An influenza virus inhibitor

Abstract

The present invention relates to an influenza virus inhibitor and the therapeutic effect of the inhibitor in patients infected with influenza virus.

Description

Technical Field

[0001] The invention belongs to the field of chemical drugs, and in particular relates to a compound with cap-dependent endonuclease inhibitory activity, and application of the compound in treating influenza. Background Art

[0002] Influenza viruses cause an infectious disease each year, killing 290,000 to 650,000 people worldwide and causing severe illness in 3 to 5 million people. In addition, epidemics caused by newly emerging recombinant viruses can have devastating global impacts. Therefore, it is necessary to continue working on improving vaccines and antiviral drugs as countermeasures. Currently, two classes of antiviral drugs are available for clinical use: neuraminidase inhibitors (NAIs: oseltamivir, zanamivir, peramivir) and M2 ion channel inhibitors (amantadine, rimantadine). However, the influenza viruses currently circulating are largely resistant to the M2 inhibitor s3. In addition, the antiviral ability of NAIs is relatively weak, and another concern for this class of drugs is the emergence of drug resistance, as was the case during the oseltamivir-resistant H1N1 influenza epidemic season from 2008 to 2009. Therefore, more effective antiviral drugs with new mechanisms of action are needed for the treatment and prevention of influenza virus infections.

[0003] The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza virus consists of PA, PB1, and PB2 subunits. It is responsible for the replication and transcription of the segmented single-stranded viral RNA genome (vRNA) in the nucleus of infected cells. Transcription of viral mRNA is carried out by a unique “cap-snatching” mechanism. This involves high hijacking of the host RNA polymerase by binding of the nascent capped transcript to the PB2 subunit, followed by cleavage at nucleotides by the cap-dependent endonuclease (CEN) in the PA subunit. Short, cap-snatching oligomers generated by the RdRp function of the PB1 subunit serve as primers for transcription of viral mRNA. Viral transcripts are polyadenylated by a mechanism at conserved enriched regions of the template vRNA for translation into functional proteins following nuclear export. As cap-snatching is essential for viral replication, cap-binding, endonuclease, and RdRp activity are all attractive targets for small molecule inhibitors, and indeed several new compounds targeting the polymerase are in active clinical development. In 2014, an RNA synthesis inhibitor, Favipiravir (Avigan), was approved in Japan, although the indication was limited to the treatment of novel influenza viruses that were unresponsive to other drugs. Pimodivir (JNJ-63623872, VX-787), a PB2 cap-binding inhibitor, has demonstrated virological efficacy alone and in combination with oseltamivir for simple influenza, but is only effective against influenza A viruses. Therefore, continued efforts are needed to discover and develop better performing influenza drugs. Summary of the invention

[0004] The object of the present invention is to provide a novel compound having cap-dependent endonuclease inhibitory activity or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound has the following structure:

[0005]

[0006] in,

[0007] The R a With R b Forming a 4-7 membered heterocyclic ring optionally substituted by substituent group A, wherein the heterocyclic ring has an exocyclic double bond sharing a carbon atom with the heterocyclic ring, preferably has an exocyclic carbon-nitrogen double bond;

[0008] P a is hydrogen, deuterium or a substituent group that forms a prodrug;

[0009] R 2 are each independently hydrogen, deuterium, halogen, hydroxy, alkyl, haloalkyl, or alkoxy;

[0010] M is an integer from 0 to 2;

[0011] Substituent Group A is independently selected from hydrogen, halogen, hydroxy, carboxyl, formyl, lower alkyl, halogenated lower alkyl, lower alkyloxy, halogenated lower alkyloxy, lower alkyloxy-lower alkyl, lower alkyloxy-lower alkyloxy, lower alkylcarbonyl, lower alkyloxycarbonyl, lower alkylamino, lower alkylcarbonylamino, lower alkylaminocarbonyl or amino-lower alkyl.

[0012] In a preferred embodiment, the compound of the present invention or its stereoisomer or salt has the following structural formula

[0013]

[0014] wherein R1 is selected from H, -CH3, -CH2-O-CH3;

[0015] R 2 are each independently hydrogen, deuterium, halogen, hydroxy, alkyl, haloalkyl, or alkoxy;

[0016] M is an integer from 0 to 2.

[0017] In a specific embodiment, the compound of the present invention or its stereoisomer or usable salt may have one of the following structures:

[0018]

[0019] Wherein P a is hydrogen or a substituent group that forms a prodrug.

[0020] Substituent group P forming a prodrug a Selected from the following structures:

[0021] (1)-C(=O)-P R0 , -C(=O)-LP R0 , -C(=O)-LOP R0 , -C(=O)-LOLP R0 ;

[0022] (2)-C(=O)-OP R1 , -C(=O)-OLP R1 、-C(=O)-NP R1 ;

[0023] (3)-C(P R2 )2-OP R3 ,-C(P R2 )2-OC(=O)-OP R3

[0024] The L is a straight chain or straight chain alkylene group, or a straight chain or chiral alkylene group;

[0025] The P R0 is hydrogen or alkyl optionally substituted by substituent Q, alkenyl optionally substituted by substituent Q, carbocyclic group optionally substituted by substituent Q, heterocyclic group optionally substituted by substituent Q, alkylamino optionally substituted by substituent Q, or alkylthio optionally substituted by substituent Q;

[0026] P R1 , P R3 each independently represents hydrogen, deuterium, or alkyl substituted by a substituent Q, carbocyclyl optionally substituted by a substituent Q, heterocyclyl optionally substituted by a substituent Q, carbocyclyl optionally substituted by a substituent Q, heterocyclyl optionally substituted by a substituent Q, or alkylsilyl optionally substituted by a substituent Q;

[0027] P R2 is hydrogen, deuterium or alkyl;

[0028] The substituent Q is hydrogen, deuterium, oxo, alkyl, hydroxyalkyl, amino, alkylamino, carbocyclyl, heterocyclyl, carbocyclylalkyl, alkylcarbonyl, halogen, hydroxy, carbonyl, alkylcarbonylamino, alkylcarbonylaminoalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkoxycarbonylalkyl, alkoxycarbonyloxy, alkylaminocarbonyloxy, alkylaminoalkyl, alkoxy, cyano, nitro, azido, alkylsulfonyl, trialkylsilyl or phosphoryl.

[0029] A more preferred embodiment is that the P R Based on:

[0030] -C(=O)-CH3, -C(=O)-O-(CH2)2-O-CH3, -CH2-OC(=O)-CH3, -CH2-OC(=O)-O-CH3, -CH2-OC(=O)-O-CH2CH3, -CH(CH3)-OC(=O)-O-CH3,

[0031] -CH2-OC(=O)-CH(CH3)-NH-C(=O)-OC(CH3)3, CH2C6H5.

[0032] Preferably, when it is a prodrug, P a The specific structure is

[0033]

[0034] For example, a prodrug having the following structure:

[0035]

[0036] The present invention also discloses a compound having the following structural formula:

[0037]

[0038]

[0039] In a specific embodiment, the compound of the present invention or its stereoisomer or pharmaceutically acceptable salt or prodrug form of the above structure, the substituent P is listed as follows: a It can be the following structure

[0040]

[0041] The exemplary structure after forming the prodrug is as follows:

[0042]

[0043]

[0044] The present invention also relates to a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0045] The pharmaceutical compositions of the present disclosure may be administered orally, parenterally or via an implanted reservoir. The term parenteral as used again includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal and intralesional injection or infusion techniques.

[0046] The pharmaceutical composition can be in the form of a sterile injectable preparation, for example, in the form of a sterile injectable aqueous or oily suspension. Such suspensions can be configured using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The details of the preparation of these compounds are known to those skilled in the art.

[0047] When administered orally, the pharmaceutical compositions of the present disclosure may be administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, useful carriers / diluents include lactose, high and low molecular weight polyethylene glycols, and dry corn starch. When aqueous suspensions are administered orally, the active ingredient is mixed with an emulsifier and a suspending agent. If desired, certain sweeteners and / or flavoring agents and / or coloring agents may be added.

[0048] Other suitable carriers for the above compositions can be found in standard pharmaceutical textbooks, for example in "Remington's Pharmaceutical Sciences", 19th ed., Mack Publishing Company, Easton, Penn., 1995. Further details regarding the design and preparation of suitable delivery forms of pharmaceutical compositions are known to those skilled in the art from the disclosure.

[0049] In the present invention, in addition to the compound of the present invention or a pharmaceutically acceptable salt thereof, other anti-influenza virus compounds may also be included, such as neuraminidase inhibitors, nucleoside drugs, PB2 inhibitors, PB1 inhibitors, M2 inhibitors or other anti-influenza drugs.

[0050] When used to prevent and / or treat influenza virus infection, the dosage level of the compound in the present disclosure is typically about 1 to about 500 milligrams per kilogram (mg / kg) of body weight per day, more specifically, about 1 to about 50 mg / kg of body weight per day. Typically, the pharmaceutical composition in the present disclosure can be administered about once to about 3 times a day, preferably once before or after the onset of influenza. Or it can be administered in the form of a continuous infusion, which can be used as a chronic or acute therapy. The amount of active ingredient that can be mixed with a carrier material to prepare a single dosage form will vary with the host being treated and the specific mode of administration.

[0051] On the other hand, the present invention relates to a product or a kit, comprising a container and a package insert, wherein the container contains a compound of formula II in crystalline form or a composition containing a compound of formula II in crystalline form, and the package insert contains instructions for use of the drug. In a preferred embodiment, the product or kit further comprises one or more containers containing one or more other antiviral drugs for preventing or treating influenza virus infection. In a preferred embodiment, the other drugs are N-neuraminidase inhibitors, nucleoside drugs, PB2 inhibitors, PB1 inhibitors, M2 inhibitors or other anti-influenza drugs.

[0052] Definition of terms:

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0054] The term "unsubstituted", when used to define a group, means that the defined group is not substituted by any group other than hydrogen atoms, and the group has the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs. For example, an unsubstituted heterocyclic group means that the hydrogen atoms on the heterocyclic ring are not substituted by any other group, such as furan, pyridine, dihydropyridine, etc.

[0055] The term "substituted", when used to define a group, means that one, two, three or more hydrogen atoms on the defined group are replaced by a substituent, and the one, two, three or more hydrogen atoms may be hydrogen atoms on the same carbon (or nitrogen) atom or on different carbons (or nitrogens). In this case, the meaning of the group should be understood in conjunction with the substituent. In the present invention, unless otherwise specified, when referring to "substituted", it means that the hydrogen atoms in the defined group are replaced by one, two, three or more substituents selected from the following:

[0056] Cyano, halogen, hydroxy, carboxyl, ester, amide, sulfonamide, amino, formyl, lower alkyl, lower hydrocarbon, lower alkynyl, halogenated lower alkyl, hydroxy-substituted lower alkyl, cycloalkyl, aryl, heterocyclic, aryl lower alkyl, heterocyclic lower alkyl, lower alkyloxy, halogenated lower alkyloxy, lower alkyloxy lower alkyl, lower alkyloxy lower alkyloxy, lower alkylcarbonyl, lower alkyloxycarbonyl, lower alkylamino, lower alkylcarbonylamino, lower alkylaminocarbonyl, lower alkylsulfonyl, lower alkylsulfonylamino, carboxy lower alkyloxy, aryl lower alkyloxy.

[0057] When it comes to specific naming, the substituent is usually placed before the substituted group. For example, "carboxymethyleneoxy" means that the oxy group is substituted by a methylene group, and the methylene group is substituted by a carboxyl group. Its structure can be represented as follows:

[0058]

[0059] The term "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformers. All stereoisomers belong to the scope of the present invention, and the compounds of the present invention may be a single stereoisomer or a mixture of other isomers, such as a racemate, or a mixture of all other stereoisomers.

[0060] The term "salt" refers to a pharmaceutically acceptable salt formed by a compound of the present invention and an acid, such as an organic or inorganic salt, for example, selected from: hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, fumaric acid, citric acid, maleic acid, benzenesulfonic acid, sulfonic acid, malic acid, succinic acid, lactic acid, acetic acid, malonic acid, etc.

[0061] The term "hydrocarbyl" includes alkyl or hydrocarbyl groups (eg, alkenyl or alkynyl).

[0062] Alkyl refers to a straight, branched or cyclic saturated substituent composed of carbon and hydrogen, and a lower alkyl refers to an alkyl composed of 1-6 carbon atoms. Alkenyl refers to a straight, branched or cyclic unsaturated substituent composed of carbon and hydrogen, and a lower alkenyl refers to an alkenyl composed of 1-6 carbon atoms; alkynyl refers to a straight or branched unsaturated substituent composed of carbon and hydrogen, and a lower alkynyl refers to an alkynyl composed of 1-6 carbon atoms. Substituted alkyl (or substituted hydrocarbon) refers to one or more hydrogen atoms on the alkyl group replaced by other groups such as halogen, hydroxyl, carboxyl, cyano, cycloalkyl, aryl, heteroaryl, oxo, heterocycloalkoxy, etc.

[0063] The term "cycloalkyl" refers to a saturated or unsaturated monocyclic hydrocarbon group, generally containing 3 to 20 carbon atoms. The cycloalkyl group may be a single ring, or may be a spiro ring, a bridged ring, a fused ring, or a fused ring.

[0064] The term aryl includes not only carbocyclic aryl but also heterocyclic aryl. Carbocyclic aryl refers to 6-10 membered all-carbocyclic or polycyclic aromatic groups, including phenyl, naphthyl, biphenyl, etc., and the carbocyclic aryl may also be substituted or unsubstituted. Heterocyclic aryl refers to a heteroaromatic system group containing at least one heteroatom, including monocyclic heterocyclic aryl or condensed heterocyclic aryl, the heteroatom is selected from oxygen, sulfur or nitrogen, including but not limited to furan, thiophene, pyrrole, thiazole, etc., and the heteroaryl may be substituted or unsubstituted.

[0065] The term "heterocyclyl" refers to a saturated or unsaturated monocyclic hydrocarbon radical containing at least one heteroatom. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a graph showing the effects of different compounds on body weight changes in mice after influenza virus infection.

[0067] Figure 2 This is a graph showing the effects of different compounds on the survival rate of mice infected with influenza virus.

[0068] Figure 3 The graph shows the effect of different compounds on the weight changes of mice after influenza virus infection (administered 24 hours after infection).

[0069] Figure 4 The graph shows the effect of different compounds on the weight changes of mice after influenza virus infection (administered 36 hours after infection).

[0070] Figure 5 The graph shows the effect of different compounds on the weight changes of mice after influenza virus infection (administered 48 hours after infection).

[0071] Figure 6This is a graph showing the effects of different compounds on the survival rate of mice after influenza virus infection (administered 24 hours after infection).

[0072] Figure 7 This is a graph showing the effects of different compounds on the survival rate of mice after influenza virus infection (administered 36 hours after infection).

[0073] Figure 8 This is a graph showing the effects of different compounds on the survival rate of mice after influenza virus infection (administered 48 hours after infection). DETAILED DESCRIPTION

[0074] The following is a general method for producing the compound of the present invention. In addition, extraction, purification, etc. may be carried out in a conventional post-chemical experiment.

[0075] The synthesis of the compounds of the present invention can be carried out by referring to procedures known in the art.

[0076] As the raw material compound, commercially available compounds, compounds described in the present specification, compounds described in the literature cited in the present specification, and other known compounds can be used.

[0077] The compounds of the present invention may exist in tautomerism, and the present invention contains these compounds, including all possible isomers thereof and mixtures thereof.

[0078] When it is desired to obtain a salt of the compound of the present invention, the compound of the present invention can be prepared in the form of a suitable salt.

[0079] What do the abbreviations mean?

[0080] DMF: N,N-dimethylformamide

[0081] DMA: N,N-dimethylacetamide

[0082] DMS: dimethyl sulfide

[0083] NMP: N-methylpyrrolidone

[0084] DMI: Dimethylimidazolinone

[0085] THF: Tetrahydrofuran

[0086] Boc: tert-Butyloxycarbonyl

[0087] PPTS: Pyridine p-toluenesulfonate

[0088] PPA: Phenylpropanolamine

[0089] PD(PPH3)4: Tetrakis(triphenylphosphine)palladium

[0090] DBU: 1,8-diazabicyclo[5.4.0]-7-undecene

[0091] T3P: Propylphosphonic anhydride

[0092] MSA: Methanesulfonic acid

[0093] TsOH: p-Toluenesulfonic acid

[0094] Tos: p-Toluenesulfonyl

[0095] Tol: p-Toluic acid

[0096] DIBALH: Diisobutylaluminum hydride

[0097] Alloc-Cl: Allyl chloroformate

[0098] HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0099] NaBH4: Sodium borohydride

[0100] n-BuLi: n-butyllithium

[0101] NBS: N-bromosuccinimide

[0102] NCS: N-chlorosuccinimide

[0103] DMAP: 4-dimethylaminopyridine

[0104] DIPEA: diisopropylethylamine

[0105] TBAF: Tetrabutylammonium fluoride

[0106] NAHMDS: Sodium Hexamethyldisilazide

[0107] TFA: trifluoroacetic acid

[0108] Preparation of compound I-2: Dissolve compound SM2 in ethylene glycol

[0109] Example 1: Synthesis of Compound I-2 (12)

[0110]

[0111] Synthesis of compound 1:

[0112]

[0113] Dissolve 2,4-piperidindione (40g, 353mmol, 1eq), ethylene glycol (44.6g, 707mmol, 2eq), p-toluenesulfonic acid monohydrate (13.4g, 70.7mmol, 0.1eq) in a reaction bottle containing 1000ml toluene, then heat to 120 degrees and remove water with a water separator. Stir at 120 degrees for 3 hours. After the reaction solution is concentrated, pour into 800.0mL of methanol, solid precipitates, filter, and concentrate the filtrate under reduced pressure to obtain a crude product. The crude product is purified by column chromatography (MeOH: DCM = 0-5%) to obtain compound 1 (32g, 203mmol, 57.7% yield) as a white solid.

[0114] Synthesis of compound 2

[0115]

[0116] Under a nitrogen atmosphere at -78 degrees, slowly add 2.5M n-butyl lithium-n-hexane solution (81 ml) to a solution of compound 1 (32 g, 203 mmol, 1.00 eq) in THF (600 ml). The mixture was stirred at -78 degrees for 2 hours. Allyl chloroformate (24.3 g, 203 mmol, 1.00 eq) was added dropwise to the reaction solution, and stirred at -78 degrees for 2 hours. The reaction solution was poured into 800 mL of ammonium chloride aqueous solution, then extracted with ethyl acetate (1000 mL), and the combined organic phase was washed with saturated brine (1000 mL) and then dried. The concentrated crude product was purified by column chromatography (DCM / EA=0-20%) to obtain compound 2 (17.3 g, 70.7 mmol, 38.5% yield), a colorless oil.

[0117] Synthesis of compound 3

[0118]

[0119] Under a nitrogen atmosphere at -78 degrees, 1M diisobutylaluminum hydride n-hexane solution (105ml, 1.5eq) was added to a solution of compound 2 (17.3g, 70.7mmol, 1.00eq) in THF (300ml). The mixture was stirred at -78 degrees for 2 hours. The reaction solution was quenched with 40ml acetone and 50ml methanol. Poured into 200mL of ammonium chloride aqueous solution, then extracted with ethyl acetate (600mL), the combined organic phase was washed with saturated brine (600mL) and then dried. The concentrated crude product was purified by column chromatography (DCM / EA=0-20%) to obtain compound 3 (12.5g, 51.4mmol, 72.7% yield), a colorless oil.

[0120] Synthesis of compound 4

[0121]

[0122] A mixture of compound 3 (12.5 g, 51.4 mmol, 1.00 eq) and acetonitrile (120 mL), cesium carbonate (34.6 g, 102 mmol, 2 eq) and iodomethane (14.5 g, 102 mmol, 2 eq) was stirred at room temperature for 16 hours. Poured into 200 mL of ammonium chloride aqueous solution, then extracted with ethyl acetate (500 mL), the combined organic phase was washed with saturated brine (500 mL) and then dried by rotation. The crude compound 4 (12.3 g, 50.4 mmol, 98.7% yield) after concentration was obtained as a colorless oil. The crude product was used directly in the next step.

[0123] Synthesis of compound 6

[0124]

[0125] Under nitrogen atmosphere, compound 5 (14.5 g, 50.4 mmol, 1.00 eq) was added to a solution of compound 4 (12.3 g, 50.4 mmol, 1.00 eq) in acetonitrile (300 ml). The mixture was stirred at -2 degrees for 10 minutes. Tin tetrachloride (19.3 g, 75 mmol, 1.50 eq) was added dropwise to the reaction solution, and the mixture was stirred at -25 degrees for 1 hour. The reaction solution was poured into 800 mL of sodium bicarbonate aqueous solution, then extracted with dichloromethane (1000 mL), and the combined organic phase was washed with saturated brine (1000 mL) and then dried. The concentrated crude product was purified by column chromatography (DCM / MeOH=0-5%) to obtain compound 6 (8.3 g, 16.1 mmol, 32.1% yield), a brown solid.

[0126] Synthesis of compound 8

[0127]

[0128] Morpholine (7 g, 80.5 mmol, 5 eq) and tetrakistriphenylphosphine palladium (3.73 g, 3.23 mmol, 0.2 eq) were added to a mixture of compound 6 (8.3 g, 16.1 mmol, 1.00 eq) and THF (100 mL), and the reaction mixture was stirred at 20-30 degrees for 16 hours. 300 ml of ether was added to the reaction mixture. After filtration, the filter cake was evaporated to dryness to obtain 5.9 g of crude product. 3 g of the crude product was prepared by chiral preparation (SFC Column: CHIRAL ART Cellulose-SB, 5*25 cm, 10 μm; Mobile Phase A: CO2, Mobile Phase B: MeOH: ACN = 1:1 (0.1% 2M NH3-MeOH); Flow rate: 250 mL / min; Gradient: isocratic 50% B; Column Temperature (℃): 35; Back Pressure (bar): 100; Wave Length: 254 nm) to obtain compound 8 (1.3 g, 3.4 mmol, 21.4% yield) as a yellow solid.

[0129] Synthesis of compound 9

[0130]

[0131] Under nitrogen atmosphere, compound A5 (0.9 g, 3.4 mmol, 1.00 eq) was added to a solution of compound 8 (1.3 g, 3.4 mmol, 1.00 eq) in ethyl acetate (10 ml). 50% wt 1-propylphosphoric anhydride in ethyl acetate (4.3 g, 6.8 mmol, 2 eq) and methanesulfonic acid (96 mg, 6.8 mmol, 2 eq) were added to the mixture, and the mixture was stirred at 70 degrees for 1 hour. The reaction solution was poured into 80 mL of sodium bicarbonate aqueous solution, then extracted with dichloromethane (100 mL), and the combined organic phase was washed with saturated brine (100 mL) and then dried. The crude product was concentrated to obtain compound 9 (1.6 g, 2.5 mmol, 74.7% yield), a brown solid.

[0132] Synthesis of compound 10

[0133]

[0134] Add 5 ml of acetonitrile, 5 ml of concentrated hydrochloric acid, and 5 ml of water to compound 9 (1.6 g, 2.5 mmol, 1.00 eq). Stir the reaction at 70 degrees for 10 minutes. Pour 20 mL of potassium carbonate aqueous solution into the reaction solution, then extract with dichloromethane (100 mL), wash the combined organic phase with saturated brine (100 mL) and spin dry. The concentrated crude product was prepared by (Column: XBridge PrepOBD C18 Column, 30*150 mm, 5 μm; Mobile Phase A: Water (0.05% TFA), Mobile Phase B: ACN; Flow rate: 80 mL / min; Gradient: 55% B to 55% Bin 14 min; Wave Length: 220 nm; RT1 (min): 13; Number Of Runs: 0) to obtain 0.8 g of yellow solid.

[0135] Synthesis of compound 11

[0136]

[0137] Methylhydroxylamine hydrochloride (0.062 g, 7.6 mmol, 10 eq) and cesium carbonate (0.13 g, 3.8 mmol, 5 eq) were added to a solution of compound 10 (0.045 g, 0.76 mmol, 1.00 eq) in acetonitrile (10 ml). The reaction was stirred at 25 degrees for 16 hours. The reaction solution was poured into 20 mL of sodium bicarbonate aqueous solution, then extracted with dichloromethane (50 mL), and the organic phase was washed with saturated brine (50 mL) and then dried. The crude product was concentrated to obtain compound 11 (0.029 g, 0.45 mmol, 59.6% yield) as a white solid.

[0138] Synthesis of Compound I-2

[0139]

[0140] Lithium chloride (0.016 g, 4.5 mmol, 10 eq) was added to a solution of compound 11 (0.029 g, 0.45 mmol, 1.00 eq) in N,N-dimethylacetamide (10 ml), and the mixture was stirred at 70 degrees for 2 hours. LCMS showed that compound 12 was generated.

[0141] According to the above method, the following compounds can be synthesized:

[0142]

[0143] Example 2: Preparation of Compound I-21

[0144]

[0145]

[0146] Example 3 Synthesis of Compound I-28

[0147]

[0148] According to the above method, the following compounds can be synthesized:

[0149]

[0150] Example 4: Synthesis of Compound I-32

[0151]

[0152] Example 5: Synthesis of Compound I-2 Prodrug (II-2)

[0153]

[0154] Compound II-1 (52.5 mg, 0.1 mmol), chloromethyl methyl carbonate (25 mg, 0.2 mmol), potassium carbonate (28 mg, 0.2 mmol) and potassium iodide (3 mg, 0.02 mmol) were reacted in N,N-dimethylformamide solution at 50 °C for 5 hours. The reaction was completed after TCL detection. Water was added to quench the reaction, and then the pH was adjusted to 3-4 with 1N dilute hydrochloric acid. The soil was filtered and dried, and compound II-2 (32.8 mg) was obtained by column chromatography.

[0155] The same method can also be used to prepare prodrugs of other drugs.

[0156] Example 6: In vitro cell inhibition assay

[0157] The CPE inhibition assay was performed as follows to evaluate the potency of test compounds in inhibiting cap-dependent endonuclease activity.

[0158] MDCK cells in 96-well tissue culture plates were cultured with test compounds and influenza A (IFV A / WSN / 33 (H1N1)) at a low infection rate at 37°C for 72 hours, the plates were fixed by adding 0.5% formaldehyde, and then stained with 0.5% crystal violet. Subsequently, the absorbance at a wavelength of 570 nm was measured using a microplate analyzer (Multiskan Ascent, Thermo). The concentration of the test compound required to reduce virus-induced CPE by 50% relative to the virus control group was represented by the 50% effective dose (EC 50 ), and CC 50 value.

[0159] The compounds were evaluated by CPE inhibition assay, where CE50 values ​​of compounds 2, 3 and 4 were less than 10 nM against IFV A / WSN / 33 influenza virus, while compound 6 was almost inactive (compounds 28-32 were almost inactive).

[0160] Table 1 Inhibitory activity of candidate compounds against IFV A / WSN / 33

[0161]

[0162]

[0163] Example 7: In vivo anti-influenza virus pharmaceutical experiment

[0164] Animals: 7-week-old female BALB / c mice with specific pathogen-free level were purchased from Shanghai Lingchang Biotechnology Co., Ltd. and arrived at the animal room of the biological department of Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd. on July 23, 2020, and were kept in independent ventilated cages. The breeding and use of mice were in accordance with the experimental protocol approved by WuXi AppTec IACUC (Animal Use Protocol No.: ID01-QD031-2020v1.0). After a 3-4-day environmental adaptation period, qualified animals were used for experiments.

[0165] Influenza A virus: WSN / 33 (H1N1), the original strain was purchased from Virapur, catalog number: F-1003A. The strain used was amplified by the biological department of WuXi AppTec, batch number: 20200518YNRP70.03. Titer: 6.80E+05PFU / mL.

[0166] Test compound: The experimental drugs are prodrug structures, namely II-00, II-01, II-1, II-2 and II-33. The test compound is powder, and the preparation method is as follows: according to the configuration concentration, accurately weigh the appropriate amount of test sample, add appropriate volume of PEG 400 and 30% Solutol HS-15 aqueous solution in turn, gradually vortex and ultrasonicate until the compound is evenly dispersed, and then add appropriate volume of 0.9% sodium chloride injection and vortex evenly to obtain a uniformly distributed solution (or suspension). The prepared suspension is stored at 4°C, and taken out and vortexed before each use for standby use.

[0167] Virus inoculation: Take out the influenza A virus WSN / 33 strain stock solution and quickly melt it in a 37℃ water bath, and dilute it with sterile DPBS to the required concentration for inoculation: 6.0*10 4 On day 0, mice were anesthetized by intraperitoneal injection of Shutai 50 / Sumenxin II anesthetics, and then the virus was inoculated by intranasal drops. The inoculation volume for each animal was 3.0*10 3pfu, and the inoculation volume was 50 μL.

[0168] Administration: Mice were treated with compounds or solvents. Oral administration was performed twice a day with a volume of 10 mL / kg. The first administration was 24 hours after virus inoculation and continued for 5 days. The control group was given an equal volume of solvent.

[0169] Grouping: According to the experimental design, the animals were divided into 6 groups, each with 6 mice, and the 6th group (solvent control) had 5 mice

[0170] Humane endpoints: According to IACUC protocol, any mouse that loses more than 35% of its body weight (based on the body weight on day 0) or / and shows signs of moribundity during the experiment will be euthanized and recorded as dead animals in the results.

[0171] Observation and results: Day 0-14: Record the weight of the animals daily, observe the health and survival of the animals, and evaluate the in vivo efficacy of the test compound in the influenza A virus mouse infection model by the weight changes and survival rates of the mice. Figure 1 and Figure 2 .

[0172] Effects of weight changes: Figure 1 The results showed that the weight of mice in the vehicle group began to decrease significantly from the third day, and then continued to decrease until death or humane end; the II-00 and II-2 drug groups were able to protect mice from weight loss caused by viral infection, and the weight of mice remained basically stable; the II-33 and II-01 drug groups were able to alleviate the weight loss caused by viral infection, and the weight of surviving mice began to recover from the 9th and 8th days, respectively; the II-1 drug group was similar to the vehicle group, and had no alleviating effect on mice with weight loss caused by the corresponding virus.

[0173] Impact on survival rate: Figure 2 The results showed that mice in the vehicle group began to die from the 6th day and all died on the 10th day, with a median survival time of 7 days and a final survival rate of 0%; mice in the II-1 group died on the 9th day and all died on the 11th day, with a median survival time of 9 days and a final survival rate of 0%; mice in the II-01 group died on the 3rd day, with a final survival rate of 66.67%; no mice in other groups died, and the final survival rates were all 100%.

[0174] Example 8: In vivo anti-influenza virus pharmacological experiment of drug administration at different time after poisoning

[0175] Test compounds: The experimental drugs are prodrug structures, namely II-00, II-1 and II-2, among which the II-2 compound component has two doses.

[0176] Administration: Mice were treated with the compound or vehicle. Oral administration was performed twice a day with a volume of 10 mL / kg. The first administration was at 24 hours, 36 hours, and 48 hours after virus inoculation, and the administration was continued for 5 days.

[0177] Grouping: According to the experimental design, the animals were divided into 13 groups, with 6 mice in each group.

[0178] Observation and results: Day 0-14: Record the weight of the animals daily, observe the health and survival of the animals, and evaluate the in vivo efficacy of the test compound in the influenza A virus mouse infection model by the weight changes and survival rates of the mice. Figure 3 , 4 and Figure 5 .

[0179] The test compound protects the body weight of mice in the model (see Figure 3 , 4 and 5)

[0180] Vehicle group: After virus inoculation, the body weight of mice began to decrease significantly from the third day and continued to decrease until the animals died naturally or were euthanized.

[0181] Test compound II-2 (0.5mpk, 5mpk) group: The low- and high-dose administration groups of compound II-2 began to be administered 24 and 36 hours after virus inoculation, and the weight of the mice remained basically stable without obvious weight loss; administration began 48 hours after virus inoculation, and the weight of the mice in the low-dose administration group decreased significantly on the 3rd day, and reached the lowest on the 7th day, with the maximum decrease of -9.2%, and then began to recover to the normal level (the effect was equivalent to the 5mpk dose group of compound II-00); the weight of the mice in the high-dose administration group decreased on the 3rd day, with the maximum decrease of -4.7%, and then began to recover and returned to normal levels on the 5th day.

[0182] Test compound II-00 (5 mpk) group: Compound II-00 was administered 24 hours after virus inoculation, and the weight of mice remained basically stable, with no significant weight loss. Administration was initiated 36 and 48 hours after virus inoculation, and the weight of mice decreased significantly on the 6th and 3rd days, respectively, and both dropped to the lowest on the 7th day of the experiment, with the maximum decrease of -5.7% and -10.3%, respectively, and then began to recover to normal levels.

[0183] Test compound II-1 (5 mpk) group: Compound II-1 was administered 24 hours after virus inoculation. The body weight of mice began to decrease significantly from the 3rd day and then continued to decrease until death or euthanasia.

[0184] The test compound protects the survival of mice in the model ( Figure 6 , Figure 7 and Figure 8 ).

[0185] Vehicle group: Mice died on the 8th day after infection, and all died naturally or were euthanized on the 9th day, with a median survival time of 8.5 days and a final survival rate of 0%.

[0186] Test compound II-2 (0.5 mpk, 5 mpk) group: No mice died during the experiment, and the terminal survival rate was 100%.

[0187] Test compound II-00 (5 mpk) group: No animal death occurred in the groups that started dosing 24 and 36 hours after virus inoculation, and the final survival rate was 100%; mice in the group that started dosing 48 hours after virus inoculation died on the 8th day of the experiment, and the terminal survival rate was 83.33%.

[0188] Test compound II-1 (5 mpk) group: All mice died on days 7 to 11, with a final survival rate of 0% and a median survival period of 8.5 days.

Claims

1. A compound or a pharmaceutically acceptable salt thereof having the following structure: P a It is hydrogen or -CH2-OC(=O)-O-CH3.

2. An antiviral pharmaceutical composition, comprising the compound or pharmaceutically acceptable salt of claim 1, and at least one selected from neuraminidase inhibitors, nucleoside compounds, PB2 inhibitors, PB1 inhibitors, M2 inhibitors or other anti-influenza virus drugs.

3. A medicament comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating influenza.

5. A product comprising a container and a package insert, wherein the container contains the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the composition of claim 2, or the medicament of claim 3, and the package insert carries instructions for use of the drug.

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