separated by silica gel chromatography column to obtain compound XII;
- the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.2 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
- the 15 neuraminidase inhibitors provided by the present invention have an inhibitory effect on various NAs, and particularly have a good inhibitory effect on zanamivir-resistant NA.
- the 15 neuraminidase inhibitors provided by the present invention also have a good virus inhibitory effect at the cellular level, and also have a good inhibitory effect on zanamivir-resistant influenza viruses, and can be used for various influenzas. Treatment of viral infections.
- the 15 neuraminidase inhibitors provided by the present invention have been verified through mouse experiments that their oral administration can achieve better anti-influenza effects, and at the same time, their lipid-water partition coefficient is dozens of times higher than that of the original drugs.
- the water solubility of the original medicine is greatly improved, and it can be made into an oral preparation, which has made a major breakthrough in the dosage form of the medicine.
- Figure 1 shows the body weight change curve and survival rate curve of experimental mice in Experimental Example 5 of the present invention.
- the purified NA proteins (N1, N5, etc.) were diluted 5 ⁇ , 25 ⁇ , 125 ⁇ , 625 ⁇ , 3125 ⁇ , 15625 ⁇ with 20/150 Tris/NaCl with a pH value of 8.0, and then placed in a black 96-well After adding 10 ⁇ L of NA solution of different concentrations and 10 ⁇ L of PBS to the microtiter plate, incubate in a constant temperature incubator at 37°C for 30min, and then add 30 ⁇ L of 167 ⁇ M 4-MUNANA (4-methylumbelliferyl-N-acetylneuraminic acid) fluorescent bottom to each well Things.
- N1, N5, etc. were diluted 5 ⁇ , 25 ⁇ , 125 ⁇ , 625 ⁇ , 3125 ⁇ , 15625 ⁇ with 20/150 Tris/NaCl with a pH value of 8.0, and then placed in a black 96-well After adding 10 ⁇ L of NA solution of different concentrations and 10 ⁇ L of PBS to the microtiter plate, incubate in a
- the inhibitor (I) was diluted with PBS in a 10-fold gradient to form a solution with a suitable concentration range, and then 10 ⁇ L of inhibitor solution of different concentration and 10 ⁇ L of NA solution of suitable concentration were added to the black 96-well plate. In addition, 10 ⁇ L of PBS and 10 ⁇ L of NA solution of appropriate concentration were added to other wells as a positive control, and 20 ⁇ L of PBS was added as a negative control.
- the Y group of compounds I-12, I-13, I-14, I-15 in the above table R in is Respective R groups Z and R ⁇ in are as listed in the above table respectively.
- the 15 influenza virus NAase inhibitors provided in the above embodiments of the present invention have inhibitory effects on the NA enzymes of various influenza viruses, especially the zanamivir-resistant influenza virus (N2( H3N2, E119V)) NA has a more prominent inhibitory effect.
- influenza viruses obtained through chicken embryo reproduction were diluted with DMEM into virus solutions of different concentrations according to a 10-fold gradient. Inoculate MDCK cells in a 96-well cell culture plate, 20h later (after the cells grow to the bottom of the culture plate), aspirate the DMEM medium containing the serum double antibody, rinse with sterilized PBS solution for 2 times, and add the pre-diluted 100 ⁇ L of good virus solution. Then the 96-well cell culture plate was placed in a 37°C cell culture incubator containing 5% CO 2 for 48 hours. Repeat 4 times for each virus concentration. Observe the cell status with an inverted microscope, and perform an ELISA test for each well. The results obtained are calculated using the Reed-Muench method to calculate the TCID 50 of the influenza virus.
- DMEM medium After filtering the 11 mM inhibitor (I) mother liquor with a 0.22 ⁇ m sterile filter, add DMEM medium and dilute it to an inhibitor solution with a suitable concentration range according to a 10-fold gradient. In addition, inoculate MDCK cells in a 96-well cell culture plate. After 20 hours, when the cells have grown to the bottom of the culture plate, aspirate the DMEM medium containing the serum double antibody, rinse with sterilized PBS solution for 2 times, and add the pre-diluted solution. 100 ⁇ L of a good virus solution of 100 times TCID 50.
- the 15 influenza virus NAase inhibitors provided by the present invention also have a good virus inhibitory effect at the cellular level. ”) It also has a more significant inhibitory effect and can be used for the treatment of various influenza virus infections.
- mice One day later, 1 cage of mice was anaesthetized and administrated 15 ⁇ L of I-3 at a dose of 6mg/kg intranasally, 1 cage of mice was given by intragastric administration 150 ⁇ L of I-3 at a dose of 60mg/kg, 1 cage of mice was given by intragastric administration 150 ⁇ L of Tamiflu at a dose of 20 mg/kg was administered.
- One cage of mice was intragastrically administered with 150 ⁇ L of 10% ethanol solution, and the last cage of mice was intragastrically administered with the same volume of 10% ethanol solution for 7 days.
- the body weight and temperature of the mice were monitored daily for 14 days. When the weight of the mouse drops to 75% of the initial weight, the mouse is deemed dead.
- the mouse body weight change curve and survival rate curve were drawn by GraphPad Prism (version 5.0) analysis.
- the compounds I1-2 and I4-15 provided by other embodiments 1-2 and 4-15 of the present invention can all obtain the anti-influenza effect of oral administration similar to that of I-3 as shown in Figure 1. In order to save the space of the present invention, I will not repeat them one by one.
- n-octanol and double distilled water were shaken with a constant temperature (37 ⁇ 1)°C shaker for 24h at room temperature to make them saturated with each other. After standing overnight for layering, the two phases are separated and stored for later use. Accurately weigh an appropriate amount of the compound to be tested in a 10 mL volumetric flask, dissolve it in n-octanol saturated with water, oscillate it ultrasonically for 30 min, and make it constant to obtain a mother liquor with a concentration of 1 mmol/L.
- the lipid-water partition coefficient (log P) is the main indicator to measure whether a drug can penetrate a biological membrane composed of a lipid bilayer, and it is related to the pharmacokinetic process of drug absorption, distribution, metabolism, and excretion in the human body.
- Log P The smaller the P value, the stronger the hydrophobicity of the compound, the easier it is to be metabolized in the body, and the higher the clearance rate (such as zanamivir).
- the larger the log P value the stronger the lipophilicity of the compound.
- lipid-water partition coefficient of the compounds designed in the present invention is generally significantly higher than that of zanamivir, which can achieve the purpose of improving the water solubility of zanamivir, and is expected to significantly improve the pharmacokinetic properties of the compound. It has a good potential as a medicine.
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Abstract
The present invention relates to an influenza virus neuraminidase inhibitor, a preparation method therefor and an application thereof, which belong to the technical field of pharmaceutical chemistry. The structural general formula of the influenza virus neuraminidase inhibitor is shown in formula I, wherein: m is any natural number selected from 2-11, n is any natural number selected from 3-12, X is selected from OCOO or O; y is selected from formula aa, formula bb and formula cc; R in formula cc is selected from H or formula dd; R' in formula dd is H or a fluorescence labeling group; and Z is any natural number selected from 3-6. The described influenza virus neuraminidase inhibitor has significant inhibitory activity on various influenza viruses, especially on zanamivir-resistant influenza viruses (H3N2, E119V). The water solubility of an original medicine is remarkably improved, the lipid-water distribution coefficient is tens of times higher than that of the original medicine, and the inhibitor can be made into an oral preparation, which is a great breakthrough in pharmaceutical dosage forms.
Description
本发明属于药物化学技术领域，涉及一种流感病毒神经氨酸酶抑制剂及其制备方法与应用。The invention belongs to the technical field of medicinal chemistry, and relates to an influenza virus neuraminidase inhibitor and a preparation method and application thereof.
流感(Infuenza)全称流行性感冒，是由流感病毒引起的急性呼吸道传染性疾病。近年来不断发生的季节性流感及不时突现的高致病性流感病毒(如H1N1，H5N1及H7N9)时刻警示着人类新一轮大流感暴发的潜在威胁性，针对流感的防控工作重要而紧迫。目前以流感病毒神经氨酸酶(NA)抑制剂为代表的oseltamivir(达菲)和zanamivir(瑞乐沙)仍然是防治流感的主要手段。但随着这些药物的广泛使用，不断出现流感病毒的耐药株，寻找新型的抗流感药物迫在眉睫。Influenza (Infuenza), the full name of influenza, is an acute respiratory infectious disease caused by influenza virus. In recent years, seasonal influenza and highly pathogenic influenza viruses that have emerged from time to time (such as H1N1, H5N1 and H7N9) have always warned of the potential threat of a new round of influenza outbreaks in humans. The prevention and control of influenza is important and urgent. . At present, oseltamivir (Tamiflu) and zanamivir (Relexa) represented by influenza virus neuraminidase (NA) inhibitors are still the main methods for preventing and treating influenza. However, with the widespread use of these drugs, drug-resistant strains of influenza viruses continue to appear, and it is urgent to find new anti-influenza drugs.
发明内容Summary of the invention
本发明的目的是提供一类流感病毒神经氨酸酶抑制剂及其制备方法与应用。本发明提供的流感病毒神经氨酸酶抑制剂较现有扎那米韦具有更加高效的抗流感病毒活性，并且水溶性有明显改善，有望制成口服药。The purpose of the present invention is to provide a class of influenza virus neuraminidase inhibitors and preparation methods and applications thereof. Compared with the existing zanamivir, the influenza virus neuraminidase inhibitor provided by the present invention has more efficient anti-influenza virus activity, and the water solubility is obviously improved, and it is expected to be made into an oral medicine.
本发明的技术方案具体如下：The technical scheme of the present invention is specifically as follows:
一种流感病毒神经氨酸酶抑制剂，其特征在于，所述流感病毒神经氨酸酶抑制剂结构通式如下：An influenza virus neuraminidase inhibitor, characterized in that the general structural formula of the influenza virus neuraminidase inhibitor is as follows:
其中：in:
m选自2-11中的任一自然数，n选自3-12中的任一自然数，X选自OCOO或O；m is selected from any natural number in 2-11, n is selected from any natural number in 3-12, and X is selected from OCOO or O;
Y选自
或，
Y is selected from or,
所述
中的R选自：
Said R in is selected from:
H、或，
H, or,
所述
中的R`为H或荧光标记基团，Z选自3-6中的任一自然数。
Said In R'is H or a fluorescent labeling group, and Z is selected from any natural number of 3-6.
当R`为荧光基团时，其主要作用是用来标记病毒，但是R`基团所在的化合物
本身也有抗病毒活性。
When R` is a fluorescent group, its main function is to label the virus, but the compound where the R` group is located It also has antiviral activity.
所述R`选自：生物素，CY3，CY5，或，FITC。The R'is selected from: biotin, CY3, CY5, or FITC.
所述的流感病毒神经氨酸酶抑制剂选自化合物I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、I-11、I-12、I-13、I-14或I-15；The influenza virus neuraminidase inhibitor is selected from compounds I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14 or I-15;
化合物I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、I-11、I-12、I-13I-14和I-15均具有如下结构通式：Compound I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I Both -13I-14 and I-15 have the following general structural formula:
化合物I-1的m为2，n为6，X为O，Y为
In compound I-1, m is 2, n is 6, X is O, Y is
I-2的m为2，n为6，X为OCOO，Y为
In I-2, m is 2, n is 6, X is OCOO, Y is
I-3的m为5，n为6，X为O，Y为
I-3’s m is 5, n is 6, X is O, Y is
I-4的m为5，n为6，X为OCOO，Y为
I-4’s m is 5, n is 6, X is OCOO, Y is
I-5的m为11，n为6，X为O，Y为
I-5’s m is 11, n is 6, X is O, Y is
I-6的m为11，n为6，X为OCOO，Y为
For I-6, m is 11, n is 6, X is OCOO, Y is
I-7的m为5，n为12，X为O，Y为
I-7’s m is 5, n is 12, X is O, Y is
I-8的m为5，n为3，X为O，Y为
I-8’s m is 5, n is 3, X is O, Y is
I-9的m为5，n为5，X为O，Y为
I-9’s m is 5, n is 5, X is O, Y is
I-10的m为5，n为6，X为O，Y为
其中，
中的R为H；
I-10’s m is 5, n is 6, X is O, Y is in, R is H;
I-11的m为5，n为6，X为OCOO，Y为
其中，
中的R为H；
I-11’s m is 5, n is 6, X is OCOO, Y is in, R is H;
I-12的m为5，n为6，X为O，Y为
其中，所述
中的R为
所述
中的Z为6，R`为生物素；
I-12’s m is 5, n is 6, X is O, Y is Among them, the R in is Said Z is 6, R` is biotin;
I-13的m为5，n为6，X为O，Y为
其中，
中的R为
所述
中的Z为6，R`为CY3；
In I-13, m is 5, n is 6, X is O, Y is in, R in is Said Z is 6, R` is CY3;
I-14的m为5，n为6，X为O，Y为
其中，
中的R为
所述
中的Z为6，R`为CY5；或，
In I-14, m is 5, n is 6, X is O, Y is in, R in is Said Where Z is 6, R` is CY5; or,
I-15的m为5，n为6，X为O，Y为
其中，
中的R为为
所述
中的Z为3，R`为生物素。
For I-15, m is 5, n is 6, X is O, Y is in, The R in is Said Z is 3 and R` is biotin.
一种流感病毒神经氨酸酶抑制剂的制备方法，其特征在于，将化合物VIII或化合物XIV经反应条件g制得所述流感病毒神经氨酸酶抑制剂；A preparation method of influenza virus neuraminidase inhibitor, characterized in that the influenza virus neuraminidase inhibitor is prepared by compound VIII or compound XIV under reaction conditions g;
所述化合物VIII的结构式为
其中，m为2-11中的任一自然数，n为3-12中的任一自然数，X为OCOO或O，Y
3为
The structural formula of the compound VIII is Among them, m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, and Y 3 is
所述化合物XIV的结构式为
其中，m为2-11中的任一自然数，n为3-12中的任一自然数，X为OCOO或O，Y
5为H或R；所述R指
其中R`为生物素、CY3或CY5；
The structural formula of the compound XIV is Wherein, m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, Y 5 is H or R; said R means Wherein R` is biotin, CY3 or CY5;
所述反应条件g指：将化合物VIII或XIV溶于溶剂中，滴加氢氧化钠水溶液，室温搅拌3小时后，用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩，浓缩后的剩余物溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液中，反应1小时后浓缩，浓缩后的剩余物经Sephadex G-15凝胶柱分离纯化；The reaction condition g refers to: dissolving compound VIII or XIV in a solvent, adding sodium hydroxide aqueous solution dropwise, stirring at room temperature for 3 hours, neutralizing the solution with Dowex-50 (H+) ion exchange resin to a pH of 7, and filtering After the solution was concentrated, the concentrated residue was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid with a volume ratio of 1/1, reacted for 1 hour and then concentrated, and the concentrated residue was separated and purified by Sephadex G-15 gel column ；
滴加氢氧化钠水溶液的作用是碱性条件下脱除甲酯基团；The effect of dropping sodium hydroxide aqueous solution is to remove the methyl ester group under alkaline conditions;
用Dowex-50(H+)离子交换树脂的作用是中和，同时把Na离子吸附到树脂上，除掉溶液中的金属离子；Dowex-50(H+) ion exchange resin is used to neutralize and at the same time adsorb Na ions on the resin to remove metal ions in the solution;
浓缩就是蒸干溶剂，因为后续反应不用该溶剂体系，具体操作就是旋转蒸发仪减压浓缩；Concentration is to evaporate the solvent, because the solvent system is not used in subsequent reactions, and the specific operation is to concentrate under reduced pressure on a rotary evaporator;
浓缩后的剩余物溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液中的作用是脱除Boc和异丙叉基团；The concentrated residue is dissolved in a mixed solution of dichloromethane and trifluoroacetic acid with a volume ratio of 1/1 to remove Boc and isopropylidene groups;
反应1小时后浓缩，是为了后面的“Sephadex G-15凝胶柱分离纯化”需要，凝胶柱上样过程中要求样品浓度不能太稀，否则分离效果不好。具体操作就是旋转蒸发仪减压浓缩；After 1 hour of reaction, concentration is required for the following "Sephadex G-15 gel column separation and purification". The sample concentration must not be too dilute during the gel column sample loading process, otherwise the separation effect will not be good. The specific operation is to concentrate under reduced pressure on a rotary evaporator;
浓缩后的剩余物经Sephadex G-15凝胶柱分离纯化，这一步的作用是纯化目标化合物，把与目标化合物分子量差距较大的杂质分离掉。The concentrated residue is separated and purified by Sephadex G-15 gel column. The function of this step is to purify the target compound and separate impurities with a large difference in molecular weight from the target compound.
所述化合物VIII由下述步骤制得：The compound VIII is prepared by the following steps:
将化合物II与化合物III经反应条件a得到化合物IV前体，再经反应条件b得到化合物IV；Compound II and compound III are subjected to reaction condition a to obtain compound IV precursor, and then reaction condition b to obtain compound IV;
化合物IV与化合物V经反应条件c得到化合物VI；Compound IV and compound V undergo reaction condition c to obtain compound VI;
将化合物VI与化合物VII经反应条件f得到化合物VIII；Compound VI and compound VII are subjected to reaction conditions f to obtain compound VIII;
所述化合物II为
其中R
1为ClCO或Ts；
The compound II is Where R 1 is ClCO or Ts;
所述化合物III为
其中n为3-12中的任一自然数，Y
1为N
3或COOMe；
The compound III is Wherein n is any natural number from 3-12, Y 1 is N 3 or COOMe;
所述化合物IV为
其中n为3-12中的任一自然数，X为OCOO或O，Y
2为NH
2或COOH；
The compound IV is Wherein n is any natural number from 3-12, X is OCOO or O, and Y 2 is NH 2 or COOH;
所述化合物V为
其中m为2-11中的任一自然数，R
2为
The compound V is Where m is any natural number from 2-11, and R 2 is
所述化合物VI为
其中m为2-11中的任一自然数，n为3-12中的任一自然数，X为OCOO或O，Y
3为
The compound VI is Where m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, and Y 3 is
所述化合物VII为
The compound VII is
当所述化合物II的R
1为Ts时，所述反应条件a指，将化合物II与化合物III溶于非极性溶剂中，100-150℃条件下回流反应过夜，溶液浓缩后经硅胶层析柱分离，得到化合物IV的前体；
When the R 1 of the compound II is Ts, the reaction condition a refers to dissolving the compound II and the compound III in a non-polar solvent, refluxing the reaction overnight at 100-150° C., the solution is concentrated and subjected to silica gel chromatography Column separation to obtain the precursor of compound IV;
回流反应过夜的温度条件100-150℃可达到1,4-二氧六环的沸点，溶剂可发生沸腾回流，是化学反应中的惯常说法；The temperature condition of reflux reaction overnight can reach the boiling point of 1,4-dioxane at 100-150℃, and the solvent can boil and reflux, which is the usual saying in chemical reactions;
浓缩的作用是为了后面硅胶层析柱分离纯化需要，硅胶层析柱分离上样过程要求样品浓度不能太稀，否则分离效果不好。具体操作就是旋转蒸发仪减压浓缩；The function of concentration is for the separation and purification needs of the silica gel chromatography column. The sample concentration of the silica gel chromatography column separation and loading process requires that the sample concentration is not too dilute, otherwise the separation effect will not be good. The specific operation is to concentrate under reduced pressure on a rotary evaporator;
当所述化合物II的R
1为ClCO时，所述反应条件a指，化合物II和化合物III溶于吡啶中，室温搅拌反应过夜；向反应液中加入甲醇，浓缩后溶于溶剂，并先后以HCl溶液和NaHCO
3溶液洗涤，有机相浓缩后经硅胶层析柱分离，得到化合物IV的前体；
When the R 1 of the compound II is ClCO, the reaction condition a means that the compound II and the compound III are dissolved in pyridine, and the reaction is stirred overnight at room temperature; methanol is added to the reaction solution, which is dissolved in the solvent after concentration, and Washing with HCl solution and NaHCO 3 solution, the organic phase is concentrated and then separated by silica gel chromatography column to obtain the precursor of compound IV;
向反应液中加入甲醇的作用是用来消耗过量的化合物II；The effect of adding methanol to the reaction solution is to consume excess compound II;
当化合物III的Y
1为N
3时，所述反应条件b指：将化合物IV前体溶于四氢呋喃中，加入去离子水，三苯基磷，反应液加热到40-60℃，优选45℃，搅拌反应1-12h，优选3h；将反应液浓缩后经硅胶层析柱分离，得到化合物IV；
When Y 1 of compound III is N 3 , the reaction condition b refers to: dissolving the precursor of compound IV in tetrahydrofuran, adding deionized water and triphenylphosphorus, and heating the reaction solution to 40-60°C, preferably 45°C , The reaction is stirred for 1-12h, preferably 3h; the reaction solution is concentrated and separated by silica gel chromatography column to obtain compound IV;
加入去离子水的作用是：使反应液为均一体系，加快反应进程，提高产率；加入三苯基磷的作用是把叠氮基团还原成氨基；The effect of adding deionized water is to make the reaction solution a homogeneous system, speed up the reaction process, and increase the yield; adding triphenylphosphorus is to reduce the azide group to an amino group;
当化合物III的Y
1为COOMe时，所述反应条件b指：将化合物IV前体溶于溶剂中，滴加氢氧化钠水溶液，室温搅拌1-6小时，优选3小时，然后用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩后经硅胶层析柱分离，得到化合物IV；
When Y 1 of compound III is COOMe, the reaction condition b refers to: dissolving the precursor of compound IV in a solvent, adding sodium hydroxide aqueous solution dropwise, stirring at room temperature for 1-6 hours, preferably 3 hours, and then using Dowex-50 The (H+) ion exchange resin is neutralized to the pH of the solution to 7, after filtration, the solution is concentrated and separated by a silica gel chromatography column to obtain compound IV;
滴加氢氧化钠水溶液起碱性作用，脱除甲酯；用Dowex-50(H+)离子交换树脂的作用是中和并吸附Na离子；Adding sodium hydroxide aqueous solution dropwise has an alkaline effect and removes methyl ester; Dowex-50 (H+) ion exchange resin is used to neutralize and adsorb Na ions;
所述反应条件c指，在氮气保护条件下，将化合物IV和化合物V溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物VI；The reaction condition c refers to dissolving compound IV and compound V in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature. After concentrating the reaction solution, it is separated by a silica gel chromatography column to obtain compound VI;
加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶的作用是，它们都是酰胺化反应试剂，促进反应正向进行；The effect of adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is that they are both amidation reagents and promote the forward progress of the reaction;
所述反应条件f指，在氮气保护条件下，将化合物VII溶于吡啶，搅拌均匀后加入化合物VI和DMAP，室温下反应2-10h，优选5h；将反应液浓缩后经硅胶层析柱分离，得到化合物VIII。The reaction condition f refers to dissolving compound VII in pyridine under nitrogen protection, stirring uniformly, adding compound VI and DMAP, and reacting at room temperature for 2-10h, preferably 5h; the reaction solution is concentrated and separated by silica gel chromatography column , Compound VIII is obtained.
所述化合物XIV通过下述步骤制得：The compound XIV was prepared by the following steps:
化合物IX与化合物X
1通过反应条件h得到化合物XI；
Compound IX and compound X 1 pass reaction conditions h to obtain compound XI;
化合物XI与化合物IV通过反应条件e得到化合物XII；Compound XI and compound IV pass reaction condition e to obtain compound XII;
化合物XII与化合物V通过反应条件i得到化合物XIII；Compound XII and compound V obtain compound XIII through reaction condition i;
化合物XIII与化合物VII通过反应条件f得到化合物XIV；Compound XIII and compound VII obtain compound XIV through reaction condition f;
所述化合物IX为
其中Y
4为H或CH
2SH；
The compound IX is Where Y 4 is H or CH 2 SH;
所述化合物X
1为
其中R`为生物素、CY3或CY5；
The compound X 1 is Wherein R` is biotin, CY3 or CY5;
所述化合物XI为
其中Y
5＝H或R，所述R指
其中R`为生物素、CY3或CY5；
The compound XI is Where Y 5 ＝H or R, and the R means Wherein R` is biotin, CY3 or CY5;
所述化合物XII为
其中Y
5为H或R，所述R指
其中R`为生物素、CY3或CY5；
The compound XII is Where Y 5 is H or R, and the R means Wherein R` is biotin, CY3 or CY5;
所述化合物XIII为
其中Y
5为H或R，所述R指
其中R`为生物素、CY3或CY5；
The compound XIII is Where Y 5 is H or R, and the R means Wherein R` is biotin, CY3 or CY5;
所述化合物IV为
其中n为3-12中的任一自然数，X为OCOO或O，Y
2为NH
2或COOH；
The compound IV is Wherein n is any natural number from 3-12, X is OCOO or O, and Y 2 is NH 2 or COOH;
所述化合物V为
其中m为2-11中的任一自然数，R
2为
The compound V is Where m is any natural number from 2-11, and R 2 is
所述化合物VII为
The compound VII is
所述反应条件h指，在氮气保护条件下，将化合物IX，化合物X
1溶于溶剂中，室温反应过夜，溶液浓缩后经硅胶层析柱分离，得到化合物XI；
The reaction condition h refers to dissolving compound IX and compound X 1 in a solvent under nitrogen protection, reacting at room temperature overnight, and then concentrating the solution and separating it on a silica gel column to obtain compound XI;
优选地，所述化合物IX、X
1、无水乙醇的用量比例为1mmol∶1mmol∶2-50ml，优选1mmol∶1mmol∶20ml；
Preferably, the dosage ratio of the compound IX, X 1 and absolute ethanol is 1mmol:1mmol:2-50ml, preferably 1mmol:1mmol:20ml;
所述溶剂选自由无水乙醇、水、甲醇组成的组，优选乙醇；The solvent is selected from the group consisting of absolute ethanol, water and methanol, preferably ethanol;
所述反应条件e指，在氮气保护条件下，将化合物IV和化合物XI溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物XII；The reaction condition e refers to dissolving compound IV and compound XI in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature. The reaction solution was concentrated and separated by silica gel chromatography column to obtain compound XII;
优选地，所述化合物IV、化合物XI、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-2mmol∶1-50ml∶1-6mmol∶0.2-1mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；Preferably, the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.2 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
所述反应条件i指，将化合物XII溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液中，反应1-3h，优选1小时，然后浓缩，剩余物经Sephadex G-15凝胶柱分离纯化后，得到处理后的化合物XII；The reaction condition i refers to dissolving compound XII in a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 1/1, reacting for 1-3h, preferably 1 hour, and then concentrating, and the residue is condensed by Sephadex G-15 After separation and purification by a gel column, the treated compound XII is obtained;
优选地，所述化合物XII、二氯甲烷与三氟乙酸体积比为1/1的混合溶液的用量比例为1mmol∶1-50mL，优选1mmol∶10mL；Preferably, the volume ratio of the mixed solution of compound XII, dichloromethane and trifluoroacetic acid with a volume ratio of 1/1 is 1mmol:1-50mL, preferably 1mmol:10mL;
将所述处理后的化合物XII与化合物V溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物XIII；The treated compound XII and compound V were dissolved in DMF, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added, and reacted overnight at room temperature. The reaction solution was concentrated and passed through a silica gel layer. Column separation to obtain compound XIII;
优选地，所述化合物IV、化合物XI、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-2mmol∶1-50ml∶1-6mmol∶0.1-1mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；Preferably, the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.1 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
所述反应条件f指：在氮气保护条件下，将化合物VII溶于吡啶，搅拌均匀后加入化合物XIII和DMAP，室温下反应1-12h，优选5h，然后将反应液浓缩后经硅胶层析柱分离，得到化合物XIV。The reaction condition f refers to: under nitrogen protection, dissolve compound VII in pyridine, stir evenly, add compound XIII and DMAP, react at room temperature for 1-12h, preferably 5h, then concentrate the reaction solution and pass it through a silica gel chromatography column After separation, compound XIV was obtained.
所述反应条件g中，所述溶剂选自由甲醇、乙醇、水组成的组；In the reaction condition g, the solvent is selected from the group consisting of methanol, ethanol, and water;
所述化合物VIII或XIV、甲醇、氢氧化钠水溶液、二氯甲烷与三氟乙酸体积比为1/1的混合溶液的用量比例为1mmol∶1-100mL∶0.5-2M∶1-100mL。The dosage ratio of the compound VIII or XIV, methanol, aqueous sodium hydroxide solution, and a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 1/1 is 1mmol:1-100mL:0.5-2M:1-100mL.
上述用量比例不是必须的，上述都是常规化学反应，溶剂和碱性用量多少对该反应没有太大影响；The above-mentioned dosage ratio is not necessary, the above-mentioned are all conventional chemical reactions, and the amount of solvent and alkalinity does not have much influence on the reaction;
当所述化合物II的R
1为Ts时，所述反应条件a中，所述化合物II、化合物III、非极性溶剂的用量比例为1mol∶1-1.5mol∶10-200ml；优选1mol∶1.2mol∶100ml；所述非极性溶剂为1,4-二氧六环；回流反应过夜的温度条件为125℃；
When R 1 of the compound II is Ts, in the reaction condition a, the dosage ratio of the compound II, the compound III, and the non-polar solvent is 1 mol: 1-1.5 mol: 10-200 ml; preferably 1 mol: 1.2 mol: 100ml; the non-polar solvent is 1,4-dioxane; the temperature condition for the reflux reaction overnight is 125°C;
非极性溶剂可以选择本领域常见的非极性溶剂，优选1,4-二氧六环的好处是可使终产物的产率达到最高；Non-polar solvents can be common non-polar solvents in the field, preferably 1,4-dioxane has the advantage that it can maximize the yield of the final product;
当所述化合物II的R
1为ClCO时，所述反应条件a中，所述溶剂选自二氯甲烷或氯仿；所述化合物II、化合物III、吡啶、甲醇、二氯甲烷、HCl溶液、NaHCO
3溶液的用量比例为1mol∶1.2mol∶100mL∶2mL∶100mL∶1M∶ 1M；
When R 1 of the compound II is ClCO, in the reaction condition a, the solvent is selected from dichloromethane or chloroform; the compound II, compound III, pyridine, methanol, dichloromethane, HCl solution, NaHCO 3 The dosage ratio of the solution is 1mol:1.2mol:100mL:2mL:100mL:1M:1M;
上述体积和浓度用量仅仅是一个大概的添加量，用量的增加或减少都可不影响最终的结果；The above-mentioned volume and concentration dosage is only an approximate dosage, and the increase or decrease of dosage does not affect the final result;
当化合物III的Y
1为N
3时，所述反应条件b中，所述化合物IV、四氢呋喃、去离子水、三苯基磷的用量比例为10mmol∶10-100ml∶10-100ml∶10-50mmol，优选10mmol∶100ml∶20ml∶20mmol；
When Y 1 of compound III is N 3 , in the reaction condition b, the dosage ratio of compound IV, tetrahydrofuran, deionized water, and triphenylphosphorus is 10mmol: 10-100ml: 10-100ml: 10-50mmol , Preferably 10mmol: 100ml: 20ml: 20mmol;
当化合物III的Y
1为COOMe时，所述反应条件b中，所述化合物IV前体、甲醇、氢氧化钠水溶液用量比例为10mmol∶10-200mL∶0.5-2M，优选10mmol∶100mL∶1M；所述溶剂选自由甲醇、乙醇、水组成的组，优选甲醇；
When Y 1 of compound III is COOMe, in the reaction condition b, the dosage ratio of the compound IV precursor, methanol, and aqueous sodium hydroxide solution is 10 mmol: 10-200 mL: 0.5-2M, preferably 10 mmol: 100 mL: 1M; The solvent is selected from the group consisting of methanol, ethanol, and water, preferably methanol;
所述反应条件c中，所述化合物IV、化合物V、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-3mmol∶1-50ml∶1-6mmol∶0.1-2mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；In the reaction condition c, the dosage ratio of the compound IV, compound V, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-3mmol:1-50ml:1 -6mmol:0.1-2mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
所述反应条件f中，所述化合物VII、吡啶、化合物VI、DMAP的用量比例为体积比1∶1-50∶1-3∶0.1-1，优选1∶10∶1.5∶0.2。In the reaction condition f, the dosage ratio of the compound VII, pyridine, compound VI, and DMAP is 1:1-50:1-3:0.1-1 by volume, preferably 1:10:1.5:0.2.
本发明所有的反应步骤和反应条件中，除特别说明的以外，所有的浓缩步骤均为了方便后续的分离纯化过柱步骤，经硅胶层析柱处理均为了分离纯化产物；N,N-二环己基碳二亚胺和4-二甲胺基吡啶均为酰胺化反应试剂，加入它们是为了促进反应正向进行；同时，所有反应条件中的各物质的用量比例范围仅仅是为了出于说明书清楚、完整的要求考虑，并不以此限制本发明的范围，各物质的用量比例是本领域技术人员可根据实际的反应需要做出常规选择和调整的，在本发明给出的数值范围比例的基础之上适当调整都不影响本发明获得最终产物。In all the reaction steps and reaction conditions of the present invention, unless otherwise specified, all the concentration steps are the column steps to facilitate the subsequent separation and purification. After the silica gel chromatography column, the products are separated and purified; N,N-bicyclic Both hexylcarbodiimide and 4-dimethylaminopyridine are reagents for amidation reaction. They are added to promote the forward progress of the reaction; at the same time, the dosage ratio range of each substance in all reaction conditions is only for the clarity of the specification. The complete requirement consideration does not limit the scope of the present invention. The dosage ratio of each substance can be routinely selected and adjusted by those skilled in the art according to actual reaction needs. The ratio is within the numerical range given by the present invention. Appropriate adjustments on this basis will not affect the final product obtained by the present invention.
所述的流感病毒神经氨酸酶抑制剂，和/或，所述的制备方法制备得到的流感病毒神经氨酸酶抑制剂在制备抗流感药物方面的应用。The application of the influenza virus neuraminidase inhibitor and/or the influenza virus neuraminidase inhibitor prepared by the preparation method in the preparation of anti-influenza drugs.
所述药物的剂型选自：口服剂、滴鼻剂、注射剂、鼻喷雾剂。The dosage form of the medicine is selected from the group consisting of oral agents, nasal drops, injections, and nasal sprays.
本发明的化合物结构通式：The general structural formula of the compound of the present invention:
R’为生物素，CY3，CY5，FITC等功能基团或荧光分子。R'is biotin, CY3, CY5, FITC and other functional groups or fluorescent molecules.
本发明还提供两种神经氨酸酶抑制剂的制备方法，反应方程式如下所示：The present invention also provides two preparation methods of neuraminidase inhibitors, and the reaction equation is as follows:
方法一：method one:
当化合物II R
1＝Ts的时候，用条件a与III反应，若得到的产物是N
3化合物，则用条件c得到IV，若得到的产物是COOMe化合物，则用条件d得到IV；当化合物II R
1＝ClCO的时候，用条件b与III反应，若得到的产物是N
3化合物，则用条件c得到IV，若得到的产物是COOMe化合物，则用条件d得到IV；得到不同的化合物IV之后，后续经过相应的反应就刻意得到不同的化合物I。
When compound II R 1 = Ts, use condition a to react with III, if the product obtained is N 3 compound, use condition c to obtain IV, if the obtained product is COOMe compound, use condition d to obtain IV; when the compound When II R 1 =ClCO, use condition b to react with III, if the product obtained is N 3 compound, use condition c to get IV, if the product obtained is COOMe compound, use condition d to get IV; get different compounds After IV, different compounds I were deliberately obtained through corresponding reactions.
具体步骤如下：Specific steps are as follows:
当所述化合物II的R
1为Ts时，经过下述反应条件a得到化合物IV前体：a.化合物II(R
1＝Ts,1mol)和化合 物III(1.2mol)溶于1,4-二氧六环(100mL)中，125℃条件下回流反应过夜。溶液浓缩后经硅胶层析柱分离，得到化合物IV的前体。
When R 1 of the compound II is Ts, the precursor of compound IV is obtained through the following reaction conditions a: a. Compound II (R 1 = Ts, 1 mol) and compound III (1.2 mol) are dissolved in 1,4-di In oxane (100 mL), the reaction was refluxed overnight at 125°C. The solution was concentrated and separated by silica gel chromatography column to obtain the precursor of compound IV.
当所述化合物II的R
1为ClCO时，经过下述反应条件a得到化合物IV前体：a:化合物II(R
1＝ClCO,1mol)和化合物III(1.2mol)溶于吡啶(100mL)中，室温搅拌反应过夜。向反应液中加入甲醇(2mL)，浓缩后溶于二氯甲烷，并先后以1M HCl溶液和1M NaHCO
3溶液洗涤，有机相浓缩后经硅胶层析柱分离，得到化合物IV的前体。
When R 1 of the compound II is ClCO, the precursor of compound IV is obtained through the following reaction conditions a: a: compound II (R 1 =ClCO, 1 mol) and compound III (1.2 mol) are dissolved in pyridine (100 mL) , The reaction was stirred overnight at room temperature. Methanol (2 mL) was added to the reaction solution, concentrated and dissolved in dichloromethane, washed with 1M HCl solution and 1M NaHCO 3 solution successively, the organic phase was concentrated and separated by silica gel chromatography column to obtain the precursor of compound IV.
当化合物III的Y
1为N
3时，经过下述反应条件b得到化合物IV：
When Y 1 of compound III is N 3 , compound IV is obtained through the following reaction condition b:
反应条件b:将叠氮化合物(N
3，10mmol)溶于四氢呋喃(100mL)中，加入去离子水20mL，三苯基磷(20mmol)，反应液加热到45℃搅拌反应3h。将反应液浓缩后经硅胶层析柱分离，得到目标化合物。此步反应为N
3还原得到NH
2的条件，所有涉及该类反应的都是此条件。若III(Y
1＝N
3)做原料就生成IV(Y
2＝NH
2),若IV+V的产物(N
3化合物)用此条件则生成VI，若XIII做原料则生成XIV的前体。
Reaction condition b: The azide compound (N 3 , 10 mmol) was dissolved in tetrahydrofuran (100 mL), 20 mL of deionized water and triphenylphosphorus (20 mmol) were added, and the reaction solution was heated to 45° C. and stirred for 3 hours. The reaction solution is concentrated and separated by a silica gel chromatography column to obtain the target compound. The reaction in this step is the condition for N 3 reduction to obtain NH 2 , and all the reactions involved in this type of reaction are under this condition. If III (Y 1 ＝N 3 ) is used as the raw material, IV (Y 2 ＝NH 2 ) will be generated. If the product of IV+V (N 3 compound) is used under this condition, VI will be generated. If XIII is used as the raw material, the precursor of XIV will be generated. .
当化合物III的Y
1为COOMe时，所述反应条件b指：将羧基甲酯化合物(COOMe，10mmol)溶于甲醇(100mL)中，滴加1M氢氧化钠的水溶液(5mL)，室温搅拌3小时后，用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩，有机相浓缩后经硅胶层析柱分离，得到目标化合物，这一步就是COOMe反应得到COOH的条件，只生成化合物IV。
When Y 1 of compound III is COOMe, the reaction condition b refers to dissolving the carboxymethyl ester compound (COOMe, 10 mmol) in methanol (100 mL), adding 1M aqueous sodium hydroxide solution (5 mL) dropwise, and stirring at room temperature for 3 After hours, use Dowex-50(H+) ion exchange resin to neutralize the solution to a pH of 7, after filtering, the solution is concentrated, and the organic phase is concentrated and separated by silica gel column to obtain the target compound. This step is the COOMe reaction to obtain COOH. Conditions, only compound IV is produced.
反应条件c指:在氮气保护条件下，将羧基化合物，例如化合物IV(COOH，1mmol)和氨基化合物，例如化合物V(NH
2，1mmol)溶于DMF(10mL)中，加入N,N-二环己基碳二亚胺(DCC，2mmol)和4-二甲胺基吡啶(DMAP，1mmol)，室温下反应过夜。将反应液浓缩后经硅胶层析柱分离，得到目标化合物(该步反应为NH
2和COOH发生酰胺化反应得到CONH的条件，所有涉及该类反应均为此条件。如果用IV+V做原料则生成IV的前体(N
3),如果IV+XI用该条件则生成XII,如果XII用条件i处理完的产物再与V反应，则生成XIII。)
Reaction condition c refers to: under the protection of nitrogen, a carboxyl compound, such as compound IV (COOH, 1 mmol) and an amino compound, such as compound V (NH 2 , 1 mmol) are dissolved in DMF (10 mL), and N,N-di Cyclohexylcarbodiimide (DCC, 2 mmol) and 4-dimethylaminopyridine (DMAP, 1 mmol) were reacted overnight at room temperature. The reaction solution is concentrated and separated by silica gel chromatography column to obtain the target compound (this step is the conditions for the amidation reaction of NH 2 and COOH to obtain CONH, and all such reactions are under this condition. If IV+V is used as the raw material The precursor of IV (N 3 ) is formed. If IV+XI is used under this condition, XII is formed. If the product treated with condition i of XII is reacted with V, XIII is formed.)
反应条件f:在氮气保护条件下，将化合物VII(1mmol)溶于吡啶(10mmol)，搅拌均匀后加入氨基化合物，例如化合物VI(NH
2，1.5mmol)和DMAP(0.2mmol)，室温下反应5h。将反应液浓缩后经硅胶层析柱分离，得到目标化合物。(VI+VII生成VIII；VII+XIV前体生成XIV)。
Reaction condition f: Under nitrogen protection, dissolve compound VII (1mmol) in pyridine (10mmol), stir well and add amino compound, such as compound VI (NH 2 , 1.5mmol) and DMAP (0.2mmol), and react at room temperature 5h. The reaction solution is concentrated and separated by a silica gel chromatography column to obtain the target compound. (VI+VII produces VIII; VII+XIV precursor produces XIV).
反应条件g:在氮气保护条件下，将化合物I的保护基前体(化合物VIII或者XIV)(1mmol)溶于甲醇(10mL)中，滴加1M氢氧化钠的水溶液(1mL)，室温搅拌3小时后，用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩，剩余物溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液(10mL)中，反应1小时后浓缩，剩余物经Sephadex G-15凝胶柱分离纯化后，得白色固体化合物I。Reaction condition g: Under nitrogen protection, the protective group precursor of compound I (compound VIII or XIV) (1 mmol) was dissolved in methanol (10 mL), and 1M aqueous sodium hydroxide solution (1 mL) was added dropwise, and stirred at room temperature for 3 After hours, neutralize the solution with Dowex-50(H+) ion exchange resin to the pH value of 7, and concentrate the solution after filtration. The remainder is dissolved in a mixed solution (10mL) with a volume ratio of dichloromethane and trifluoroacetic acid of 1/1. After reacting for 1 hour, it was concentrated, and the residue was separated and purified by Sephadex G-15 gel column to obtain compound I as a white solid.
方法二：Method Two:
具体步骤如下(其中部分反应步骤与方法一相同)：The specific steps are as follows (part of the reaction steps are the same as method one):
所述反应条件e指，在氮气保护条件下，将化合物IV和化合物XI溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物XII；The reaction condition e refers to dissolving compound IV and compound XI in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature. The reaction solution was concentrated and separated by silica gel chromatography column to obtain compound XII;
优选地，所述化合物IV、化合物XI、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-2mmol∶1-50ml∶1-6mmol∶0.2-1mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；Preferably, the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.2 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;
h:在氮气保护条件下，将化合物IX(Y
4＝CH
2SH，1mmol)，化合物X(1mmol)溶于无水乙醇(20mL)中，室温反应过夜，溶液浓缩后经硅胶层析柱分离，得到化合物XI。
h: Under nitrogen protection, dissolve compound IX (Y 4 =CH 2 SH, 1 mmol) and compound X (1 mmol) in absolute ethanol (20 mL), react overnight at room temperature, and separate the solution by silica gel chromatography after concentration , Compound XI is obtained.
i:将Boc保护的氨基化合物(就是化合物XII)(1mmol)溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液(10mL)中，反应1小时后浓缩，剩余物经Sephadex G-15凝胶柱分离纯化后，得到氨基化合物XIII。i: Dissolve the Boc-protected amino compound (compound XII) (1mmol) in a mixed solution (10mL) with a volume ratio of dichloromethane and trifluoroacetic acid of 1/1, react for 1 hour and concentrate, and then pass the remainder to Sephadex G After -15 gel column separation and purification, the amino compound XIII is obtained.
本发明的有益效果在于：The beneficial effects of the present invention are:
(1)本发明提供的15种神经氨酸酶抑制剂对各种NA均具有抑制效果，特别是对耐扎那米韦的NA仍有较好的抑制效果。(1) The 15 neuraminidase inhibitors provided by the present invention have an inhibitory effect on various NAs, and particularly have a good inhibitory effect on zanamivir-resistant NA.
(2)本发明提供的15种神经氨酸酶抑制剂在细胞水平同样具有很好的病毒抑制效果，对耐扎那米韦的流感病毒也具有很好的抑制效果，可以用于各种流感病毒感染的治疗。(2) The 15 neuraminidase inhibitors provided by the present invention also have a good virus inhibitory effect at the cellular level, and also have a good inhibitory effect on zanamivir-resistant influenza viruses, and can be used for various influenzas. Treatment of viral infections.
(3)本发明提供的15种神经氨酸酶抑制剂通过小鼠实验验证了其口服给药能获得较好的抗流感效果，同时其脂水分配系数较原有药物高出数十倍，极大地改善了原有药物的水溶性，可制成口服剂，在药物剂型上有重大突破。(3) The 15 neuraminidase inhibitors provided by the present invention have been verified through mouse experiments that their oral administration can achieve better anti-influenza effects, and at the same time, their lipid-water partition coefficient is dozens of times higher than that of the original drugs. The water solubility of the original medicine is greatly improved, and it can be made into an oral preparation, which has made a major breakthrough in the dosage form of the medicine.
图1为本发明实验例5的实验小鼠体重变化曲线和生存率曲线。Figure 1 shows the body weight change curve and survival rate curve of experimental mice in Experimental Example 5 of the present invention.
下面结合具体实施例，进一步阐述本发明，这些实施例仅用于说明本发明而不用于限制本发明的范围。The present invention will be further described below with reference to specific examples, which are only used to illustrate the present invention and not to limit the scope of the present invention.
本发明所有实验例涉及的试剂均可商购获得，如无特别说明，所有实验操作均为有机化学领域技术人员通常理解的常规操作。All the reagents involved in the experimental examples of the present invention are commercially available. Unless otherwise specified, all experimental operations are routine operations commonly understood by those skilled in the organic chemistry field.
实验例1、以合成化合物I-1为例，其结构式如下：Experimental example 1. Take the synthetic compound I-1 as an example, and its structural formula is as follows:
I-1(m＝2,n＝6,X＝O,Y＝
):
I-1(m=2,n=6,X=O,Y= ):
a:化合物II(R
1＝Ts,1mmol)和化合物III(n＝6,Y
1＝N
3,1.2mmol)溶于1,4-二氧六环(10mL)中，125℃条件下回流反应过夜。溶液浓缩后经硅胶层析柱分离，得到化合物IV的前体(就是化合物IV中的Y
2＝N
3)。
a: Compound II (R 1 = Ts, 1 mmol) and Compound III (n = 6, Y 1 = N 3 , 1.2 mmol) were dissolved in 1,4-dioxane (10 mL), and reacted under reflux at 125°C overnight. The solution is concentrated and separated by silica gel chromatography to obtain the precursor of compound IV (that is, Y 2 =N 3 in compound IV).
c:将叠氮化合物(上一步产物IV的前体N
3化合物，1mmol)溶于四氢呋喃(10mL)中，加入去离子水2mL，三苯基磷(2mmol)，反应液加热到45℃搅拌反应3h。将反应液浓缩后经硅胶层析柱分离，得到目标化合物IV(n＝6,X＝O,Y
2＝NH
2)。
c: Dissolve the azide compound (precursor N 3 compound of product IV in the previous step, 1 mmol) in tetrahydrofuran (10 mL), add 2 mL of deionized water, and triphenylphosphorus (2 mmol), and heat the reaction solution to 45°C and stir to react. 3h. The reaction solution was concentrated and separated by silica gel chromatography to obtain the target compound IV (n=6, X=O, Y 2 =NH 2 ).
e:在氮气保护条件下，将羧基化合物(COOH，1mmol)和氨基化合物(NH
2，1mmol)溶于DMF(10mL)中，加入N,N-二环己基碳二亚胺(DCC，2mmol)和4-二甲胺基吡啶(DMAP，1mmol)，室温下反应过夜。将反应液浓缩后经硅胶层析柱分离，得到目标化合物。
e: Under nitrogen protection, dissolve carboxyl compound (COOH, 1mmol) and amino compound (NH 2 , 1mmol) in DMF (10mL), and add N,N-dicyclohexylcarbodiimide (DCC, 2mmol) React with 4-dimethylaminopyridine (DMAP, 1 mmol) overnight at room temperature. The reaction solution is concentrated and separated by a silica gel chromatography column to obtain the target compound.
f:在氮气保护条件下，将化合物VII(1mmol)溶于吡啶(10mmol)，搅拌均匀后加入氨基化合物(NH
2，1.5mmol)和DMAP(0.2mmol)，室温下反应5h。将反应液浓缩后经硅胶层析柱分离，得到目标化合物。
f: Under nitrogen protection, dissolve compound VII (1 mmol) in pyridine (10 mmol), stir well, add amino compound (NH 2 , 1.5 mmol) and DMAP (0.2 mmol), and react at room temperature for 5 hours. The reaction solution is concentrated and separated by a silica gel chromatography column to obtain the target compound.
g:在氮气保护条件下，将化合物I的保护基前体(1mmol)溶于甲醇(10mL)中，滴加1M氢氧化钠的水溶液(1mL)，室温搅拌3小时后，用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩，剩余物溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液(100mL)中，反应1小时后浓缩，剩余物经Sephadex G-15凝胶柱分离纯化后，得白色固体化合物I。g: Under nitrogen protection, the protective group precursor (1 mmol) of compound I was dissolved in methanol (10 mL), and 1M aqueous sodium hydroxide solution (1 mL) was added dropwise. After stirring at room temperature for 3 hours, use Dowex-50 ( The H+) ion exchange resin was neutralized to the pH of the solution to 7, and the solution was concentrated after filtration. The residue was dissolved in a mixed solution (100 mL) with a volume ratio of dichloromethane and trifluoroacetic acid of 1/1. After reacting for 1 hour, it was concentrated. After the residue was separated and purified by Sephadex G-15 gel column, a white solid compound I was obtained.
所得化合物：I-1，白色固体，
1H NMR(500MHz,CDCl
3:MeOD＝1:1):δ5.62-5.63(m,1H),5.32(d,J＝4.9Hz,1H),4.94(d,J＝7.4Hz,1H),4.52–4.39(m,2H),4.19(d,J＝8.0Hz,1H),3.99-3.94(m,3H),3.67-3.49(m,11H),3.38-3.16(m,4H),2.39(dd,J＝13.0,2.4Hz,1H),2.14(t,J＝12.0Hz,1H),1.98-1.82(m,10H),1.65–0.76(m,41H),0.71(s,3H).ESI-HRMS:m/z calculated forC
51H
87N
6O
12[M+H]
+:975.63820,Found:975.63751.
Obtained compound: I-1, white solid, 1 H NMR (500MHz, CDCl 3 : MeOD=1:1): δ5.62-5.63 (m, 1H), 5.32 (d, J=4.9 Hz, 1H), 4.94 (d,J=7.4Hz,1H),4.52–4.39(m,2H), 4.19(d,J=8.0Hz,1H), 3.99-3.94(m,3H), 3.67-3.49(m,11H), 3.38-3.16 (m, 4H), 2.39 (dd, J = 13.0, 2.4 Hz, 1H), 2.14 (t, J = 12.0 Hz, 1H), 1.98-1.82 (m, 10H), 1.65-0.76 (m, 41H),0.71(s,3H).ESI-HRMS: m/z calculated for C 51 H 87 N 6 O 12 [M+H] + :975.63820,Found: 975.63751.
实验例2、采用类似于实施例1的方法合成化合物I-2～I-15，(化合物I-1～I-9用合成路线一合成，化合物I-10～I-15用合成路线二合成)以下为化合物结构和数据。Experimental example 2. Compounds I-2～I-15 were synthesized by a method similar to Example 1. (Compounds I-1～I-9 were synthesized by synthetic route one, and compounds I-10～I-15 were synthesized by synthetic route two. ) The following is the compound structure and data.
I-2(m＝2,n＝6,X＝OCOO,Y＝
):
1H NMR(500MHz,CDCl
3:MeOD＝1:1):δ5.63-5.61(m,1H),5.40(d,J＝4.4Hz,1H),4.94(d,J＝8.0Hz,1H),4.40–4.28(m,3H),4.17(t,J＝8.4Hz,1H),4.10(t,J＝6.4Hz,2H),3.99-3.91(m,13),3.60(m,9H),3.34–3.23(m,4H),2.37-2.30(m,2H),2.09–1.82(m,9H),1.77–0.80(m,41H),0.71(s,3H).ESI-HRMS:m/z calculated forC
53H
89N
6O
14[M+H]
+:1433.64368,Found:1433.64259.
I-2 (m=2, n=6, X=OCOO, Y= ): 1 H NMR(500MHz, CDCl 3 : MeOD=1:1): δ5.63-5.61(m,1H), 5.40(d,J=4.4Hz,1H), 4.94(d,J=8.0Hz, 1H), 4.40–4.28 (m, 3H), 4.17 (t, J = 8.4 Hz, 1H), 4.10 (t, J = 6.4 Hz, 2H), 3.99-3.91 (m, 13), 3.60 (m, 9H ), 3.34–3.23(m, 4H), 2.37-2.30(m, 2H), 2.09–1.82(m, 9H), 1.77–0.80(m, 41H), 0.71(s, 3H).ESI-HRMS: m /z calculated forC 53 H 89 N 6 O 14 [M+H] + :1433.64368,Found:1433.64259.
I-3(m＝5,n＝6,X＝O,Y＝
):
1H NMR(500MHz,CDCl
3:MeOD＝1:1):δ5.48-5.47(m,1H),5.26(d,J＝4.5Hz,1H),4.90(d,J＝7.7Hz,1H),4.36–4.31(m,2H),4.01-3.89(m,4H),3.69–3.40(m,22H),3.32–3.14(m,4H),3.09(ddd,J＝11.0,7.8,4.0Hz,1H),2.21–2.03(m,2H),1.96–1.62(m,10H),1.55–0.64(m,41H),0.60(s,3H).ESI-HRMS:m/z calculated forC
58H
101N
6O
15[M+H]
+:1121.73249,Found:1121.73201.
I-3 (m=5, n=6, X=O, Y= ): 1 H NMR(500MHz, CDCl 3 : MeOD=1:1): δ5.48-5.47(m,1H), 5.26(d,J=4.5Hz,1H), 4.90(d,J=7.7Hz, 1H), 4.36–4.31(m,2H),4.01-3.89(m,4H), 3.69–3.40(m,22H), 3.32–3.14(m,4H), 3.09(ddd,J=11.0,7.8,4.0 Hz,1H),2.21-2.03(m,2H),1.96-1.62(m,10H),1.55-0.64(m,41H),0.60(s,3H).ESI-HRMS: m/z calculated for C 58 H 101 N 6 O 15 [M+H] + :1121.73249,Found:1121.73201.
I-4(m＝5,n＝6,X＝OCOO,Y＝
):
1H NMR(500MHz,CDCl
3:MeOD＝1:1):δ5.49-5.46(m,1H),5.30(s,1H),4.84(s,1H),4.40–3.87(m,9H),3.76–3.03(m,24H),2.28(d,J＝8.4Hz,2H),2.01–1.71(m,10H),1.69–0.67(m,41H),0.62(s,3H).ESI-HRMS:m/z calculated forC
59H
101N
6O
17[M+H]
+:1165.72232,Found:1165.72347.
I-4 (m=5, n=6, X=OCOO, Y= ): 1 H NMR (500MHz, CDCl 3 : MeOD=1:1): δ5.49-5.46 (m, 1H), 5.30 (s, 1H), 4.84 (s, 1H), 4.40-3.87 (m, 9H ),3.76-3.03(m,24H),2.28(d,J=8.4Hz,2H),2.01-1.71(m,10H),1.69-0.67(m,41H),0.62(s,3H).ESI- HRMS: m/z calculated for C 59 H 101 N 6 O 17 [M+H] + :1165.72232,Found:1165.72347.
I-5(m＝11,n＝6,X＝O,Y＝
):
1H NMR(500MHz,CDCl
3:MeOD＝1:1):δ5.51-5.50(m,1H),5.28(d,J＝4.0Hz,1H),4.94(d,J＝8.1Hz,1H),4.36–4.25(m,4H),4.14(t,J＝9.2Hz,1H),3.98-3.93(m,3H),3.74–3.39(m,46H),3.36–3.02(m,7H),2.22–2.12(m,2H),1.92–0.69(m,51H),0.65(s,3H).ESI-HRMS:m/z calculated forC
70H
125N
6O
21[M+H]
+:1385.88978,Found:1385.88900.
I-5 (m=11, n=6, X=O, Y= ): 1 H NMR(500MHz, CDCl 3 : MeOD=1:1): δ5.51-5.50(m,1H), 5.28(d,J=4.0Hz,1H), 4.94(d,J=8.1Hz, 1H), 4.36–4.25(m,4H), 4.14(t,J=9.2Hz,1H), 3.98-3.93(m,3H), 3.74–3.39(m,46H), 3.36–3.02(m,7H) ,2.22–2.12(m,2H),1.92–0.69(m,51H),0.65(s,3H).ESI-HRMS: m/z calculated for C 70 H 125 N 6 O 21 [M+H] + :1385.88978 ,Found:1385.88900.
I-6(m＝11,n＝6,X＝OCOO,Y＝
):
1H NMR(500MHz,CDCl
3:MeOD＝1:1):δ5.44-5.43(m,1H),5.30-5.29(m,1H),4.88-4.83(m,1H),4.42–3.82(m,9H),3.79–2.89(m,58H),2.27(d,J＝8.1Hz,2H),2.04–1.79(m,10H),1.74–0.67(m,41H),0.59(s,3H).ESI-HRMS:m/z calculated forC
71H
125N
6O
23[M+H]
+:1429.87961,Found:1429.87860.
I-6 (m=11, n=6, X=OCOO, Y= ): 1 H NMR (500MHz, CDCl 3 : MeOD=1:1): δ5.44-5.43 (m, 1H), 5.30-5.29 (m, 1H), 4.88-4.83 (m, 1H), 4.42-3.82 (m,9H),3.79–2.89(m,58H),2.27(d,J=8.1Hz,2H),2.04–1.79(m,10H),1.74–0.67(m,41H),0.59(s,3H) ).ESI-HRMS: m/z calculated for C 71 H 125 N 6 O 23 [M+H] + :1429.87961,Found:1429.87860.
I-7(m＝5,n＝12,X＝O,Y＝
):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.48-5.47(m,1H),5.26(d,J＝4.4Hz,1H),4.90(d,J＝8.0Hz,1H),4.36–4.29(m,2H),4.09(t,J＝8.8Hz,1H),3.92-3.86(m,3H),3.66–3.40(m,20H),3.36–3.03(m,7H),2.21(ddd,J＝13.0,4.4,2.0Hz,1H),2.11–2.06(m,1H),1.95–0.68(m,53H),0.64(s,3H).ESI-HRMS:m/z calculated for C64H113N6O15[M+H]+:1205.82639,Found:1205.82797.
I-7 (m = 5, n = 12, X = O, Y = ): 1H NMR(500MHz, CDCl3: MeOD=1:1): δ5.48-5.47(m,1H), 5.26(d,J=4.4Hz,1H), 4.90(d,J=8.0Hz,1H) , 4.36–4.29(m,2H),4.09(t,J=8.8Hz,1H),3.92-3.86(m,3H),3.66–3.40(m,20H),3.36–3.03(m,7H),2.21 (ddd,J=13.0,4.4,2.0Hz,1H),2.11–2.06(m,1H),1.95–0.68(m,53H),0.64(s,3H).ESI-HRMS: m/z calculated for C64H113N6O15 [M+H]+:1205.82639,Found:1205.82797.
I-8(m＝5,n＝3,X＝O,Y＝
):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.49-5.47(m,1H),5.24(d,J＝4.4Hz,1H),4.92(d,J＝7.6Hz,1H),4.35–4.31(m,2H),4.00–3.89(m,4H),3.66–3.41(m,22H),3.31–3.15(m,4H),3.04(ddd,J＝11.1,7.8,4.1Hz,1H),2.20–2.01(m,2H),1.94–1.64(m,10H),1.56–0.66(m,35H),0.60(s,3H).ESI-HRMS:m/z calculated forC55H95N6O15[M+H]+:1079.68554,Found:1079.68475.
I-8 (m=5, n=3, X=O, Y= ): 1H NMR(500MHz, CDCl3: MeOD=1:1): δ5.49-5.47(m,1H), 5.24(d,J=4.4Hz,1H), 4.92(d,J=7.6Hz,1H) ,4.35–4.31(m,2H),4.00–3.89(m,4H),3.66–3.41(m,22H),3.31–3.15(m,4H),3.04(ddd,J=11.1,7.8,4.1Hz, 1H),2.20--2.01(m,2H),1.94-1.64(m,10H),1.56--0.66(m,35H),0.60(s,3H).ESI-HRMS: m/z calculated for C55H95N6O15(M+H ]+:1079.68554,Found:1079.68475.
I-9(m＝5,n＝6,X＝O,Y＝
):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.51-5.50(m,1H),5.26(d,J＝4.4Hz,1H),4.90(d,J＝8.0Hz,1H),4.37–4.30(m,2H),4.08(t,J＝9.1Hz,1H),3.89(s,1H),3.69–3.41(m,24H),3.32–3.15(m,4H),3.06(ddd,J＝11.0,7.8,4.4Hz,1H),2.74-2.69(m,2H),2.25(ddd,J＝13.0,4.4,2.1Hz,1H),2.14–2.04(m,1H),1.98–1.59(m,10H),1.55–0.68(m,41H),0.60(s,3H).ESI-HRMS:m/z calculated for C58H101N6O15[M+H]+:1121.73249,Found:1121.73396.
I-9 (m=5, n=6, X=O, Y= ): 1H NMR(500MHz, CDCl3: MeOD=1:1): δ5.51-5.50(m,1H), 5.26(d,J=4.4Hz,1H), 4.90(d,J=8.0Hz,1H) ,4.37–4.30(m,2H),4.08(t,J=9.1Hz,1H), 3.89(s,1H), 3.69–3.41(m,24H), 3.32–3.15(m,4H),3.06(ddd ,J=11.0,7.8,4.4Hz,1H),2.74-2.69(m,2H),2.25(ddd,J=13.0,4.4,2.1Hz,1H),2.14-2.04(m,1H),1.98-1.59 (m,10H),1.55--0.68(m,41H),0.60(s,3H).ESI-HRMS:m/z calculated for C58H101N6O15(M+H)+:1121.73249,Found:1121.73396.
I-10(m＝5,n＝6,X＝O,Y＝
R＝H):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.49-5.47(m,1H),5.25 (d,J＝4.4Hz,1H),4.92(d,J＝7.8Hz,1H),4.35–4.30(m,2H),4.04-3.89(m,6H),3.69–3.43(m,22H),3.31–3.07(m,5H),2.20–2.03(m,2H),1.94–0.60(m,54H).ESI-HRMS:m/z calculated for C60H104N7O16[M+H]+:1178.75396,Found:1178.75439.
I-10 (m=5, n=6, X=O, Y= R=H): 1H NMR (500MHz, CDCl3: MeOD=1:1): δ5.49-5.47(m,1H), 5.25 (d,J=4.4Hz,1H), 4.92(d,J=7.8Hz ,1H), 4.35--4.30(m,2H),4.04-3.89(m,6H),3.69--3.43(m,22H),3.31-3.07(m,5H),2.20--2.03(m,2H),1.94 –0.60(m,54H).ESI-HRMS:m/z calculated for C60H104N7O16[M+H]+:1178.75396,Found:1178.75439.
I-11(m＝5,n＝6,X＝OCOO,Y＝
R＝H):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.47-5.46(m,1H),5.33(s,1H),4.82(s,1H),4.41–3.87(m,11H),3.75–3.03(m,24H),2.22(d,J＝8.5Hz,2H),2.01–1.71(m,10H),1.69–0.62(m,44H).ESI-HRMS:m/z calculated for C61H104N7O18[M+H]+:1222.74378,Found:1222.74501.
I-11 (m=5, n=6, X=OCOO, Y= R＝H):1H NMR(500MHz,CDCl3:MeOD=1:1):δ5.47-5.46(m,1H),5.33(s,1H),4.82(s,1H),4.41-3.87(m, 11H),3.75–3.03(m,24H),2.22(d,J=8.5Hz,2H),2.01–1.71(m,10H),1.69–0.62(m,44H).ESI-HRMS: m/z calculated for C61H104N7O18[M+H]+:1222.74378,Found:1222.74501.
I-12(m＝5,n＝6,X＝O,Y＝
Z＝6,R’＝Biotin):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.89(s,1H),5.72(d,J＝2.0Hz,1H),5.33–5.31(m,1H),5.02(s,1H),4.58–4.44(m,4H),4.33-4.29(m,2H),4.15(s,2H),4.04(ddd,J＝9.0,6.4,2.5Hz,1H),3.94-3.10(m,65H),2.94–2.87(m,3H),2.72(d,J＝12.0Hz,1H),2.59-2.53(m,3H),2.33(ddd,J＝13.0,4.5,2.3Hz,1H),2.22–2.13(m,2H),2.10(s,3H),2.01–1.94(m,2H),1.90–0.61(s,53H).ESI-HRMS:m/z calculated for C92H157N12O27S2[M+H]+:1926.07225,Found:1926.07108.
I-12 (m=5, n=6, X=O, Y= Z=6, R'=Biotin): 1H NMR (500MHz, CDCl3:MeOD=1:1): δ5.89(s,1H), 5.72(d,J=2.0Hz,1H),5.33-5.31(m ,1H),5.02(s,1H),4.58–4.44(m,4H),4.33-4.29(m,2H),4.15(s,2H),4.04(ddd,J=9.0,6.4,2.5Hz,1H ), 3.94-3.10 (m, 65H), 2.94-2.87 (m, 3H), 2.72 (d, J = 12.0 Hz, 1H), 2.59-2.53 (m, 3H), 2.33 (ddd, J = 13.0, 4.5 ,2.3Hz,1H),2.22–2.13(m,2H),2.10(s,3H),2.01–1.94(m,2H),1.90–0.61(s,53H).ESI-HRMS: m/z calculated for C92H157N12O27S2[M+H]+:1926.07225,Found:1926.07108.
I-13(m＝5,n＝6,X＝O,Y＝
Z＝6,R’＝CY3):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ8.31(t,1H,J＝13.0Hz),7.70–7.64(m,4H),7.16–7.13(m,2H),6.21–6.17(m,2H),5.39–5.38(m,1H),4.55–4.52(m,1H),4.15(s,2H),3.94–3.09(m,55H),2.92–2.85(m,4H),2.52-2.48(m,1H),2.30–1.77(m,11H),1.67–0.62(m,65H).ESI-HRMS:m/z calculated for C114H180N12O32S3[M+H]+:2325.19887,Found:2325.19705.
I-13 (m=5, n=6, X=O, Y= Z=6, R'=CY3): 1H NMR (500MHz, CDCl3:MeOD=1:1): δ8.31(t,1H,J=13.0Hz), 7.70–7.64(m,4H), 7.16–7.13 (m, 2H), 6.21--6.17 (m, 2H), 5.39--5.38 (m, 1H), 4.55--4.52 (m, 1H), 4.15 (s, 2H), 3.94-3.09 (m, 55H), 2.92 –2.85(m,4H),2.52-2.48(m,1H),2.30–1.77(m,11H),1.67–0.62(m,65H).ESI-HRMS:m/z calculated for C114H180N12O32S3[M+H] +:2325.19887,Found:2325.19705.
I-14(m＝5,n＝6,X＝O,Y＝
Z＝6,R’＝CY5):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ7.82–7.71(m,6H),7.27–7.25(m,2H),6.38(dd,1H,J＝12.4,12.0Hz),6.12–6.04(m,2H),5.74(d,J＝2.2Hz,1H),5.33–5.29(m,1H),4.50-4.46(m,2H),4.27(dd,J＝10.3,9.1Hz,1H),4.15(s,2H),4.07–3.02(m,61H),2.91–2.85(m,2H),2.59-2.55(m,1H),2.30–1.80(m,14H),1.70–0.64(m,65H).ESI-HRMS:m/z calculated for C115H182N12O32S3[M+H]+:2339.21452,Found:2339.21549.
I-14 (m=5, n=6, X=O, Y= Z=6, R'=CY5): 1H NMR (500MHz, CDCl3:MeOD=1:1): δ7.82–7.71(m,6H), 7.27–7.25(m,2H), 6.38(dd,1H, J = 12.4, 12.0 Hz), 6.12-6.04 (m, 2H), 5.74 (d, J = 2.2 Hz, 1H), 5.33-5.29 (m, 1H), 4.50-4.46 (m, 2H), 4.27 (dd ,J=10.3,9.1Hz,1H),4.15(s,2H),4.07–3.02(m,61H), 2.91–2.85(m,2H), 2.59-2.55(m,1H), 2.30–1.80(m ,14H),1.70--0.64(m,65H).ESI-HRMS:m/z calculated for C115H182N12O32S3[M+H]+:2339.21452,Found:2339.21549.
I-15(m＝5,n＝6,X＝O,Y＝
Z＝3,R’＝Biotin):1H NMR(500MHz,CDCl3:MeOD＝1:1):δ5.85(s,1H),5.72(d,J＝1.9Hz,1H),5.31–5.30(m,1H),5.02(s,1H),4.56–4.46(m,4H),4.34-4.26(m,2H),4.17(s,2H),4.04-3.10(m,54H),2.96–2.90(m,3H),2.71(d,J＝12.0Hz,1H),2.59-2.53(m,3H),2.34–2.19(m,3H),2.10(s,3H),2.00–1.96(m,2H),1.91–0.60(s,53H).ESI-HRMS:m/z calculated for C86H145N12O24S2[M+H]+:1793.99361,Found:1793.99429.
I-15 (m=5, n=6, X=O, Y= Z = 3, R'= Biotin): 1H NMR (500MHz, CDCl3: MeOD = 1:1): δ 5.85 (s, 1H), 5.72 (d, J = 1.9 Hz, 1H), 5.31-5.30 (m ,1H),5.02(s,1H),4.56-4.46(m,4H),4.34-4.26(m,2H),4.17(s,2H),4.04-3.10(m,54H),2.96-2.90(m ,3H), 2.71(d,J=12.0Hz,1H), 2.59-2.53(m,3H), 2.34–2.19(m,3H), 2.10(s,3H), 2.00–1.96(m,2H), 1.91–0.60(s,53H).ESI-HRMS: m/z calculated for C86H145N12O24S2[M+H]+:1793.99361,Found:1793.99429.
实验例3、NA酶活抑制实验：Experimental example 3. NA enzyme activity inhibition experiment:
将纯化后得到的NA蛋白(N1、N5等)分别用20/150的PH值8.0的Tris/NaCl稀释5×、25×、125×、625×、3125×、15625×后，在黑色96孔酶标板中分别加入10μL不同浓度的NA溶液和10μL PBS后，在37℃恒温培养箱中孵育30min，每个孔再加入30μL的167μM 4-MUNANA(4-methylumbelliferyl-N-acetylneuraminic acid)荧光底物。用酶标仪测量每分钟的荧光值(excitation wavelengths 355nm、emission wavelengths 460nm)，共测量30min。从结果中选取荧光值在30min内成线性增长且未超过5000RU的NA浓度作为酶活抑制实验的浓度。将抑制剂(I)分别用PBS按照10倍梯度稀释成合适浓度范围的溶液，然后在黑色96孔板分别加入10μL不同浓度的抑制剂溶液、10μL合适浓度的NA溶液。另外，在其他孔加入10μL的PBS和10μL的合适浓度的NA溶液作为阳性对照，加入20μL的PBS作为阴性对照。将黑色酶标板在37℃恒温培养箱中孵育30min后，每个孔再加入30μL的167μM 4-MUNANA荧光底物后，立即用酶标仪测量30min内荧光值。每个实验重复3次，所得到结果用GraphPad Prism(version 5.0)分析作图后得到不同抑制剂对NA的IC
50值。
The purified NA proteins (N1, N5, etc.) were diluted 5×, 25×, 125×, 625×, 3125×, 15625× with 20/150 Tris/NaCl with a pH value of 8.0, and then placed in a black 96-well After adding 10μL of NA solution of different concentrations and 10μL of PBS to the microtiter plate, incubate in a constant temperature incubator at 37°C for 30min, and then add 30μL of 167μM 4-MUNANA (4-methylumbelliferyl-N-acetylneuraminic acid) fluorescent bottom to each well Things. Measure the fluorescence value per minute (excitation wavelengths 355nm, emission wavelengths 460nm) with a microplate reader for a total of 30 minutes. From the results, select the concentration of NA whose fluorescence value increases linearly within 30 min and does not exceed 5000 RU as the concentration of enzyme activity inhibition experiment. The inhibitor (I) was diluted with PBS in a 10-fold gradient to form a solution with a suitable concentration range, and then 10 μL of inhibitor solution of different concentration and 10 μL of NA solution of suitable concentration were added to the black 96-well plate. In addition, 10 μL of PBS and 10 μL of NA solution of appropriate concentration were added to other wells as a positive control, and 20 μL of PBS was added as a negative control. After incubating the black ELISA plate in a constant temperature incubator at 37°C for 30 min, add 30 μL of 167 μM 4-MUNANA fluorescent substrate to each well, and immediately measure the fluorescence value within 30 min with a plate reader. Each experiment was repeated 3 times, and the results obtained were analyzed and graphed with GraphPad Prism (version 5.0) to obtain the IC 50 values of different inhibitors against NA.
上表中的I-12、I-13、I-14、I-15化合物的Y基团
中的R为
各自的R基团
中的Z和R`分别如上表所列示。
The Y group of compounds I-12, I-13, I-14, I-15 in the above table R in is Respective R groups Z and R` in are as listed in the above table respectively.
由上表可以看出，本发明上述各实施例提供的15种流感病毒NA酶抑制剂对各种流感病毒的NA酶均具有抑制效果，特别是对耐扎那米韦的流感病毒(N2(H3N2,E119V))的NA有着更为突出的抑制效果。It can be seen from the above table that the 15 influenza virus NAase inhibitors provided in the above embodiments of the present invention have inhibitory effects on the NA enzymes of various influenza viruses, especially the zanamivir-resistant influenza virus (N2( H3N2, E119V)) NA has a more prominent inhibitory effect.
实验例4、细胞实验Experimental example 4. Cell experiment
将通过鸡胚繁殖得到的流感病毒(包括H1N1，H3N2，H3N2(E119V突变株)，H5N1等)按照10倍梯度用DMEM稀释成不同浓度的病毒溶液。在96孔细胞培养板中接种MDCK细胞，20h后(待细胞长满培养板底部) 后，吸除含有血清双抗的DMEM培养基，用灭菌后的PBS溶液冲洗2遍后，加入事前稀释好的病毒溶液100μL。然后将96孔细胞培养板放入37℃含有5％CO
2的细胞培养箱中培养48h。每个病毒浓度重复4次。倒置显微镜观察细胞状态，并对每个孔进行ELISA检测试验，所得结果利用Reed-Muench法计算出流感病毒的TCID
50。
The influenza viruses (including H1N1, H3N2, H3N2 (E119V mutant strain), H5N1, etc.) obtained through chicken embryo reproduction were diluted with DMEM into virus solutions of different concentrations according to a 10-fold gradient. Inoculate MDCK cells in a 96-well cell culture plate, 20h later (after the cells grow to the bottom of the culture plate), aspirate the DMEM medium containing the serum double antibody, rinse with sterilized PBS solution for 2 times, and add the pre-diluted 100μL of good virus solution. Then the 96-well cell culture plate was placed in a 37°C cell culture incubator containing 5% CO 2 for 48 hours. Repeat 4 times for each virus concentration. Observe the cell status with an inverted microscope, and perform an ELISA test for each well. The results obtained are calculated using the Reed-Muench method to calculate the TCID 50 of the influenza virus.
将11mM的抑制剂(I)母液用0.22μm的无菌滤器过滤后，加入DMEM培养基按照10倍梯度稀释成合适浓度范围的抑制剂溶液。另外，在96孔细胞培养板中接种MDCK细胞，20h后待细胞长满培养板底部后，吸除含有血清双抗的DMEM培养基，用灭菌后的PBS溶液冲洗2遍后，加入事前稀释好的100倍TCID
50的病毒溶液100μL。然后将96孔细胞培养板放入37℃含有5％CO
2的细胞培养箱中孵浴1h后，吸除病毒溶液，用灭菌后的PBS溶液冲洗1遍后，加入不同浓度的抑制剂溶液，然后将96孔细胞培养板放入37℃含有5％CO
2的细胞培养箱中培养72h。每个抑制剂浓度重复4次。倒置显微镜观察细胞状态，并对每个孔进行ELISA检测试验，所得结果利用Reed-Muench法计算出不同抑制剂分子针对不同流感病毒的EC
50。
After filtering the 11 mM inhibitor (I) mother liquor with a 0.22 μm sterile filter, add DMEM medium and dilute it to an inhibitor solution with a suitable concentration range according to a 10-fold gradient. In addition, inoculate MDCK cells in a 96-well cell culture plate. After 20 hours, when the cells have grown to the bottom of the culture plate, aspirate the DMEM medium containing the serum double antibody, rinse with sterilized PBS solution for 2 times, and add the pre-diluted solution. 100μL of a good virus solution of 100 times TCID 50. Then put the 96-well cell culture plate in a 37°C cell culture incubator containing 5% CO 2 and incubate for 1 hour, aspirate the virus solution, wash it with sterilized PBS solution once, and add inhibitor solutions of different concentrations Then put the 96-well cell culture plate into a 37°C cell culture incubator containing 5% CO 2 for 72 hours. Repeat 4 times for each inhibitor concentration. Observe the cell status with an inverted microscope, and perform an ELISA test for each well. The results obtained are calculated using the Reed-Muench method to calculate the EC 50 of different inhibitor molecules against different influenza viruses.
由上表可以看出，本发明提供的15种流感病毒NA酶抑制剂在细胞水平同样具有很好的病毒抑制效果，对耐扎那米韦的流感病毒(如上表中的“mutH3N2(E119V)”)也具有更显著的抑制效果，可以用于各种流感病毒感染的治疗。It can be seen from the above table that the 15 influenza virus NAase inhibitors provided by the present invention also have a good virus inhibitory effect at the cellular level. ") It also has a more significant inhibitory effect and can be used for the treatment of various influenza virus infections.
实验例5、动物实验(连续给药保护小鼠实验)Experimental example 5, animal experiment (continuous administration to protect mice experiment)
将30只雌性Balb/c小鼠分成5笼，每笼6只。所有小鼠用5％水合氯醛注射麻醉后，其中4笼每只滴鼻接种10
4PFU的A/Puerto Rico/8/34(H1N1)流感病毒30μL，另外1笼滴鼻接种PBS 30μL。另外，使用10％乙醇水溶液分别配置6mg/mL的I-3(m＝5,n＝6,X＝O,Y＝
)溶液与2mg/mL的达菲溶液。
Thirty female Balb/c mice were divided into 5 cages, 6 in each cage. After all mice were anesthetized with 5% chloral hydrate injection, 4 cages were inoculated intranasally with 10 4 PFU A/Puerto Rico/8/34 (H1N1) influenza virus 30 μL, and the other 1 cage was intranasally inoculated with 30 μL PBS. In addition, a 10% ethanol aqueous solution was used to prepare 6 mg/mL I-3 (m=5, n=6, X=O, Y= ) Solution and 2mg/mL Tamiflu solution.
1天后，1笼小鼠麻醉后滴鼻给药15μL剂量为6mg/kg的I-3，1笼小鼠灌胃给药150μL剂量为60mg/kg的I-3，1笼小鼠灌胃给药150μL剂量为20mg/kg的达菲，1笼小鼠灌胃给药150μL 10％乙醇溶液，最后1笼小鼠灌胃相同体积的10％乙醇溶液，持续给药7天。每天监测小鼠的体重和体温，持续14天。当小鼠体重下降到起初体重的75％时，视为该小鼠死亡。小鼠体重变化曲线和生存率曲线用GraphPad Prism(version 5.0)分析绘制得到。One day later, 1 cage of mice was anaesthetized and administrated 15μL of I-3 at a dose of 6mg/kg intranasally, 1 cage of mice was given by intragastric administration 150μL of I-3 at a dose of 60mg/kg, 1 cage of mice was given by intragastric administration 150 μL of Tamiflu at a dose of 20 mg/kg was administered. One cage of mice was intragastrically administered with 150 μL of 10% ethanol solution, and the last cage of mice was intragastrically administered with the same volume of 10% ethanol solution for 7 days. The body weight and temperature of the mice were monitored daily for 14 days. When the weight of the mouse drops to 75% of the initial weight, the mouse is deemed dead. The mouse body weight change curve and survival rate curve were drawn by GraphPad Prism (version 5.0) analysis.
由图1的结果可以看出，本发明实验例3提供的化合物I-3在小鼠实验中通过滴鼻和口服给药的方式均可百分百保护感染小鼠，因此本发明的化合物具有开发为口服抗流感药物的潜力。It can be seen from the results in Fig. 1 that the compound I-3 provided in Experimental Example 3 of the present invention can protect the infected mice 100% by nasal drip and oral administration in the mouse experiment. Therefore, the compound of the present invention has Develop the potential for oral anti-flu drugs.
本发明其它实施例1-2、4-15提供的化合物I1-2、I4-15均能获得如图1所示的类似I-3口服给药抗流感效果，为节约本发明的篇幅，在此不一一赘述。The compounds I1-2 and I4-15 provided by other embodiments 1-2 and 4-15 of the present invention can all obtain the anti-influenza effect of oral administration similar to that of I-3 as shown in Figure 1. In order to save the space of the present invention, I will not repeat them one by one.
实验例6、本发明流感病毒NA酶抑制剂的水溶性效果验证Experimental Example 6. Verification of the water-soluble effect of the influenza virus NAase inhibitor of the present invention
本发明流感病毒NA酶抑制剂的水溶性效果通过测定脂水分配系数进行验证：The water solubility effect of the influenza virus NAase inhibitor of the present invention is verified by measuring the fat-water partition coefficient:
将正辛醇和二次蒸馏水在室温下用恒温(37±1)℃摇床振荡24h,使其相互饱和。静置过夜分层后，两相分离，保存备用。精密称取适量待测化合物于10mL容量瓶中，用水饱和的正辛醇溶解，超声振荡30min，定容，得到浓度为1mmol/L的母液。分别用移液枪精密量取一定量总溶液于10mL容量瓶中，用水饱和的正辛醇稀释并定容为10～100μmol/L系列浓度，浓度增加梯度为10μmol/L，分别在200～400nm波长范围内扫描，取最大吸收峰处吸光度绘制化合物的标准回归方程。The n-octanol and double distilled water were shaken with a constant temperature (37±1)℃ shaker for 24h at room temperature to make them saturated with each other. After standing overnight for layering, the two phases are separated and stored for later use. Accurately weigh an appropriate amount of the compound to be tested in a 10 mL volumetric flask, dissolve it in n-octanol saturated with water, oscillate it ultrasonically for 30 min, and make it constant to obtain a mother liquor with a concentration of 1 mmol/L. Use a pipette to accurately measure a certain amount of the total solution in a 10mL volumetric flask, dilute with water-saturated n-octanol and dilute to a series concentration of 10-100μmol/L, with a concentration increase gradient of 10μmol/L, respectively at 200-400nm Scan within the wavelength range and draw the standard regression equation of the compound by taking the absorbance at the maximum absorption peak.
分别用移液枪精密量取500μL和1000μL待测化合物母液于250mL容量瓶中，以水饱和的正辛醇溶解，超声振荡30min，定容，分别取3份5mL的该溶液与5mL正辛醇饱和的水混合后置于恒温摇床中，室温下振荡24h，然后离心使两相充分分离，紫外可见分光光度计室温下分别测定化合物在有机相和水相的紫外吸收光谱，根据标准回归方程即可计算有机相、水相中待测化合物的浓度，然后再按公式log P
o/w＝log(c
o/c
w)计算化合物的脂水分配系数，每种浓度测定3次，取平均值计得该化合物的脂水分配系数。
Use a pipette to accurately measure 500 μL and 1000 μL of the mother solution of the compound to be tested in a 250 mL volumetric flask, dissolve it with water-saturated n-octanol, sonicate for 30 min, and dilute to volume. Take 3 portions of 5 mL of this solution and 5 mL of n-octanol, respectively. Saturated water was mixed and placed in a constant temperature shaker, oscillated at room temperature for 24 hours, and then centrifuged to fully separate the two phases. UV-Vis spectrophotometer was used to determine the UV absorption spectra of the compound in the organic phase and the water phase at room temperature, according to the standard regression equation You can calculate the concentration of the test compound in the organic phase and the water phase, and then calculate the lipid-water partition coefficient of the compound according to the formula log P o/w ＝log(c o /c w ), measure each concentration 3 times and take the average The value is calculated to obtain the fat-water partition coefficient of the compound.
脂水分配系数(log P)是衡量药物能否透过由脂质双分子层构成的生物膜的主要指标，关系到药物在人体的吸收、分配、代谢、排泄等药代动力学过程，log P数值越小，说明化合物的疏水性越强，在体内越容易被代谢，具有较高的清除率(例如扎那米韦)，log P数值越大说明化合物的亲脂性越强。通过上述数据说明，本发明所设计化合物的脂水分配系数普遍较扎那米韦较有显著提高，能够达到改善扎那米韦水溶性的目的，预期能够显著改善化合物的药代动力学性质，具有很好的成药潜力。The lipid-water partition coefficient (log P) is the main indicator to measure whether a drug can penetrate a biological membrane composed of a lipid bilayer, and it is related to the pharmacokinetic process of drug absorption, distribution, metabolism, and excretion in the human body. Log The smaller the P value, the stronger the hydrophobicity of the compound, the easier it is to be metabolized in the body, and the higher the clearance rate (such as zanamivir). The larger the log P value, the stronger the lipophilicity of the compound. The above data shows that the lipid-water partition coefficient of the compounds designed in the present invention is generally significantly higher than that of zanamivir, which can achieve the purpose of improving the water solubility of zanamivir, and is expected to significantly improve the pharmacokinetic properties of the compound. It has a good potential as a medicine.
以下为本发明涉及的其他中间体化合物数据：The following is the data of other intermediate compounds involved in the present invention:
IV-1(n＝6,X＝O,Y
2＝NH
2):
1H NMR(500MHz,CDCl
3):δ5.39–5.37(m,1H),3.42(td,J＝6.7,2.4Hz,2H),3.17(tt,J＝11.0,4.0Hz,1H),2.76(t,J＝7.1Hz,2H),2.35(ddd,J＝13.0,4.4,2.0Hz,1H),2.20–2.15(m,1H),2.03–1.92(m,2H),1.90–1.77(m,3H),1.67–0.79(m,41H),0.68(s,3H).ESI:m/z calculated for C
33H
60NO[M+H]
+:486.5,found:486.4.
IV-1(n=6,X=O,Y 2 ＝NH 2 ): 1 H NMR (500MHz, CDCl 3 ): δ5.39–5.37(m,1H),3.42(td,J=6.7,2.4Hz , 2H), 3.17 (tt, J = 11.0, 4.0 Hz, 1H), 2.76 (t, J = 7.1 Hz, 2H), 2.35 (ddd, J = 13.0, 4.4, 2.0 Hz, 1H), 2.20-2.15 ( m,1H),2.03–1.92(m,2H),1.90–1.77(m,3H),1.67–0.79(m,41H),0.68(s,3H).ESI: m/z calculated for C 33 H 60 NO[M+H] + :486.5,found:486.4.
IV-2(n＝6,X＝OCOO,Y
2＝NH
2):
1H NMR(500MHz,CDCl
3):δ5.44–5.41(m,1H),4.44(ddd,J＝16.2,10.8,5.3Hz,1H),4.11(t,J＝6.6Hz,2H),2.67(t,J＝6.9Hz,2H),2.44–2.31(m,2H),2.06–1.79(m,5H),1.77–0.80(m,41H),0.68(s,3H).ESI:m/z calculated for C
34H
60NO
3[M+H]
+:530.4,found:530.5.
IV-2 (n=6, X=OCOO, Y 2 ＝NH 2 ): 1 H NMR (500MHz, CDCl 3 ): δ5.44-5.41 (m, 1H), 4.44 (ddd, J=16.2, 10.8, 5.3Hz, 1H), 4.11 (t, J = 6.6 Hz, 2H), 2.67 (t, J = 6.9 Hz, 2H), 2.44–2.31 (m, 2H), 2.06–1.79 (m, 5H), 1.77– 0.80(m,41H),0.68(s,3H).ESI:m/z calculated for C 34 H 60 NO 3 [M+H] + :530.4,found:530.5.
IV-3(n＝12,X＝O,Y
2＝NH
2):δ5.32–5.31(m,1H),3.46(td,J＝6.4,2.0Hz,2H),3.17(tt,J＝11.2,4.4Hz,1H),2.76(t,J＝7.0Hz,2H),2.34(ddd,J＝13.0,4.4,2.0Hz,1H),2.27–2.17(m,1H),2.04–1.96(m,2H),1.90–1.76(m,3H),1.60–0.70(m,53H),0.65(s,3H).ESI:m/z calculated for C
39H
72NO[M+H]
+:570.6,found:570.5.
IV-3 (n=12, X=O, Y 2 ＝NH 2 ): δ5.32–5.31(m,1H), 3.46(td,J=6.4,2.0Hz,2H), 3.17(tt,J= 11.2,4.4Hz,1H),2.76(t,J=7.0Hz,2H), 2.34(ddd,J=13.0,4.4,2.0Hz,1H), 2.27–2.17(m,1H),2.04–1.96(m ,2H),1.90–1.76(m,3H),1.60–0.70(m,53H),0.65(s,3H).ESI:m/z calculated for C 39 H 72 NO[M+H] + :570.6, found: 570.5.
IV-4(n＝3,X＝O,Y
2＝NH
2):
1H NMR(500MHz,CDCl
3):δ5.49–5.47(m,1H),4.46(ddd,J＝16.0,10.2,5.0Hz,1H),4.14(t,J＝6.2Hz,2H),2.66(t,J＝6.7Hz,2H),2.42–2.29(m,2H),2.14–1.79(m,5H),1.75–0.74(m,35H),0.64(s,3H).ESI:m/z calculated for C
30H
54NO[M+H]
+:444.4,found:444.5.
IV-4 (n=3, X=O, Y 2 ＝NH 2 ): 1 H NMR (500MHz, CDCl 3 ): δ5.49–5.47 (m, 1H), 4.46 (ddd, J=16.0, 10.2, 5.0Hz, 1H), 4.14 (t, J = 6.2 Hz, 2H), 2.66 (t, J = 6.7 Hz, 2H), 2.42–2.29 (m, 2H), 2.14–1.79 (m, 5H), 1.75– 0.74(m,35H),0.64(s,3H).ESI:m/z calculated for C 30 H 54 NO[M+H] + :444.4,found:444.5.
IV-5(n＝6,X＝O,Y
2＝COOH):
1H NMR(500MHz,CDCl
3):δ5.35–5.34(m,1H),3.45(td,J＝6.6,2.3Hz,2H),3.12 (tt,J＝11.3,4.4Hz,1H),2.42-2.35(m,3H),2.22–2.15(m,1H),2.05–1.94(m,2H),1.92–1.77(m,3H),1.64–0.77(m,41H),0.68(s,3H).ESI:m/z calculated for C
33H
57O
3[M+H]
+:501.4,found:501.4.
IV-5(n=6,X=O,Y 2 ＝COOH): 1 H NMR (500MHz, CDCl 3 ): δ5.35-5.34(m,1H), 3.45(td,J=6.6,2.3Hz, 2H), 3.12 (tt,J=11.3,4.4Hz,1H),2.42-2.35(m,3H),2.22-2.15(m,1H),2.05-1.94(m,2H),1.92-1.77(m, 3H),1.64-0.77(m,41H),0.68(s,3H).ESI:m/z calculated for C 33 H 57 O 3 [M+H] + :501.4,found:501.4.
VI-1(m＝2,n＝6,X＝O,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ6.98(br s,1H),5.39–5.37(m,1H),3.98(s,2H),3.89-3.72(m,6H),3.42(td,J＝6.7,2.4Hz,2H),3.22-3.19(m,3H),2.76(t,J＝7.1Hz,2H),2.35(ddd,J＝13.0,4.4,2.0Hz,1H),2.20–2.15(m,1H),2.03–1.92(m,2H),1.90–1.77(m,3H),1.67–0.79(m,41H),0.68(s,3H).ESI:m/z calculated for C
38H
69N
2O
4[M+H]
+:617.5,found:617.5.
VI-1 (m = 2, n = 6, X = O, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ 6.98 (br s, 1H), 5.39-5.37 (m, 1H), 3.98 (s, 2H), 3.89-3.72 (m, 6H), 3.42 (td ,J=6.7,2.4Hz,2H),3.22-3.19(m,3H),2.76(t,J=7.1Hz,2H), 2.35(ddd,J=13.0,4.4,2.0Hz,1H), 2.20– 2.15(m,1H),2.03-1.92(m,2H),1.90-1.77(m,3H),1.67-0.79(m,41H),0.68(s,3H).ESI: m/z calculated for C 38 H 69 N 2 O 4 [M+H] + :617.5,found:617.5.
VI-2(m＝2,n＝6,X＝OCOO,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ6.96(br s,1H),5.44–5.41(m,1H),4.44(ddd,J＝16.2,10.8,5.3Hz,1H),4.11(t,J＝6.6Hz,2H),3.98(s,2H),3.89-3.72(m,6H),3.22-3.19(m,2H),2.67(t,J＝6.9Hz,2H),2.44–2.31(m,2H),2.06–1.79(m,5H),1.77–0.80(m,41H),0.68(s,3H).ESI:m/z calculated for C
40H
71N
2O
6[M+H]
+:675.5,found:675.5.
VI-2 (m=2, n=6, X=OCOO, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ 6.96 (br s, 1H), 5.44-5.41 (m, 1H), 4.44 (ddd, J = 16.2, 10.8, 5.3 Hz, 1H), 4.11 (t ,J=6.6Hz,2H),3.98(s,2H),3.89-3.72(m,6H),3.22-3.19(m,2H),2.67(t,J=6.9Hz,2H),2.44-2.31( m,2H),2.06–1.79(m,5H),1.77–0.80(m,41H),0.68(s,3H).ESI:m/z calculated for C 40 H 71 N 2 O 6 [M+H] + :675.5,found:675.5.
VI-3(m＝5,n＝6,X＝O,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ6.97(br s,1H),5.38–5.36(m,1H),3.92(s,2H),3.79-3.42(m,20H),3.21-3.17(m,3H),2.78(t,J＝7.0Hz,2H),2.35-2.31(m,1H),2.22–2.17(m,1H),2.03–1.92(m,2H),1.90–1.72(m,3H),1.64–0.79(m,44H).ESI:m/z calculated for C
45H
83N
2O
7[M+H]
+:763.6,found:763.6.
VI-3 (m = 5, n = 6, X = O, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ 6.97 (br s, 1H), 5.38-5.36 (m, 1H), 3.92 (s, 2H), 3.79-3.42 (m, 20H), 3.21-3.17 (m,3H),2.78(t,J=7.0Hz,2H),2.35-2.31(m,1H),2.22-2.17(m,1H),2.03-1.92(m,2H),1.90-1.72(m ,3H),1.64-0.79(m,44H).ESI:m/z calculated for C 45 H 83 N 2 O 7 [M+H] + :763.6,found:763.6.
VI-4(m＝5,n＝6,X＝OCOO,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ6.98(br s,1H),5.44–5.42(m,1H),4.46(ddd,J＝16.0,10.6,5.0Hz,1H),4.13(t,J＝6.8Hz,2H),3.88(s,2H),3.74-3.42(m,18H),3.22-3.19(m,2H),2.67(t,J＝6.8Hz,2H),2.44–2.36(m,2H),2.02–1.81(m,5H),1.76–0.80(m,41H),0.68(s,3H).ESI:m/z calculated for C
46H
83N
2O
9[M+H]
+:807.6,found:807.6.
VI-4 (m=5, n=6, X=OCOO, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ 6.98 (br s, 1H), 5.44-5.42 (m, 1H), 4.46 (ddd, J = 16.0, 10.6, 5.0 Hz, 1H), 4.13 (t ,J=6.8Hz,2H),3.88(s,2H),3.74-3.42(m,18H),3.22-3.19(m,2H),2.67(t,J=6.8Hz,2H),2.44-2.36( m,2H),2.02–1.81(m,5H),1.76–0.80(m,41H),0.68(s,3H).ESI:m/z calculated for C 46 H 83 N 2 O 9 [M+H] + :807.6,found:807.6.
VI-5(m＝11,n＝6,X＝O,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ6.96(br s,1H),5.38–5.37(m,1H),3.96(s,2H),3.72-3.42(m,44H),3.22-3.17(m,3H),2.74(t,J＝7.2Hz,2H),2.31-2.26(m,1H),2.19–2.15(m,1H),2.03–1.96(m,2H),1.91–1.77(m,3H),1.67–0.72(m,41H),0.64(s,3H).ESI:m/z calculated for C
57H
103N
2O
13[M+H]
+:1023.7,found:1023.8.
VI-5 (m = 11, n = 6, X = O, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ 6.96 (br s, 1H), 5.38-5.37 (m, 1H), 3.96 (s, 2H), 3.72-3.42 (m, 44H), 3.22-3.17 (m,3H),2.74(t,J=7.2Hz,2H),2.31-2.26(m,1H),2.19-2.15(m,1H),2.03-1.96(m,2H),1.91-1.77(m ,3H),1.67–0.72(m,41H),0.64(s,3H).ESI:m/z calculated for C 57 H 103 N 2 O 13 [M+H] + :1023.7,found:1023.8.
VI-6(m＝11,n＝6,X＝OCOO,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ7.01(br s,1H),5.44–5.42(m,1H),4.44-4.41(m,1H),4.15(t,J＝6.8Hz,2H),3.89(s,2H),3.74-3.22(m,44H),2.64(t,J＝6.7Hz,2H),2.44–2.31(m,2H),2.04–1.79(m,5H),1.75–0.80(m,41H),0.66(s,3H).ESI:m/z calculated for C
58H
106N
2O
15[M+H]
+:1070.8,found:1070.7.
VI-6 (m = 11, n = 6, X = OCOO, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ7.01 (br s, 1H), 5.44-5.42 (m, 1H), 4.44-4.41 (m, 1H), 4.15 (t, J = 6.8 Hz, 2H ), 3.89(s, 2H), 3.74-3.22(m, 44H), 2.64(t, J = 6.7Hz, 2H), 2.44–2.31(m, 2H), 2.04–1.79(m, 5H), 1.75– 0.80(m,41H),0.66(s,3H).ESI:m/z calculated for C 58 H 106 N 2 O 15 [M+H] + :1070.8,found:1070.7.
VI-7(m＝5,n＝12,X＝O,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ7.01(br s,1H),5.35–5.33(m,1H),3.88(s,2H),3.79-3.62(m,18H),3.42-3.40(m,2H),3.22-3.16(m,3H),2.79(t,J＝7.2Hz,2H),2.34-2.30(m,1H),2.26–2.17(m,1H),2.03–1.96(m,2H),1.92–1.76(m,3H),1.62–0.70(m,56H).ESI:m/z calculated for C
51H
95N
2O
7[M+H]
+:847.7,found:847.7.
VI-7 (m = 5, n = 12, X = O, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ7.01 (br s, 1H), 5.35-5.33 (m, 1H), 3.88 (s, 2H), 3.79-3.62 (m, 18H), 3.42-3.40 (m,2H),3.22-3.16(m,3H),2.79(t,J=7.2Hz,2H),2.34-2.30(m,1H),2.26-2.17(m,1H),2.03-1.96(m ,2H),1.92–1.76(m,3H),1.62–0.70(m,56H).ESI:m/z calculated for C 51 H 95 N 2 O 7 [M+H] + :847.7,found:847.7.
VI-8(m＝5,n＝3,X＝O,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ6.94(br s,1H),5.37–5.35(m,1H),3.94(s,2H),3.76-3.42(m,20H),3.20-3.17(m,3H),2.75(t,J＝7.2Hz,2H),2.36-2.31(m,1H),2.22–2.16(m,1H),2.02–1.95(m,2H),1.90–1.74(m,3H),1.66–0.75(m,38H).ESI:m/z calculated for C
42H
77N
2O
7[M+H]
+:721.6,found:721.5.
VI-8 (m = 5, n = 3, X = O, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ 6.94 (br s, 1H), 5.37-5.35 (m, 1H), 3.94 (s, 2H), 3.76-3.42 (m, 20H), 3.20-3.17 (m,3H),2.75(t,J=7.2Hz,2H),2.36-2.31(m,1H),2.22–2.16(m,1H),2.02–1.95(m,2H),1.90–1.74(m ,3H),1.66–0.75(m,38H).ESI:m/z calculated for C 42 H 77 N 2 O 7 [M+H] + :721.6,found:721.5.
VI-9(m＝5,n＝6,X＝O,Y
3＝
):
1H NMR(500MHz,CDCl
3):δ7.09(br s,1H),5.36–5.34(m,1H),3.89-3.72(m,18H),3.44-3.12(m,6H),2.49-2.35(m,3H),2.20–2.12(m,1H),2.02–1.94(m,2H),1.92–1.77(m,3H),1.67–0.72(m,41H),0.64(s,3H).ESI:m/z calculated for C
45H
83N
2O
7[M+H]
+:763.6,found:763.6.
VI-9 (m = 5, n = 6, X = O, Y 3 = ): 1 H NMR (500MHz, CDCl 3 ): δ7.09 (br s, 1H), 5.36-5.34 (m, 1H), 3.89-3.72 (m, 18H), 3.44-3.12 (m, 6H), 2.49 -2.35(m,3H), 2.20–2.12(m,1H), 2.02–1.94(m,2H), 1.92–1.77(m,3H), 1.67–0.72(m,41H), 0.64(s,3H) .ESI:m/z calculated for C 45 H 83 N 2 O 7 [M+H] + :763.6,found:763.6.
XII-1(n＝6,X＝O,Y
5＝H):
1H NMR(500MHz,CDCl
3):δ6.29(br s,1H),5.39–5.32(m,2H),3.46-3.22(m,6H),3.10(tt,J＝11.0,4.4Hz,1H),2.31(ddd,J＝13.0,4.4,2.0Hz,1H),2.22–2.15(m,1H),2.02–1.94(m,2H),1.90–1.77(m,3H), 1.64–0.64(m,53H).ESI:m/z calculated for C
40H
71N
2O
4[M+H]
+:643.5,found:643.6.
XII-1(n=6,X=O,Y 5 ＝H): 1 H NMR (500MHz, CDCl 3 ): δ6.29(br s,1H),5.39–5.32(m,2H),3.46-3.22 (m, 6H), 3.10 (tt, J = 11.0, 4.4 Hz, 1H), 2.31 (ddd, J = 13.0, 4.4, 2.0 Hz, 1H), 2.22-2.15 (m, 1H), 2.02-1.94 (m ,2H),1.90–1.77(m,3H), 1.64–0.64(m,53H).ESI:m/z calculated for C 40 H 71 N 2 O 4 [M+H] + :643.5,found:643.6.
XII-2(n＝6,X＝OCOO,Y
5＝H):
1H NMR(500MHz,CDCl
3):δ6.36(br s,1H),5.49–5.39(m,2H),4.41(ddd,J＝16.0,10.4,5.4Hz,1H),4.17(t,J＝6.8Hz,2H),3.44-3.29(m,4H),2.41–2.30(m,2H),2.04–1.78(m,5H),1.73–0.68(m,53H).ESI:m/z calculated for C
41H
71N
2O
6[M+H]
+:687.5,found:687.5.
XII-2(n=6,X=OCOO,Y 5 ＝H): 1 H NMR (500MHz, CDCl 3 ): δ6.36(br s,1H), 5.49–5.39(m,2H), 4.41(ddd ,J=16.0,10.4,5.4Hz,1H),4.17(t,J=6.8Hz,2H),3.44-3.29(m,4H),2.41–2.30(m,2H),2.04–1.78(m,5H ),1.73-0.68(m,53H).ESI:m/z calculated for C 41 H 71 N 2 O 6 [M+H] + :687.5,found:687.5.
XII-3(n＝6,X＝O,Y
5＝R,Z＝6,R’＝Biotin):
1H NMR(500MHz,MeOD):δ5.86(s,1H),5.35–5.34(m,1H),5.06(s,1H),4.58–4.51(m,2H),4.33-4.30(m,1H),3.84-3.80(m,3H),3.66–3.62(m,24H),3.53-3.40(m,8H),3.46-3.10(m,7H),2.94–2.90(m,3H),2.73(d,J＝12.6Hz,1H),2.55-2.52(m,3H),2.31(ddd,J＝13.0,4.4,2.0Hz,1H),2.24–2.15(m,2H),2.02–1.94(m,2H),1.90–1.77(m,3H),1.64–0.64(s,59H).ESI:m/z calculated for C
72H
124N
7O
15S
2[M+H]
+:1390.9,found:1390.9.
XII-3 (n = 6, X = O, Y 5 = R, Z = 6, R '= Biotin): 1 H NMR (500MHz, MeOD): δ5.86 (s, 1H), 5.35-5.34 (m ,1H),5.06(s,1H),4.58-4.51(m,2H),4.33-4.30(m,1H),3.84-3.80(m,3H),3.66-3.62(m,24H),3.53-3.40 (m, 8H), 3.46-3.10 (m, 7H), 2.94-2.90 (m, 3H), 2.73 (d, J = 12.6 Hz, 1H), 2.55-2.52 (m, 3H), 2.31 (ddd, J =13.0,4.4,2.0Hz,1H),2.24–2.15(m,2H),2.02–1.94(m,2H),1.90–1.77(m,3H),1.64–0.64(s,59H).ESI:m /z calculated for C 72 H 124 N 7 O 15 S 2 [M+H] + :1390.9,found:1390.9.
XII-4(n＝6,X＝O,Y
5＝R,Z＝6,R’＝CY3):
1H NMR(500MHz,MeOD):δ8.30(t,1H,J＝13.4Hz),7.70–7.62(m,4H),7.18–7.15(m,2H),6.20–6.15(m,2H),5.39–5.38(m,1H),4.56–4.52(m,1H),3.94–3.09(m,35H),2.94–2.85(m,4H),2.50-2.46(m,1H),2.31–1.77(m,11H),1.64–0.64(m,74H).ESI:m/z calculated for C
93H
147N
7O
20S
3[M+H]
+:1778.0,found:1778.0.
XII-4 (n = 6, X = O, Y 5 = R, Z = 6, R '= CY3): 1 H NMR (500MHz, MeOD): δ8.30 (t, 1H, J = 13.4Hz), 7.70–7.62(m,4H), 7.18–7.15(m,2H), 6.20–6.15(m,2H), 5.39–5.38(m,1H), 4.56–4.52(m,1H), 3.94–3.09(m ,35H),2.94-2.85(m,4H),2.50-2.46(m,1H),2.31-1.77(m,11H),1.64-0.64(m,74H).ESI:m/z calculated for C 93 H 147 N 7 O 20 S 3 [M+H] + :1778.0,found:1778.0.
XII-5(n＝6,X＝O,Y
5＝R,Z＝6,R’＝CY5):
1H NMR(500MHz,MeOD):δ7.88–7.72(m,6H),7.27–7.25(m,2H),6.32(dd,1H,J＝12.4,12.0Hz),6.10–6.02(m,2H),5.31–5.26(m,1H),4.07–3.02(m,37H),2.92–2.86(m,2H),2.55-2.52(m,1H),2.30–1.82(m,11H),1.72–0.64(m,74H).ESI:m/z calculated for C
95H
149N
7O
20S
3[M+H]
+:1804.0,found:1804.0.
XII-5 (n = 6, X = O, Y 5 = R, Z = 6, R '= CY5): 1 H NMR (500MHz, MeOD): δ7.88-7.72 (m, 6H), 7.27-7.25 (m,2H),6.32(dd,1H,J=12.4,12.0Hz),6.10–6.02(m,2H),5.31–5.26(m,1H),4.07–3.02(m,37H),2.92–2.86 (m,2H),2.55-2.52(m,1H),2.30-1.82(m,11H),1.72-0.64(m,74H).ESI:m/z calculated for C 95 H 149 N 7 O 20 S 3 [M+H] + :1804.0,found:1804.0.
XII-6(n＝6,X＝O,Y
5＝R,Z＝3,R’＝Biotin):
1H NMR(500MHz,MeOD):δ5.89(s,1H),5.34–5.32(m,1H),5.02(s,1H),4.56–4.50(m,2H),4.33-4.28(m,1H),3.83-3.77(m,3H),3.66–3.62(m,12H),3.53-3.10(m,15H),2.97–2.92(m,3H),2.71(d,J＝12.0Hz,1H),2.58-2.54(m,3H),2.31–2.19(m,3H),2.00–1.93(m,2H),1.90–0.64(s,62H).ESI:m/z calculated for C
66H
112N
7O
12S
2[M+H]
+:1258.8,found:1258.8.
XII-6 (n = 6, X = O, Y 5 = R, Z = 3, R '= Biotin): 1 H NMR (500MHz, MeOD): δ5.89 (s, 1H), 5.34-5.32 (m ,1H),5.02(s,1H),4.56-4.50(m,2H),4.33-4.28(m,1H),3.83-3.77(m,3H),3.66-3.62(m,12H),3.53-3.10 (m,15H), 2.97–2.92(m,3H), 2.71(d, J=12.0Hz, 1H), 2.58-2.54(m, 3H), 2.31–2.19(m, 3H), 2.00–1.93(m ,2H),1.90–0.64(s,62H).ESI:m/z calculated for C 66 H 112 N 7 O 12 S 2 [M+H] + :1258.8,found:1258.8.
XIII-1(m＝5,n＝6,X＝O,Y
5＝H):
1H NMR(500MHz,CDCl
3):δ6.75(br s,1H),6.42(br s,1H),5.39–5.37(m,1H),4.12(s,2H),3.76-3.22(m,27H),3.12(tt,J＝11.2,4.5Hz,1H),2.30(ddd,J＝13.2,4.7,2.1Hz,1H),2.20–2.15(m,1H),2.00–1.93(m,2H),1.90–1.79(m,3H),1.64–0.65(m,44H).ESI:m/z calculated for C
47H
84N
5O
8[M+H]
+:846.6,found:846.6.
XIII-1(m=5,n=6,X=O,Y 5 =H): 1 H NMR(500MHz,CDCl 3 ):δ6.75(br s,1H),6.42(br s,1H), 5.39–5.37(m,1H),4.12(s,2H),3.76-3.22(m,27H),3.12(tt,J=11.2,4.5Hz,1H),2.30(ddd,J=13.2,4.7,2.1 Hz, 1H), 2.20-2.15 (m, 1H), 2.00-1.93 (m, 2H), 1.90-1.79 (m, 3H), 1.64-0.65 (m, 44H). ESI: m/z calculated for C 47 H 84 N 5 O 8 [M+H] + :846.6,found:846.6.
XIII-2(m＝5,n＝6,X＝OCOO,Y
5＝H):
1H NMR(500MHz,CDCl
3):δ6.68(br s,1H),6.46(br s,1H),5.41–5.39(m,1H),4.40(ddd,J＝15.8,10.2,5.3Hz,1H),4.19-4.15(m,3H),3.76-3.29(m,24H),2.40–2.31(m,2H),2.02–0.68(m,49H).ESI:m/z calculated for C
48H
84N
5O
10[M+H]
+:890.6,found:890.6.
XIII-2 (m=5, n=6, X=OCOO, Y 5 ＝H): 1 H NMR (500MHz, CDCl 3 ): δ6.68 (br s, 1H), 6.46 (br s, 1H), 5.41–5.39(m,1H), 4.40(ddd,J=15.8,10.2,5.3Hz,1H), 4.19-4.15(m,3H),3.76-3.29(m,24H), 2.40–2.31(m,2H) ),2.02–0.68(m,49H).ESI:m/z calculated for C 48 H 84 N 5 O 10 [M+H] + :890.6,found:890.6.
XIII-3(m＝5,n＝6,X＝O,Y
5＝R,Z＝6,R’＝Biotin):
1H NMR(500MHz,MeOD):δ5.89(s,1H),5.34–5.32(m,1H),5.04(s,1H),4.56–4.50(m,2H),4.34-4.32(m,1H),4.18(s,2H),3.84-3.10(m,62H),2.92–2.87(m,3H),2.74(d,J＝12.4Hz,1H),2.58-2.53(m,3H),2.30(ddd,J＝13.1,4.4,2.2Hz,1H),2.22–2.13(m,2H),2.02–1.94(m,2H),1.91–1.78(m,3H),1.64–0.61(s,50H).ESI:m/z calculated for C
79H
137N
10O
19S
2[M+H]
+:1594.0,found:1593.9.
XIII-3 (m = 5, n = 6, X = O, Y 5 = R, Z = 6, R'= Biotin): 1 H NMR (500MHz, MeOD): δ 5.89 (s, 1H), 5.34 --5.32(m,1H),5.04(s,1H),4.56--4.50(m,2H),4.34-4.32(m,1H),4.18(s,2H),3.84-3.10(m,62H),2.92 –2.87(m,3H),2.74(d,J＝12.4Hz,1H),2.58-2.53(m,3H), 2.30(ddd,J＝13.1,4.4,2.2Hz,1H),2.22-2.13(m ,2H),2.02–1.94(m,2H),1.91–1.78(m,3H),1.64–0.61(s,50H).ESI:m/z calculated for C 79 H 137 N 10 O 19 S 2 (M +H] + :1594.0,found:1593.9.
XIII-4(m＝5,n＝6,X＝O,Y
5＝R,Z＝6,R’＝CY3):
1H NMR(500MHz,MeOD):δ8.31(t,1H,J＝13.0Hz),7.70–7.64(m,4H),7.16–7.13(m,2H),6.21–6.17(m,2H),5.39–5.38(m,1H),4.55–4.52(m,1H),4.15(s,2H),3.94–3.09(m,55H),2.92–2.85(m,4H),2.52-2.48(m,1H),2.30–1.77(m,11H),1.67–0.62(m,65H).ESI:m/z calculated for C
100H
160N
10O
24S
3[M+H]
+:1981.1,found:1981.1.
XIII-4 (m = 5, n = 6, X = O, Y 5 = R, Z = 6, R'= CY3): 1 H NMR (500MHz, MeOD): δ8.31 (t, 1H, J = 13.0Hz), 7.70--7.64 (m, 4H), 7.16--7.13 (m, 2H), 6.21--6.17 (m, 2H), 5.39--5.38 (m, 1H), 4.55--4.52 (m, 1H), 4.15 (s,2H),3.94-3.09(m,55H),2.92-2.85(m,4H),2.52-2.48(m,1H),2.30-1.77(m,11H),1.67-0.62(m,65H) .ESI:m/z calculated for C 100 H 160 N 10 O 24 S 3 [M+H] + :1981.1,found:1981.1.
XIII-5(m＝5,n＝6,X＝O,Y
5＝R,Z＝6,R’＝CY5):
1H NMR(500MHz,MeOD):δ7.86–7.70(m,6H),7.27–7.25(m,2H),6.39(dd,1H,J＝12.4,12.0Hz),6.14–6.06(m,2H),5.30–5.28(m,1H),4.19(s,2H),4.07–3.02(m,57H),2.90–2.85(m,2H),2.57-2.53(m,1H),2.30–1.80(m,11H),1.70–0.61(m,65H).ESI:m/z calculated for C
102H
162N
10O
24S
3[M+H]
+:2007.1,found:2007.1.
XIII-5 (m = 5, n = 6, X = O, Y 5 = R, Z = 6, R '= CY5): 1 H NMR (500MHz, MeOD): δ7.86-7.70 (m, 6H) ,7.27–7.25(m,2H),6.39(dd,1H,J=12.4,12.0Hz),6.14–6.06(m,2H),5.30–5.28(m,1H),4.19(s,2H),4.07 --3.02(m,57H),2.90–2.85(m,2H), 2.57-2.53(m,1H), 2.30–1.80(m,11H),1.70–0.61(m,65H).ESI: m/z calculated for C 102 H 162 N 10 O 24 S 3 [M+H] + :2007.1,found:2007.1.
XIII-6(m＝5,n＝6,X＝O,Y
5＝R,Z＝3,R’＝Biotin):
1H NMR(500MHz,MeOD):δ5.88(s,1H),5.33–5.32(m,1H),5.00(s,1H),4.58–4.53(m,2H),4.32-4.28(m,1H),4.16(s,2H),3.83-3.10(m,50H),2.97–2.92(m,3H),2.70(d,J＝12.2Hz,1H),2.58-2.54(m,3H),2.33–2.19(m,3H),2.00–1.93(m,2H),1.90–0.63(s,53H).ESI:m/z calculated for C
73H
125N
10O
16S
2[M+H]
+:1461.9,found:1461.9.
XIII-6 (m = 5, n = 6, X = O, Y 5 = R, Z = 3, R '= Biotin): 1 H NMR (500MHz, MeOD): δ5.88 (s, 1H), 5.33 --5.32(m,1H),5.00(s,1H),4.58--4.53(m,2H),4.32-4.28(m,1H),4.16(s,2H),3.83-3.10(m,50H),2.97 –2.92(m,3H),2.70(d,J=12.2Hz,1H),2.58-2.54(m,3H),2.33–2.19(m,3H),2.00–1.93(m,2H),1.90–0.63 (s,53H).ESI:m/z calculated for C 73 H 125 N 10 O 16 S 2 [M+H] + :1461.9,found:1461.9.
Claims (10)
- 一种流感病毒神经氨酸酶抑制剂，其特征在于，所述流感病毒神经氨酸酶抑制剂结构通式如下：An influenza virus neuraminidase inhibitor, characterized in that the general structural formula of the influenza virus neuraminidase inhibitor is as follows:
其中：in:m选自2-11中的任一自然数，n选自3-12中的任一自然数，X选自OCOO或O；m is selected from any natural number in 2-11, n is selected from any natural number in 3-12, and X is selected from OCOO or O;Y选自Y is selected from
所述中的R选自： Said
R in is selected from:H、或，H, or,
所述中的R`为H或荧光标记基团，Z选自3-6中的任一自然数。 Said
In R'is H or a fluorescent labeling group, and Z is selected from any natural number from 3-6. - 根据权利要求1所述的流感病毒神经氨酸酶抑制剂，其特征在于，所述R`选自：生物素，CY3，CY5，或，FITC。The influenza virus neuraminidase inhibitor according to claim 1, wherein the R'is selected from the group consisting of biotin, CY3, CY5, or FITC.
- 根据权利要求1所述的流感病毒神经氨酸酶抑制剂，其特征在于，其选自化合物I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、I-11、I-12、I-13、I-14或I-15；The influenza virus neuraminidase inhibitor according to claim 1, characterized in that it is selected from compounds I-1, I-2, I-3, I-4, I-5, I-6, I- 7. I-8, I-9, I-10, I-11, I-12, I-13, I-14 or I-15;化合物I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-10、I-11、I-12、I-13I-14和I-15均具有如下结构通式：Compound I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I Both -13I-14 and I-15 have the following general structural formula:
化合物I-1的m为2，n为6，X为O，Y为In compound I-1, m is 2, n is 6, X is O, Y is
I-2的m为2，n为6，X为OCOO，Y为In I-2, m is 2, n is 6, X is OCOO, Y is
I-3的m为5，n为6，X为O，Y为I-3’s m is 5, n is 6, X is O, Y is
I-4的m为5，n为6，X为OCOO，Y为I-4’s m is 5, n is 6, X is OCOO, Y is
I-5的m为11，n为6，X为O，Y为I-5’s m is 11, n is 6, X is O, Y is
I-6的m为11，n为6，X为OCOO，Y为For I-6, m is 11, n is 6, X is OCOO, Y is
I-7的m为5，n为12，X为O，Y为I-7’s m is 5, n is 12, X is O, Y is
I-8的m为5，n为3，X为O，Y为I-8’s m is 5, n is 3, X is O, Y is
I-9的m为5，n为5，X为O，Y为I-9’s m is 5, n is 5, X is O, Y is
I-10的m为5，n为6，X为O，Y为其中，
中的R为H； I-10’s m is 5, n is 6, X is O, Y is
in,R is H;I-11的m为5，n为6，X为OCOO，Y为其中，
中的R为H； I-11’s m is 5, n is 6, X is OCOO, Y is
in,R is H;I-12的m为5，n为6，X为O，Y为其中，所述
中的R为
所述
中的Z为6，R`为生物素； I-12’s m is 5, n is 6, X is O, Y is
Among them, theR in isSaidZ is 6, R` is biotin;I-13的m为5，n为6，X为O，Y为其中，
中的R为
所述
中的Z为6，R`为CY3； In I-13, m is 5, n is 6, X is O, Y is
in,R in isSaidZ is 6, R` is CY3;I-14的m为5，n为6，X为O，Y为其中，
中的R为
所述
中的Z为6，R`为CY5；或， In I-14, m is 5, n is 6, X is O, Y is
in,R in isSaidWhere Z is 6, R` is CY5; or,I-15的m为5，n为6，X为O，Y为其中，
中的R为为
所述
中的Z为3，R`为生物素。 For I-15, m is 5, n is 6, X is O, Y is
in,The R in isSaidZ is 3 and R` is biotin. - 一种流感病毒神经氨酸酶抑制剂的制备方法，其特征在于，将化合物VIII或化合物XIV经反应条件g制得所述流感病毒神经氨酸酶抑制剂；A preparation method of influenza virus neuraminidase inhibitor, characterized in that the influenza virus neuraminidase inhibitor is prepared by compound VIII or compound XIV under reaction conditions g;所述化合物VIII的结构式为其中，m为2-11中的任一自然数，n为3-12中的任一自然数，X为OCOO或O，Y 3为
The structural formula of the compound VIII is
Among them, m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, and Y 3 is所述化合物XIV的结构式为其中，m为2-11中的任一自然数，n为3-12中的任一自然数，X为OCOO或O，Y 5为H或R；所述R指
其中R`为生物素、CY3或CY5； The structural formula of the compound XIV is
Wherein, m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, Y 5 is H or R; said R meansWherein R` is biotin, CY3 or CY5;所述反应条件g指：将化合物VIII或XIV溶于溶剂中，滴加氢氧化钠水溶液，室温搅拌3小时后，用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩，浓缩后的剩余物溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液中，反应1小时后浓缩，浓缩后的剩余物经Sephadex G-15凝胶柱分离纯化。The reaction condition g refers to: dissolving compound VIII or XIV in a solvent, adding sodium hydroxide aqueous solution dropwise, stirring at room temperature for 3 hours, neutralizing the solution with Dowex-50 (H+) ion exchange resin to a pH of 7, and filtering After the solution was concentrated, the concentrated residue was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid with a volume ratio of 1/1, reacted for 1 hour and then concentrated, and the concentrated residue was separated and purified by Sephadex G-15 gel column . - 根据权利要求4所述的一种流感病毒神经氨酸酶抑制剂的制备方法，其特征在于，所述化合物VIII由下述步骤制得：The method for preparing an influenza virus neuraminidase inhibitor according to claim 4, wherein the compound VIII is prepared by the following steps:将化合物II与化合物III经反应条件a得到化合物IV前体，再经反应条件b得到化合物IV；Compound II and compound III are subjected to reaction condition a to obtain compound IV precursor, and then reaction condition b to obtain compound IV;化合物IV与化合物V经反应条件c得到化合物VI；Compound IV and compound V undergo reaction condition c to obtain compound VI;将化合物VI与化合物VII经反应条件f得到化合物VIII；Compound VI and compound VII are subjected to reaction conditions f to obtain compound VIII;所述化合物II为其中R 1为ClCO或Ts； The compound II is
Where R 1 is ClCO or Ts;所述化合物III为其中n为3-12中的任一自然数，Y 1为N 3或COOMe； The compound III is
Wherein n is any natural number from 3-12, Y 1 is N 3 or COOMe;所述化合物IV为其中n为3-12中的任一自然数，X为OCOO或O，Y 2为NH 2或COOH； The compound IV is
Wherein n is any natural number from 3-12, X is OCOO or O, and Y 2 is NH 2 or COOH;所述化合物V为其中m为2-11中的任一自然数，R 2为
The compound V is
Where m is any natural number from 2-11, and R 2 is所述化合物VI为其中m为2-11中的任一自然数，n为3-12中的任一自然数，X为OCOO或O，Y 3为
The compound VI is
Where m is any natural number from 2-11, n is any natural number from 3-12, X is OCOO or O, and Y 3 is所述化合物VII为The compound VII is
当所述化合物II的R 1为Ts时，所述反应条件a指，将化合物II与化合物III溶于非极性溶剂中，100-150℃条件下回流反应过夜，溶液浓缩后经硅胶层析柱分离，得到化合物IV的前体； When the R 1 of the compound II is Ts, the reaction condition a refers to dissolving the compound II and the compound III in a non-polar solvent, refluxing the reaction overnight at 100-150° C., the solution is concentrated and subjected to silica gel chromatography Column separation to obtain the precursor of compound IV;当所述化合物II的R 1为ClCO时，所述反应条件a指，化合物II和化合物III溶于吡啶中，室温搅拌反应过夜；向反应液中加入甲醇，浓缩后溶于溶剂，并先后以HCl溶液和NaHCO 3溶液洗涤，有机相浓缩后经硅胶层析柱分离，得到化合物IV的前体； When the R 1 of the compound II is ClCO, the reaction condition a means that the compound II and the compound III are dissolved in pyridine, and the reaction is stirred overnight at room temperature; methanol is added to the reaction solution, which is dissolved in the solvent after concentration, and Washing with HCl solution and NaHCO 3 solution, the organic phase is concentrated and then separated by silica gel chromatography column to obtain the precursor of compound IV;当化合物III的Y 1为N 3时，所述反应条件b指：将化合物IV前体溶于四氢呋喃中，加入去离子水，三苯基磷，反应液加热到40-60℃，优选45℃，搅拌反应1-12h，优选3h；将反应液浓缩后经硅胶层析柱分离，得到化合物IV； When Y 1 of compound III is N 3 , the reaction condition b refers to: dissolving the precursor of compound IV in tetrahydrofuran, adding deionized water and triphenylphosphorus, and heating the reaction solution to 40-60°C, preferably 45°C , The reaction is stirred for 1-12h, preferably 3h; the reaction solution is concentrated and separated by silica gel chromatography column to obtain compound IV;当化合物III的Y 1为COOMe时，所述反应条件b指：将化合物IV前体溶于溶剂中，滴加氢氧化钠水溶液，室温搅拌1-6小时，优选3小时，然后用Dowex-50(H+)离子交换树脂中和至溶液pH值为7，过滤后溶液浓缩后经硅胶层析柱分离，得到化合物IV； When Y 1 of compound III is COOMe, the reaction condition b refers to: dissolving the precursor of compound IV in a solvent, adding sodium hydroxide aqueous solution dropwise, stirring at room temperature for 1-6 hours, preferably 3 hours, and then using Dowex-50 The (H+) ion exchange resin is neutralized to the pH of the solution to 7, after filtration, the solution is concentrated and separated by a silica gel chromatography column to obtain compound IV;所述反应条件c指，在氮气保护条件下，将化合物IV和化合物V溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物VI；The reaction condition c refers to dissolving compound IV and compound V in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature. After concentrating the reaction solution, it is separated by a silica gel chromatography column to obtain compound VI;所述反应条件f指，在氮气保护条件下，将化合物VII溶于吡啶，搅拌均匀后加入化合物VI和DMAP，室温下反应2-10h，优选5h；将反应液浓缩后经硅胶层析柱分离，得到化合物VIII。The reaction condition f refers to dissolving compound VII in pyridine under nitrogen protection, stirring uniformly, adding compound VI and DMAP, and reacting at room temperature for 2-10h, preferably 5h; the reaction solution is concentrated and separated by silica gel chromatography column , Compound VIII is obtained. - 根据权利要求4所述的一种流感病毒神经氨酸酶抑制剂的制备方法，其特征在于，所述化合物XIV通过下述步骤制得：The method for preparing an influenza virus neuraminidase inhibitor according to claim 4, wherein the compound XIV is prepared by the following steps:化合物IX与化合物X 1通过反应条件h得到化合物XI； Compound IX and compound X 1 pass reaction conditions h to obtain compound XI;化合物XI与化合物IV通过反应条件e得到化合物XII；Compound XI and compound IV pass reaction condition e to obtain compound XII;化合物XII与化合物V通过反应条件i得到化合物XIII；Compound XII and compound V obtain compound XIII through reaction condition i;化合物XIII与化合物VII通过反应条件f得到化合物XIV；Compound XIII and compound VII obtain compound XIV through reaction condition f;所述化合物IX为其中Y 4为H或CH 2SH； The compound IX is
Where Y 4 is H or CH 2 SH;所述化合物X 1为其中R`为生物素、CY3或CY5； The compound X 1 is
Wherein R` is biotin, CY3 or CY5;所述化合物XI为其中Y 5＝H或R，所述R指
其中R`为生物素、CY3或CY5； The compound XI is
Where Y 5 ＝H or R, and the R meansWherein R` is biotin, CY3 or CY5;所述化合物XII为其中Y 5为H或R，所述R指
其中R`为生物素、CY3或CY5； The compound XII is
Where Y 5 is H or R, and the R meansWherein R` is biotin, CY3 or CY5;所述化合物XIII为其中Y 5为H或R，所述R指
其中R`为生物素、CY3或CY5； The compound XIII is
Where Y 5 is H or R, and the R meansWherein R` is biotin, CY3 or CY5;所述化合物IV为其中n为3-12中的任一自然数，X为OCOO或O，Y 2为NH 2或COOH； The compound IV is
Wherein n is any natural number from 3-12, X is OCOO or O, and Y 2 is NH 2 or COOH;所述化合物V为其中m为2-11中的任一自然数，R 2为
The compound V is
Where m is any natural number from 2-11, and R 2 is所述化合物VII为The compound VII is
所述反应条件h指，在氮气保护条件下，将化合物IX，化合物X 1溶于溶剂中，室温反应过夜，溶液浓缩后经硅胶层析柱分离，得到化合物XI； The reaction condition h refers to dissolving compound IX and compound X 1 in a solvent under nitrogen protection, reacting at room temperature overnight, and then concentrating the solution and separating it on a silica gel column to obtain compound XI;优选地，所述化合物IX、X 1、无水乙醇的用量比例为1mmol∶1mmol∶2-50ml，优选1mmol∶1mmol∶20ml； Preferably, the dosage ratio of the compound IX, X 1 and absolute ethanol is 1mmol:1mmol:2-50ml, preferably 1mmol:1mmol:20ml;所述溶剂选自由无水乙醇、水、甲醇组成的组，优选乙醇；The solvent is selected from the group consisting of absolute ethanol, water and methanol, preferably ethanol;所述反应条件e指，在氮气保护条件下，将化合物IV和化合物XI溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物XII；The reaction condition e refers to dissolving compound IV and compound XI in DMF under nitrogen protection, adding N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and reacting overnight at room temperature. The reaction solution was concentrated and separated by silica gel chromatography column to obtain compound XII;优选地，所述化合物IV、化合物XI、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-2mmol∶1-50ml∶1-6mmol∶0.2-1mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；Preferably, the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.2 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;所述反应条件i指，将化合物XII溶于二氯甲烷与三氟乙酸体积比为1/1的混合溶液中，反应1-3h，优选1小时，然后浓缩，剩余物经Sephadex G-15凝胶柱分离纯化后，得到处理后的化合物XII；The reaction condition i refers to dissolving compound XII in a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 1/1, reacting for 1-3h, preferably 1 hour, and then concentrating, and the residue is condensed by Sephadex G-15 After separation and purification by a gel column, the treated compound XII is obtained;优选地，所述化合物XII、二氯甲烷与三氟乙酸体积比为1/1的混合溶液的用量比例为1mmol∶1-50mL，优选1mmol∶10mL；Preferably, the volume ratio of the mixed solution of compound XII, dichloromethane and trifluoroacetic acid with a volume ratio of 1/1 is 1mmol:1-50mL, preferably 1mmol:10mL;将所述处理后的化合物XII与化合物V溶于DMF中，加入N,N-二环己基碳二亚胺和4-二甲胺基吡啶，室温 下反应过夜，将反应液浓缩后经硅胶层析柱分离，得到化合物XIII；The treated compound XII and compound V were dissolved in DMF, N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added, and reacted overnight at room temperature. The reaction solution was concentrated and passed through a silica gel layer. Column separation to obtain compound XIII;优选地，所述化合物IV、化合物XI、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-2mmol∶1-50ml∶1-6mmol∶0.1-1mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；Preferably, the dosage ratio of the compound IV, compound XI, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-2mmol:1-50ml:1-6mmol:0.1 -1mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;所述反应条件f指：在氮气保护条件下，将化合物VII溶于吡啶，搅拌均匀后加入化合物XIII和DMAP，室温下反应1-12h，优选5h，然后将反应液浓缩后经硅胶层析柱分离，得到化合物XIV；The reaction condition f refers to: under nitrogen protection, dissolve compound VII in pyridine, stir evenly, add compound XIII and DMAP, react at room temperature for 1-12h, preferably 5h, then concentrate the reaction solution and pass it through a silica gel chromatography column Separated to obtain compound XIV;优选地，所述化合物VII、化合物XIII、吡啶、DMAP的用量比例为体积比1∶2-50∶1-3∶0.1-1，优选1∶10∶1.5∶0.2。Preferably, the dosage ratio of the compound VII, compound XIII, pyridine, and DMAP is 1:2-50:1-3:0.1-1 by volume, preferably 1:10:1.5:0.2. - 根据权利要求4所述的一种流感病毒神经氨酸酶抑制剂的制备方法，其特征在于，所述反应条件g中，所述溶剂选自由甲醇、乙醇、水组成的组；The method for preparing an influenza virus neuraminidase inhibitor according to claim 4, wherein in the reaction conditions g, the solvent is selected from the group consisting of methanol, ethanol, and water;所述化合物VIII或XIV、甲醇、氢氧化钠水溶液、二氯甲烷与三氟乙酸体积比为1/1的混合溶液的用量比例为1mmol∶1-100mL∶0.5-2M∶1-100mL。The dosage ratio of the compound VIII or XIV, methanol, aqueous sodium hydroxide solution, and a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 1/1 is 1mmol:1-100mL:0.5-2M:1-100mL.
- 根据权利要求5所述的一种流感病毒神经氨酸酶抑制剂的制备方法，其特征在于，The method for preparing an influenza virus neuraminidase inhibitor according to claim 5, characterized in that:当所述化合物II的R 1为Ts时，所述反应条件a中，所述化合物II、化合物III、非极性溶剂的用量比例为1mol∶1-1.5mol∶10-200ml；优选1mol∶1.2mol∶100ml；所述非极性溶剂为1,4-二氧六环；回流反应过夜的温度条件为125℃； When R 1 of the compound II is Ts, in the reaction condition a, the dosage ratio of the compound II, the compound III, and the non-polar solvent is 1 mol: 1-1.5 mol: 10-200 ml; preferably 1 mol: 1.2 mol: 100ml; the non-polar solvent is 1,4-dioxane; the temperature condition for the reflux reaction overnight is 125°C;当所述化合物II的R 1为ClCO时，所述反应条件a中，所述溶剂选自二氯甲烷或氯仿；所述化合物II、化合物III、吡啶、甲醇、二氯甲烷、HCl溶液、NaHCO 3溶液的用量比例为1mol∶1.2mol∶100mL∶2mL∶100mL∶1M∶1M； When R 1 of the compound II is ClCO, in the reaction condition a, the solvent is selected from dichloromethane or chloroform; the compound II, compound III, pyridine, methanol, dichloromethane, HCl solution, NaHCO 3 The dosage ratio of the solution is 1mol:1.2mol:100mL:2mL:100mL:1M:1M;当化合物III的Y 1为N 3时，所述反应条件b中，所述化合物IV、四氢呋喃、去离子水、三苯基磷的用量比例为10mmol∶10-100ml∶10-100ml∶10-50mmol，优选10mmol∶100ml∶20ml∶20mmol； When Y 1 of compound III is N 3 , in the reaction condition b, the dosage ratio of compound IV, tetrahydrofuran, deionized water, and triphenylphosphorus is 10mmol: 10-100ml: 10-100ml: 10-50mmol , Preferably 10mmol: 100ml: 20ml: 20mmol;当化合物III的Y 1为COOMe时，所述反应条件b中，所述化合物IV前体、甲醇、氢氧化钠水溶液用量比例为10mmol∶10-200mL∶0.5-2M，优选10mmol∶100mL∶1M；所述溶剂选自由甲醇、乙醇、水组成的组，优选甲醇； When Y 1 of compound III is COOMe, in the reaction condition b, the dosage ratio of the compound IV precursor, methanol, and aqueous sodium hydroxide solution is 10 mmol: 10-200 mL: 0.5-2M, preferably 10 mmol: 100 mL: 1M; The solvent is selected from the group consisting of methanol, ethanol, and water, preferably methanol;所述反应条件c中，所述化合物IV、化合物V、DMF、N,N-二环己基碳二亚胺、4-二甲胺基吡啶用量比例为1mmol∶1-3mmol∶1-50ml∶1-6mmol∶0.1-2mmol，优选1mmol∶1mmol∶10ml∶2mmol∶1mmol；In the reaction condition c, the dosage ratio of the compound IV, compound V, DMF, N,N-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 1mmol:1-3mmol:1-50ml:1 -6mmol:0.1-2mmol, preferably 1mmol:1mmol:10ml:2mmol:1mmol;所述反应条件f中，所述化合物VII、吡啶、化合物VI、DMAP的用量比例为体积比1∶1-50∶1-3∶0.1-1，优选1∶10∶1.5∶0.2。In the reaction condition f, the dosage ratio of the compound VII, pyridine, compound VI, and DMAP is 1:1-50:1-3:0.1-1 by volume, preferably 1:10:1.5:0.2.
- 权利要求1-3任一所述的流感病毒神经氨酸酶抑制剂，和/或，权利要求5-8任一所述的制备方法制备得到的流感病毒神经氨酸酶抑制剂在制备抗流感药物方面的应用。The influenza virus neuraminidase inhibitor according to any one of claims 1 to 3, and/or the influenza virus neuraminidase inhibitor prepared by the preparation method according to any one of claims 5-8 is used in the preparation of anti-influenza virus neuraminidase inhibitors. Drug application.
- 根据权利要求9所述的应用，其特征在于，所述药物的剂型选自：口服剂、滴鼻剂、注射剂、鼻喷雾剂。The application according to claim 9, wherein the dosage form of the medicine is selected from the group consisting of oral agents, nasal drops, injections, and nasal sprays.
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