CN117924284A - High-efficiency antiviral compound and application thereof - Google Patents

High-efficiency antiviral compound and application thereof Download PDF

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CN117924284A
CN117924284A CN202410071449.0A CN202410071449A CN117924284A CN 117924284 A CN117924284 A CN 117924284A CN 202410071449 A CN202410071449 A CN 202410071449A CN 117924284 A CN117924284 A CN 117924284A
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请求不公布姓名
李海德
侯雯
姜龙
潘伟
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Nanjing Zhihe Medical Technology Co ltd
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/503—Pyridazines; Hydrogenated pyridazines spiro-condensed
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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Abstract

The invention provides a compound shown as the following formula (I-0) or hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt and a preparation method thereof. The compound of the present invention can be used for preparing medicines for preventing/treating viral infections including influenza viruses.

Description

High-efficiency antiviral compound and application thereof
The application relates to a split application of a high-efficiency antiviral compound and application of the high-efficiency antiviral compound, wherein the application date is 2022, 11, 1, and the application number is 202211354886.0.
Technical Field
The present invention relates to compounds having anti-influenza virus activity or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, processes for their preparation and their use in anti-influenza virus.
Background
Influenza viruses mainly include four types of influenza a virus, influenza b virus, influenza c virus and influenza d virus, among which influenza a virus and influenza b virus are the main human influenza viruses, influenza a virus is the strongest among them, the number of people infected in seasons of influenza is the largest, and serious respiratory tract infection diseases can be induced, resulting in death of 30 or more tens of thousands of people worldwide each year. In China, tens of millions of people are infected with influenza virus each year, especially the prevalence rate and the mortality rate of infants and the elderly are high, and diseases such as pneumonia and the like can be caused.
Currently, the major anti-influenza drugs on the market are: amantadine (AMANTADINE), the neuraminidase inhibitor oseltamivir (Oseltamivir) or Zanamivir (Zanamivir).
RNA polymerase of influenza virus contains Cap-dependent endonuclease (Cap-DEPENDENT ENDONUCLEASE), and inhibition of the activity of the Cap-dependent endonuclease can inhibit proliferation of viruses, which has become a promising target for development of antiviral drugs, and various heterocyclic compounds have been used as Cap-dependent endonuclease inhibitors.
However, these compounds exhibit poor physicochemical properties, which in turn affect the relevant pharmacokinetic properties, making them undesirable as influenza therapeutics. Thus, there is still an urgent need to develop new cap-dependent endonuclease inhibitors for treating influenza.
Disclosure of Invention
Unless specifically stated otherwise herein, the terms used herein are all the basic meanings commonly understood by those skilled in the art.
The invention provides a compound with an anti-influenza virus effect. The existing medicines have the function of resisting influenza viruses through targeting neuraminidase, and the compound has the function of inhibiting virus replication through inhibiting cap-dependent endonuclease in influenza viruses. The targeting virus at an earlier stage of the replication cycle has better effects of preventing and treating influenza, shows better anti-influenza virus effects in vitro or in vivo experiments, and is expected to treat strains resistant to oseltamivir and avian influenza virus strains (H7N 9, H5N 1).
The compound of the invention has better anti-influenza virus effect in-vitro and in-vivo pharmacodynamic tests and better in-vivo exposure in-vivo pharmacodynamic tests of animals. During the compound optimisation process it was unexpectedly found that: (1) After the compound is treated, the lung virus titer of the test animal is lower, and the lesions of lung tissues are lighter, which shows that the anti-influenza virus activity of the compound is obviously improved; (2) It has further been found that the compounds of the present invention have good lung tissue distribution in animal experiments, lower clearance rate in the lung, longer half-life; (3) Enantiomers of the compounds of the invention have similar antiviral activity in vitro cell assays, but only the S-configuration has higher antiviral activity in vivo animal assays, and the R-configuration is substantially absent. The cognition of the difference of the optical isomer in the aspect of pharmacodynamic tests at the cellular level and in vivo in animals is overturned; (4) The ester compound has remarkable advantages over other various forms of esters, shows better and higher in vivo exposure, and particularly has optimal performance of the compound M19. These characteristics are more favorable for exerting the anti-influenza virus effect of the drug for the influenza treatment which is usually transmitted through respiratory tract, and are expected to have great clinical value and therapeutic advantage.
The invention provides a compound shown as the following formula (I-0) or hydrate, solvate, optical isomer, polymorph, isotope derivative and pharmaceutically acceptable salt thereof:
in the formula (I-0), n is 0,1, 2,3 and 4;
R 0 is fluorine, chlorine, bromine, iodine, trifluoromethyl or cyano;
R a、Rb and R c are each independently selected from hydrogen, deuterium or methyl;
Q is hydrogen,
Wherein,
X 1 is an O atom or an S atom;
n1 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n2 is 0 or 1, and n1 and n2 are not both 0;
n3 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n4 is 0 or 1, and n3 and n4 are not both 0;
n5 is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; n6 is 0 or 1;
Each R 1、R2、R3 or R 4 is independently selected from hydrogen or methyl;
R 5 is the following group, substituted or unsubstituted with one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C8 alkylamino;
The group A is the following group: halogen, cyano, amino, hydroxy, carboxyl, nitro, trifluoromethyl, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 carbocyclyl, C2-C8 heterocyclyl, C1-C8 alkylamino.
In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (I-1):
The definition of the substituent in the formula (I-1) is defined as the formula (I-0).
In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (I-2):
the definition of the substituent in the formula (I-2) is defined as the formula (I-0).
In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (I-3):
The definition of the substituent in the formula (I-3) is defined as the formula (I-0).
In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (I-4):
in the formula (I-4), other substituents are defined as in the formula (I-0).
In some embodiments, the present invention provides compounds, or hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (I-5):
The definition of the substituent in the formula (I-5) is defined as the formula (I-0).
In some embodiments, the present invention provides compounds, or hydrates, solvates, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof:
in the formula (I), n1 is 0,1, 2, 3, 4, 5, 6, 7, 8 or 9;
n2 is 0 or 1;
x 1 is O or S;
X 2 is S or Se;
Each R 1、R2、R3 or R 4 is independently hydrogen or methyl;
R 5 is hydrogen or the following substituted or unsubstituted with one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C18 alkylamino;
When X 1 is O and X 2 is S, R 5 is not C1-C8 alkoxy;
The group A is the following group: halogen, cyano, amino, hydroxy, carboxyl, nitro, trifluoromethyl, C1-C8 alkyl, C1-C8 alkoxy, C3-C8 carbocyclyl, C2-C8 heterocyclyl, C1-C8 alkylamino.
In some embodiments, the present invention provides compounds, or hydrates, solvates, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (II):
The definition of the substituent in the formula (II) is defined as the formula (I).
In some embodiments, the present invention provides compounds, or hydrates, solvates, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (III):
the definition of the substituent in the formula (III) is defined as in the formula (I).
In some embodiments, the present invention provides compounds, or hydrates, solvates, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, as shown in formula (iv):
the definition of the substituent in the formula (IV) is defined as the formula (I).
In an embodiment of the application, the halogen is fluorine, chlorine, bromine, or iodine.
In an embodiment of the application, the heteroatom is N, O or S.
In embodiments of the present application, the solvate refers to a complex formed by the interaction of a compound with a pharmaceutically acceptable solvent, including ethanol, isopropanol, acetic acid, ethanolamine.
In an embodiment of the present application, the C1-C8 alkyl group means a straight or branched saturated aliphatic hydrocarbon group having 1 to 8 carbon atoms in the molecule. Including but not limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, and the like.
In the embodiments of the present application, the alkoxy group of C1-C8 and the alkylthio group of C1-C8 refer to a group in which a saturated aliphatic hydrocarbon group having 1-8 carbon atoms is inserted into an oxygen atom or a sulfur atom at any reasonable position, and include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, isobutoxy, 2-ethylethoxy, methylthio, ethylthio, propylthio, isopropylthio, isobutylthio and the like.
In the embodiment of the application, the C1-C8 alkylamino refers to a group in which a saturated aliphatic hydrocarbon group containing 1-8 carbon atoms is inserted into an-NH-or-NH 2 group at any reasonable position, and the saturated aliphatic hydrocarbon group comprises mono-, di-and naphthene amino groups, including but not limited to methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, di-n-propylamino, diisopropylamino and the like.
In embodiments of the present application, the C3-C8 carbocyclyl group refers to a monocyclic or fused polycyclic saturated or unsaturated cyclic hydrocarbon group having 3 to 8 carbon atoms, including, but not limited to, cyclopropyl, cyclopentyl, bicyclo [3.1.0] hexyl, bicyclo [3.2.0] heptyl, cyclopentadienyl, and the like.
In an embodiment of the present application, the C2-C8 heterocyclic group refers to a saturated or unsaturated cyclic group having 2 to 8 carbon atoms and 1 to 4 hetero atoms in the molecule. Including, but not limited to, cyclic ethoxy, aziridinyl, tetrahydrothienyl, tetrahydropyrrolyl, piperidinyl, hexahydropyridazinyl, dihydropyridinyl, cyclopentenyl sulfide, morpholinyl, and the like.
In some embodiments, in formula (I-0), n is 0; in some embodiments, in formula (I-0), n is 1,2, 3, or 4; in some specific embodiments, in formula (I-0), n is 1; in some specific embodiments, in formula (I-0), n is 2.
In some embodiments, in formula (I-0), R 0 is fluoro, chloro, bromo or iodo, preferably R 0 is fluoro or chloro; in some embodiments, in formula (I-0), R 0 is trifluoromethyl or cyano.
In some embodiments, in formula (I-0), R a is hydrogen; in some embodiments, in formula (I-0), R a is deuterium; in some embodiments, in formula (I-0), R a is methyl.
In some embodiments, R b and R c are both hydrogen; in some embodiments, R b and R c are both deuterium; in some embodiments, R b is hydrogen and R c is deuterium; in some embodiments, R b and R c are both methyl; in some embodiments, R b is hydrogen and R c is methyl.
In some embodiments, in formula (I-0), Q is hydrogen; in some embodiments, in formula (I-0), Q isIn some embodiments, in formula (I-0), Q is/>In some embodiments, in formula (I-0), Q is/>
In an embodiment of the invention, n1 and n2 are not both 0; wherein,
In some embodiments, n1 is 0; in some embodiments, n1 is 1, 2, 3, 4, 5, 6, 7, 8, or 9; in some specific embodiments, n1 is 1; in some specific embodiments, n1 is 2;
in some embodiments, n2 is 0; in some embodiments, n2 is 1.
In an embodiment of the invention, n3 and n4 are not both 0; wherein,
In some embodiments, n3 is 0; in some embodiments, n3 is 1, 2, 3, 4, 5, 6, 7, 8, or 9; in some specific embodiments, n3 is 1; in some specific embodiments, n3 is 2;
In some embodiments, n4 is 0; in some embodiments, n4 is 1.
In some embodiments, n5 is 0; in some embodiments, n5 is 1, 2, 3, 4, 5, 6, 7, 8, or 9; in some specific embodiments, n5 is 1; in some specific embodiments, n5 is 2.
In some embodiments, n6 is 0; in some embodiments, n6 is 1.
In some embodiments, X 1 is an O atom; in some embodiments, X 1 is an S atom.
In embodiments of the invention, each R 1 or R 2 is independently hydrogen or methyl, meaning that the carbon to which R 1 and R 2 are attached will have n1 or n3 or n5, and when n1 or n3 or n5 is greater than 1, the n1 or n3 or n 5R 1 or R 2 do not affect each other and may be the same or different.
In embodiments of the invention, each R 3 or R 4 is independently hydrogen or methyl, i.e., may or may not be substituted with methyl on the carbon atom to which R 1、R2、R3 or R 4 is attached.
In some embodiments, R 5 is the following group, substituted or unsubstituted with one or more groups a: C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkylthio, C1-C18 alkylamino; preferably, R 5 is the following group, substituted or unsubstituted with one or more groups a: C1-C8 alkyl, C1-C8 alkoxy; more preferably, R 5 is C1-C8 alkoxy substituted or unsubstituted with one or more groups A.
In some embodiments, group a is halogen, trifluoromethyl or cyano; in some embodiments, group a is amino, hydroxy, carboxyl, nitro; in some embodiments, group a is the following group: C1-C8 alkyl, C1-C8 alkoxy, C3-C8 carbocyclyl, C2-C8 heterocyclyl, C1-C8 alkylamino.
In an embodiment of the present invention, formula (I-0) contains a chiral center (chiral carbon atom at the position of the tag), and the optical isomers refer to optical isomers caused by different configurations of the carbon atoms shown in the figures, and the compound or the intermediate thereof can obtain a single-configuration compound through chiral separation.
In embodiments of the invention, compounds of the S-and R-configuration are subjected to absolute configuration determination by electron circular dichroism. In some embodiments, the compounds of the invention are racemates; in some embodiments, the compounds of the invention are in the S-configuration.
In an embodiment of the present invention, compounds of the S-configuration and R-configuration were subjected to optical rotation testing (according to the chinese pharmacopoeia 2020 edition-quarter-0621 optical rotation assay with methanol as solvent). In some specific embodiments, the compounds of the invention are racemates; in some specific embodiments, the compounds of the invention are the levorotatory forms.
In an embodiment of the invention, the S-configuration compound is the levorotatory form.
The compounds provided by the present invention include, but are not limited to, the following:
Or a hydrate, solvate, optical isomer, polymorph, isotopic derivative, pharmaceutically acceptable salt thereof.
Further, the pharmaceutically acceptable salts include inorganic acid salts and organic acid salts thereof.
In some specific embodiments, the pharmaceutically acceptable salts of the compounds include inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like, and organic acid salts include trifluoroacetate, methanesulfonate, tartrate, p-toluenesulfonate, and the like.
In a second aspect, the present invention provides a preparation route of a polycyclic compound represented by structural formula (I), which comprises the following steps:
The definition of the groups in the above scheme is as defined for the group of formula (I) wherein L 1 and L 2 are activating leaving groups, and the groups of formula (II-3) are synthesized according to the existing literature reporting method (J.Med. Chem.52, (3), 2009,771-778; WO 2019/136112), and formulas (II-1) and (II-2) can yield formula (II-3) under basic conditions, followed by formulas (II-3) and (II-4) under basic conditions.
In embodiments of the invention, references or patents to materials or intermediates (e.g., formulae SM1, SM2, SM3-0 and SM 5) required for the synthesis of formula (I-0) or comparative examples are made, the definition of the relevant substituents in formula SM5 being as described for formula (I-0),
The formula SM1 is synthesized by reference to the synthesis method described in patent CN 110300753A; formulae SM3 and SM5 are synthesized by the synthesis method described in patent CN 113226327A. The formula SM2 is synthesized by one-step synthesis of commercial product SM2-0 and methanesulfonyl chloride, and is specifically synthesized by referring to the synthesis method in patent CN 113226327A.
In a third aspect, the present invention provides a pharmaceutical composition comprising the above compound or a hydrate, solvate, optical isomer, polymorph, isotopic derivative, pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include one or a combination of several fillers, binders, diluents, lubricants, preservatives, taste masking agents or co-solvents. The pharmaceutical composition can be used for resisting influenza virus.
Further, the dosage forms of the pharmaceutical composition are tablets, capsules, powder, granules, pills, suspensions, syrups and injection.
In a fourth aspect of the invention, the compounds of the invention, including hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, are useful for anti-influenza virus use.
The compounds of the present invention have enhanced antiviral activity in vivo or in vitro. Studies have shown that S-configuration products have better anti-influenza virus activity in vivo, while R-configuration has relatively poorer activity.
In the research of the in-vivo drug effect of the compound, the pulmonary virus titer of the animal is lower, and the lesions of the pulmonary tissues are lighter; in tissue distribution studies, the compounds of the invention exhibit good lung tissue distribution; in pharmacokinetic studies, the ester compounds of the present invention exhibit higher in vivo exposure and less peak time than other ester compounds.
Stronger antiviral activity, high in-vivo exposure, rapid peak concentration and high lung distribution, which are more favorable for exerting the anti-influenza virus effect of the medicine and are expected to have great clinical value and therapeutic advantage.
The invention provides application of the compound, including hydrate, solvate, optical isomer, polymorph, isotope derivative, pharmaceutically acceptable salt or pharmaceutical composition thereof in preparing anti-influenza virus drugs.
The present invention provides a method for preventing or treating influenza virus infection comprising administering to a subject in need thereof a therapeutically effective amount of a compound as described above, including hydrates, solvates, optical isomers, polymorphs, isotopic derivatives, pharmaceutically acceptable salts thereof.
Drawings
FIG. 1 is a comparison of quantitative calculated predictive theory electron circular dichroism spectrum with experimental ECD spectrum to determine absolute configuration.
Figure 2 shows the body weight change of each group of animals in the anti-influenza efficacy experiment.
Figure 3 shows the change in survival rate of animals in each group in an anti-influenza efficacy experiment.
FIG. 4 shows pulmonary viral titers in anti-influenza efficacy experiments.
Fig. 5 is a lung tissue distribution.
Detailed Description
The following examples will allow one skilled in the art to more fully understand the invention, but are not intended to limit the invention in any way, the structure of all compounds being determined by MS or 1 H-NMR, all optical isomers of the single configuration involved being configuration determined by optical rotation testing or electron circular dichroism.
In this example, unless otherwise specified, the solvents and reagents used were all commercially available. The starting materials were all commercially available.
Embodiment one: synthesis of M01 and M11
Synthesis of Compound 2:
Compound 1 (100 g,793mmol,1 eq) was dissolved in DMF (1.5L), potassium carbonate (219 g,1.59mol,2 eq) was added, the ice water bath was cooled to 0℃and benzyl bromide (203 g,1.19mol,1.5 eq) was added dropwise, and after the addition was completed, the reaction was carried out at 0℃for 30 minutes, and then transferred to an oil bath pot for 5 hours at 80 ℃. TLC (EA/PE=1/2, EA is ethyl acetate, PE is petroleum ether) detects that there is only a small amount of remaining raw material, after-treatment, the system is cooled to room temperature, poured into water (3L), extracted with ethyl acetate (300 ml x 3), the organic phases are combined and washed with water (100 ml x 3), saturated brine (200 ml x 1), dried over anhydrous sodium sulfate for 20 minutes, and the organic phase is filtered, concentrated and purified by column chromatography (EA/PE=1/5 to 1/1) to obtain 157g of product. Yellow oil, yield 91.8%.
Synthesis of Compound 3:
Compound 2 (100 g,115mmol,1 eq) was dissolved in bromobenzene (1L) at room temperature and reacted with SeO 2 (152 g,347mmol,3 eq) in an oil bath at 180deg.C for 16 hours, and the reaction was complete as determined by TLC (EA/PE=1/2). The system was cooled to room temperature, celite was added to a buchner funnel and filtered off with celite (100 ml x 3), the combined organic phases were water-pumped to concentrate the dichloromethane and the bromobenzene was oil-pumped to give crude (100 g, red oil).
Synthesis of Compound 4:
Compound 3 (100 g, 433 mmol,1 eq) was dissolved in DMSO (1.5L) at room temperature, cyclopropylaldehyde (91 g,1.3mol,3 eq) was added, the temperature was reduced to 0℃in an ice-water bath, pyrrolidine (31 g, 433 mmol,1 eq) was added dropwise under nitrogen protection, the reaction was continued for 30 minutes at 15℃after the dropwise addition was completed, and the mixture was transferred to an oil bath at 50℃for 5 hours. TLC (DCM/MeOH=20/1) checked complete reaction of starting material, cooled the system to room temperature, poured into water (3L), extracted with ethyl acetate (250 ml x 3), combined organic phases washed with water (100 ml x 3), saturated brine (100 ml x 1), dried over anhydrous sodium sulfate for 30min, and concentrated column chromatography purification of the organic phase (EA/PE=1/10-1/1) gave the product (32 g, red oil, yield 24.6％).ESI-MS(+):m/z＝301.1;1H-NMR(CDCl3,400MHz)：δ8.85(s,1H),7.69-7.67(d,1H),7.28-7.43(m,5H),6.42-6.43(d,1H),5.28(s,2H),4.93-4.95(d,1H),2.79-2.81(d,1H),1.15-1.27(m,4H).
Synthesis of Compound 5:
Compound 4 (32 g,107mmol,1 eq) was dissolved in methanol (300 ml)/water (150 ml) under ice-water bath, trifluoroacetyl hydrazine (27.3 g,214mmol,2 eq) was added and reacted in ice-water bath for 30 min after the addition, oil bath 50℃for 4 hours, TLC (DCM/MeOH=20/1) detected complete reaction of starting material. The reaction system was cooled to room temperature, methanol in the system was concentrated, water (150 ml) was added, extraction was performed with ethyl acetate (100 ml x 3), the combined organic phases were washed with saturated brine (500 ml x 1), dried over anhydrous sodium sulfate for 10 minutes, and the organic phases were purified by filtration, concentration and column chromatography (EA/pe=1/10 to 1/1) to give a product (14 g, yellow solid, yield) 45％).ESI-MS(+):m/z＝297.1;1H-NMR(DMSO-D6,400MHz)：δ7.90-7.92(d,1H),7.31-7.43(m,5H),7.14-7.15(s,1H),6.28-6.30(d,1H),5.78-5.79(d,1H),5.27-5.30(d,1H),5.03-5.06(d,1H),4.09-4.10(m,1H),1.19-1.28(m,2H),0.85-0.90(m,1H),0.61-0.64(m,1H).
Compound 6 synthesis:
Compound 5 (14 g,47.2mmol,1 eq) was dissolved in THF (150 ml) under an ice-water bath and triethylamine (9.6 g,94.5mmol,2 eq) and DMAP (1.73 g,14.2mmol,0.3 eq) were added. Acetic anhydride (9.6 g,94.5mmol,2 eq) was added dropwise at 0℃and after the addition was completed, the reaction was continued at 0℃for 30 minutes and then allowed to proceed to room temperature for 2 hours. TLC (EA/pe=1/1) checked the completion of the starting material reaction. The system was poured into water (150 ml), extracted with ethyl acetate (50 ml x 3), the combined organic phases were saturated brine (100 ml x 2), dried over anhydrous sodium sulfate for 10 min, and the organic phases were purified by filtration, concentration, column chromatography (EA/pe=1/10 to 1/1) to give product 6 (15 g, yellow solid, yield 94%).
Compound 7 synthesis:
Compound 6 (15 g,44.3mmol,1 eq) was dissolved in THF (150 ml) and methanol (50 ml) under ice-water bath, sodium borohydride (3.35 g,133mmol,3 eq) was added in portions at 0℃and the reaction was carried out for 30 minutes at 0℃after the addition was completed, and the reaction was allowed to proceed to room temperature for 2 hours. TLC (EA/PE=1/1) checked for disappearance of starting material, the system was poured into an aqueous solution of ammonium chloride (150 ml), extracted with ethyl acetate (50 ml x 3), the combined organic phases saturated brine (50 ml x 3), dried over anhydrous sodium sulfate for 10 min, the organic phases filtered and concentrated to give the crude product (15 g, yellow solid) which was taken directly to the next step without purification.
Compound 8 synthesis:
Compound 7 (15 g,44mmol,1 eq) was dissolved in THF (150 ml), DMAP (0.54 g,4.4mmol,0.1 eq), triethylamine (8.92 g,88mmol,2 eq) were added dropwise at 0℃and Boc 2 O (19.2 g,88mmol,2 eq) was added and reacted for 30 minutes at 0℃after the addition was completed and then allowed to react for 2 hours at room temperature. TLC (DCM/MeOH=20/1) checked for disappearance of starting material, poured the system into water (150 ml), extracted with ethyl acetate (50 ml x 3), combined with saturated brine (50 ml x 3), dried over anhydrous sodium sulfate for 10 min, and concentrated by filtration and column chromatography purification of the organic phase (EA/PE=1/10-1/1) gave the product (15 g, yellow solid, yield 78%).
Compound 9 synthesis:
Compound 8 (15 g,34mmol,1 eq) was dissolved in methanol (150 ml) under an ice-water bath, and potassium carbonate (4.7 g,34mmol,1 eq) was added in portions and reacted at 0℃for 30 minutes at room temperature for 6 hours. TLC (DCM/meoh=20/1) checked complete reaction of the starting materials, the system was poured into water (150 ml), extracted with ethyl acetate (50 ml x 3), the organic phases were combined and washed with saturated brine (30 ml x 3), dried over anhydrous sodium sulfate for 10 min, and the organic phase was concentrated by filtration to give crude (10 g, yellow solid).
Compound 10 synthesis:
Compound 9 (10 g,25mmol,1 eq) was dissolved in DCM (100 ml) under ice-water bath and Dess-Martin oxidant (16 g,37.6mmol,1.5 eq) was added in portions at 0deg.C and reacted at 0deg.C for 30 min at room temperature for 5 hours. TLC (DCM/meoh=15/1) checked complete reaction of starting material, the system was poured into aqueous sodium bicarbonate (100 ml), DCM extracted (30 ml x 3) and the organic phase was combined with saturated brine (30 ml x 1). Drying with anhydrous sodium sulfate for 10 min, filtering, concentrating, column chromatography purifying (EA/PE=1/10-1/0) the organic phase to obtain crude product 10g, pulping the crude product with ethyl acetate to obtain pure product 5.4g, concentrating the mother liquor to obtain crude product 4.6g.ESI-MS(+):m/z＝397.3;1H-NMR(DMSO-D6,400MHz)：δ7.84-7.86(d,1H),7.28-7.46(m,5H),6.30-6.32(d,1H),5.07-5.20(m,2H),3.85-4.20(dd,2H),1.43(s,9H),1.35-1.37(m,1H),1.18-1.28(m,2H),1.08-1.12(m,1H).
Synthesis of Compound 12
After nitrogen is replaced in the three-mouth bottle, 226ml of phenylmagnesium bromide (2.8 mol/L) is added, the temperature is reduced to below 10 ℃ under ice water bath, 49.9g of selenium powder is added in batches, the reaction temperature is controlled to be not more than 30 ℃, and the reaction is carried out for 2 hours after the selenium powder is added. Adding 2mol/L hydrochloric acid under ice-water bath, reacting to release heat obviously, adding EA for extraction, separating liquid, and spin-drying organic phase to obtain the product 12 as brown oily substance with strong odor. The next reaction was directly carried out.
Synthesis of Compound 14
After nitrogen is replaced in the three-port bottle, 81.18g of LDA (lithium diisopropylamide) is added, the temperature is reduced to minus 30 ℃ under dry ice-ethanol solution, 50g of THF solution of 3, 4-difluorobenzoic acid is dropwise added, the heat release is obvious in the dropwise adding process, the temperature of a reaction system is controlled to be lower than minus 20 ℃, the reaction is carried out for 2 hours, DMF is added, the heat release is obvious, the temperature of the reaction system is controlled to be lower than minus 20 ℃, the temperature is not required to be controlled, the reaction is gradually carried out to room temperature, the reaction is carried out overnight, and the sampling detection reaction is finished. HCl is added into the reaction system, the heat release is obvious, EA is added for extraction, the solution is separated, the organic phase is dried by spinning, 72g of crude product of the compound 14 is obtained, the obtained product is directly used for the next reaction, the obtained crude product is yellow solid, and the yield is 122% (DMF in the material is not dried by spinning).
Synthesis of Compound 15
After nitrogen was replaced in the three-port flask, 58.81g of compound 12, 49.6g of compound 14, 300ml of toluene, 17.6g of camphorsulfonic acid were added, and the temperature was raised to 70℃and the reaction was carried out overnight. Cooling the reaction system to room temperature, adding NaOH solution, separating liquid, adding EA (ethylene oxide) into water phase for extraction, combining organic phases, washing the organic phases with saturated NaCl, and spin-drying to obtain a crude product: 123.08g, pulping the obtained crude product by PE, and carrying out suction filtration to obtain 34.4g of product, and recovering 14.83g of pulping filtrate. Compound 15 was an orange solid in 47.9% yield.
Synthesis of Compound 16
16.9G of AlCl 3 and 250ml of toluene are added into a three-mouth bottle, the system is cooled in an ice water bath, 17.1g of tetramethyl disiloxane is added, the mixture is stirred uniformly, 150ml of toluene solution of compound 15 (34.4) is added, the reaction is slightly exothermic, alCl 3 is gradually dissolved, and the temperature is heated to 80 ℃ for 1 hour; stopping the reaction, adding sulfuric acid solution (16.2mL+240 mL of water), separating, extracting the water phase with EA, spin-drying the organic phase, pulping the crude product with PE, and suction-filtering to obtain 22g of solid, wherein the obtained solid is yellow powdery solid with the theoretical amount of the product: 34.68g, yield 22g total of solid product compound 16: 63.4%. Directly used in the next reaction.
Synthesis of Compound 17:
Adding 429g of polyphosphoric acid into a three-mouth bottle, heating to 80 ℃, adding 42g of compound 16, heating to 120 ℃, changing the color of a reaction system from yellow to dark purple, reacting for 1 hour, sampling, adding water, and treating by EA; reducing the reaction temperature to below 100 ℃, adding water, stirring uniformly, adding EA for extraction, separating an organic phase, spin-drying, pulping a crude product by PE, filtering to obtain 3.4g of a product, separating filtrate by column chromatography to obtain 10g of the product, wherein the compound 17 is colorless to pale yellow flocculent solid, and the theoretical yield is as follows: 39.69g, actual yield: 13.4g, yield: 33.76%.
Synthesis of Compound 18:
2.12g of Compound 17 and 20ml of ethanol were added to a three-necked flask, and the system was stirred. Dropwise adding an ethanol solution of sodium borohydride (0.26 g) under an ice water bath, slightly heating a reaction system, transferring to room temperature for reaction after dropwise adding, gradually dissolving materials, sampling and detecting after the reaction solution is dissolved, and processing after the reaction is finished; adding 2mol/L hydrochloric acid until no bubbles are generated, wherein the pH value is=4-6, a large amount of solids are separated out, filtering to obtain solids, extracting filtrate with EA, separating an organic phase, and spin-drying the organic phase to obtain a product 18 which is brown solid and directly used for the next reaction.
Synthesis of Compound 19
2.5G of compound 10, 2.13g of compound 18, 5.44g of compound T3P (propylphosphoric anhydride), 1.1g of methanesulfonic acid and 30ml of EA are added into a single-port bottle, the mixture is heated to reflux, reacted overnight, sampled and detected, and the mixture is treated after the reaction is completed; post-treatment: saturated aqueous sodium bicarbonate was added until no bubbles evolved, the solution was separated, the aqueous phase was extracted with EA, the organic phases were combined, dried by spinning, and the crude product was slurried with PE to give product 19 as a pale brown powdery solid, theoretical yield: 3.36g, actual yield: 2.2g, yield: 65.48%.
Synthesis of Compound M01:
2.6g of compound 19, 0.94g of compound LiCl and 20ml of DMA (N, N-dimethylacetamide) are added into a single-port bottle, the temperature is raised to 100 ℃, the reaction liquid is yellow and turbid, the reaction is carried out for 2 hours, sampling detection is carried out, and the treatment is carried out after the reaction is completed; adding saturated sodium bicarbonate aqueous solution, separating out solid, filtering, extracting filtrate with EA, spin-drying organic phase, pulping crude product with PE to obtain 2.28g of compound M01, wherein the product is brown solid powder, and the yield is: 104% (yield slightly higher than theoretical yield, judged to be solvent DMA residual). Directly used in the next reaction. ESI-MS (+): m/z=501.1.
Synthesis of compound M11:
To a single vial was added 1.1g of compound M01, 0.61g of K 2CO3, 0.55g of KI, and 0.55g of chloromethyl dimethyl carbonate. And adding 20mL of DMA (direct memory access) into the system, heating to 60 ℃, reacting overnight, sampling and detecting, and processing after the reaction is finished. Cooling the reaction system to room temperature, adding 2N HCl, adding water, precipitating solid, suction filtering, extracting filtrate with EA, separating organic phase, dissolving filter cake with EA, mixing with organic phase, drying organic phase with anhydrous sodium sulfate, spin-drying, separating by column chromatography to obtain brown solid powder 0.766g of compound M11, yield ：58.9％.ESI-MS(+):m/z＝589.5;1H-NMR(DMSO-D6,400MHz)：δ7.26-7.41(m,5H),7.10-7.13(m,1H),6.92-6.98(m,2H),5.90-5.92(d,1H),5.74-5.86(d,1H),5.56-5.58(d,1H),5.38-5.42(m,2H),4.12-4.16(m,2H),4.01-4.07(m,1H),3.74(s,3H),2.79(s,1H),1.73-1.75(m,1H),1.16-1.24(m,2H),0.87-0.90(m,1H),0.72-0.74(m,1H).
Embodiment two: resolution of M19
The compound M11 is prepared and separated to obtain M19 and enantiomer M19-1, which are white solids. The preparation conditions are as follows: using a supercritical fluid chromatograph; chromatographic column: CHIRALPAK IB-N4.6 x 100mm,3 μm; the solvent is methanol, and the carrier is liquid CO 2; the pressure was 1500psi, the flow rate was set at 2.0ml/min, the column temperature 25℃and the elution was gradient. Wherein the retention time of M19 is 2.86min, and the result shows that the optical rotation is performed by taking methanol as a solvent. The retention time of M19-1 was 2.55min, and the result was shown as a D-isomer by optical rotation measurement using methanol as a solvent. M19 and M19-1 adopt the method of determining absolute configuration of chiral compound widely adopted internationally at present, quantitatively calculate and predict theoretical electron circular dichroism (ECD, namely commonly referred to as circular dichroism) spectrum, and compare with experimental ECD spectrum (comparison result is shown in figure 1), experimental ECD signal accords with theoretical calculation result, thus finally determining absolute configuration, M19 is S configuration, and M19-1 is R configuration.
Embodiment III: synthesis of PX90-02
Synthesis of Compound 25:
2g of compound SM1, 2.54g of compound SM2, 0.6g of pyridine and 30ml of 1, 4-dioxane were added to the reaction flask, and the system was heated at 60℃to react for 6 hours. After the reaction was completed, the system was concentrated to dryness, extracted with dichloromethane and water, and the organic phase was concentrated to dryness, and purified by a silica gel column to obtain 2.16g of compound 25 in 59% yield. ESI-MS (+): m/z= 542.48.
Synthesis of compound N01:
2g of the compound (25), 20ml of ethyl acetate and 0.88g of 5% palladium on charcoal were charged in the reaction flask, and the system was stirred at room temperature for 2 hours after being filled with a hydrogen balloon and replaced with hydrogen (1 atm). After filtration through celite the system was concentrated to dryness under reduced pressure and purified by a flash column to give 1.48g of compound N01 in 89% yield as ESI-MS (+): m/z= 452.78.
Synthesis of Compound PX 90-02-01:
1.2 g of Compound N01 was dissolved in acetonitrile (15 mL), 0.6mL of diisopropylethylamine was added, and 0.4 g of 2-bromoethanol was added. Then heated to 90℃and TLC checked for completion of the reaction. After cooling to room temperature, evaporating to dryness under reduced pressure, adding water and dichloromethane, shaking, collecting the organic phase, washing with 10% NaOH (10 ml), water (10 ml) and brine (10 ml), drying with sodium sulfate, filtering, evaporating to dryness under reduced pressure, purifying with silica gel column to obtain 0.95 g of compound PX90-02-01, yield 72%. M+h + = 496.13.
Synthesis of Compound PX 90-02:
To the reaction flask, 0.5 g of compound PX90-02-01,0.66 g of cesium carbonate, 33 mg of potassium iodide, 8ml of N, N-dimethylacetamide and 20ml of water were added, and the system was stirred and heated to 60℃and 5ml of an N, N-dimethylacetamide solution of 0.2 g of chloromethyl methyl carbonate was added. The system reacts for 8 hours at 55 ℃, is cooled to room temperature, is added with ethyl acetate, is washed by saturated ammonium chloride, water and saturated saline water in sequence, is separated into liquid and is dried by anhydrous sodium sulfate; the organic phase was concentrated and evaporated to dryness, and the residue was purified by silica gel column to give 0.46 g of compound PX90-02 in 46% yield. M+h + = 584.73.
Embodiment four: synthesis of M10
Synthesis of Compound 22:
synthesis of Compound 22 (Pharmaceutical Research,2005, vol.22, #3, p.390-396) was performed. 2.9g of Compound 21 in 10ml of dichloromethane was cooled to 0℃and 12ml of dimethylamine tetrahydrofuran was slowly added dropwise thereto, followed by reaction at room temperature for 24 hours. Concentrated to dryness, dichloromethane and water were added and the organic phase was separated and washed 3 times with 5% nahco 3 solution. The organic phase was concentrated to dryness to give 1.31g of compound 22 in 43% yield. M+h + = 137.92.
Synthesis of compound M10:
To the reaction flask, 0.5 g of Compound M01,0.65 g of cesium carbonate, 33 mg of potassium iodide, 8ml of N, N-dimethylacetamide and 20ml of water were added, and the system was stirred and heated to 60℃and 5ml of a solution of 0.15 g of Compound 22 in N, N-dimethylacetamide was added. The system reacts for 8 hours at 55 ℃, is cooled to room temperature, is added with ethyl acetate, is washed by saturated ammonium chloride, water and saturated saline water in sequence, is separated into liquid and is dried by anhydrous sodium sulfate; the organic phase was concentrated and evaporated to dryness, and the residue was purified by silica gel column to give 0.32 g of compound M10 in 53% yield. M+h + = 600.73.
Fifth embodiment: synthesis of M03 and M14
Synthesis of Compound 23
Under the protection of nitrogen, 1g of compound 17 is dissolved in 15ml of THF, 140mg of lithium aluminum hydride-D4 is slowly added at 0 ℃, and the system is heated to 25 ℃ to react for 8 hours. The system is cooled to 0 ℃, and water is added for quenching reaction. The system was extracted with 2N hydrochloric acid and EA. The organic phase was concentrated to dryness. Column chromatography gave compound 23 in a total of 0.58g, 57% yield, ESI-MS (+): m/z=314.0.
Synthesis of Compound 24
Referring to the synthetic method for compound 19, 0.94g of compound 24 was synthesized, ESI-MS (+): m/z= 592.1.
Synthesis of Compound M03
Referring to the synthesis of compound M01, 0.51g of compound M03 was synthesized, ESI-MS (+): M/z=502.1.
Synthesis of Compound M14
Referring to the synthesis of compound M11, 0.32g of compound M14 was synthesized, ESI-MS (+): M/z= 590.1.
Example six: synthesis of M07 and M08
Synthesis of Compound M08-1:
Under the protection of nitrogen, 1g of compound 17 is dissolved in 15ml of THF, 4ml of methyl lithium reagent (1.6M diethyl ether solution) is slowly added dropwise at-20 ℃, and the system is naturally warmed to 25 ℃ for reaction for 8 hours. The system is cooled to 0 ℃, and water is added for quenching reaction. Concentrating to dryness, and extracting with EA and water. The organic phase was separated and concentrated to dryness. Column chromatography gave 0.77g total of compound M08-1 in 73% yield, ESI-MS (+): M/z=327.1.
Synthesis of Compound M08-2
Referring to the synthesis of compound 19, 0.94g of compound M08-2 was synthesized, ESI-MS (+): M/z=605.2.
Synthesis of Compound M07
Referring to the synthesis of compound M01, 0.51g of compound M07 was synthesized, ESI-MS (+): M/z=515.1.
Synthesis of Compound M08
Referring to the preparation and separation method of the compound M01, the compound M08 is obtained, and the absolute configuration is S configuration after confirming that M08 is a levorotatory body.
Embodiment seven: synthesis of M24
Synthesis of Compound M04
The compound M01 is prepared and separated to obtain M04 and enantiomer, which are white solids. The preparation conditions are as follows: using a supercritical fluid chromatograph; chromatographic column: CHIRALPAK IB-N4.6 x 100mm,3 μm; the solvent is methanol, and the carrier is liquid CO 2; the pressure was 1500psi, the flow rate was set at 2.0ml/min, the column temperature 25℃and the elution was gradient. Optical rotation was performed using methanol as solvent, M04 being shown as the levorotatory form and the enantiomer of M04 being shown as the dextrorotatory form. As can be seen from comparison with M19 and M19-1, the absolute configuration of M04 is the S configuration.
Synthesis of Compound M24
100Mg of compound M04 are dissolved in 3ml of DMF in an ice-water bath, 16mg of sodium hydride (60%) are added and stirred for 30 minutes, 60mg of chloromethyl tert-butyrate are added, the reaction is allowed to slowly warm to room temperature for half an hour, the reaction is continued for 12 hours, after quenching with water, extraction is carried out 3 times with EA and water, the organic phases are combined, dried over anhydrous sodium sulfate, the product is isolated by chromatography and concentrated to give 41.8mg of compound M24 in a yield of 34% and ESI-MS (+): M/z=615.1.
Example eight: synthesis of Compound M26
Synthesis of Compound M26-3
4.7Ml of Compound M26-1 and 3ml of pyridine were dissolved in 30ml of dichloromethane, and the system was cooled to 0 ℃.3ml of compound M26-2 was slowly dropped into the above system, and the internal temperature was controlled to be not higher than 10 ℃. The system was reacted at room temperature for 16 hours, saturated sodium carbonate solution was added to the system for extraction, and the organic phase was concentrated to dryness under reduced pressure to give an oil, which was used directly in the next step.
Synthesis of Compound M26
Referring to the synthesis of compound M11, compound M04 and compound M26-3 reacted to give compound M26 in a total of 48mg, yield 42%, ESI-MS (+): M/z= 633.1.
Comparative example one: synthesis of N02
According to the preparation and separation method of the reference compound M19, the compound N02 is prepared and separated from the compound N01, and is subjected to optical rotation test and is compared with the optical rotation of the M19, and NO2 is a levorotatory body.
Comparative example two: synthesis of N11 and N12
Referring to the synthesis method of the compound M19, N01 is used as a material to synthesize N11 in one step, and then the compound N12 is obtained by a similar preparation and separation method of M19. And through optical rotation test and comparison with the optical rotation of M19, N12 is a levorotatory body and S configuration.
Comparative example three: synthesis of Compound N13
Synthesis of Compound N13-2
1G of compound N13-1 and 60ml of methylene chloride were added to the reaction flask, 1g of imidazole and 1.55g of t-butyldimethylchlorosilane (TBSCl) were added to the system at room temperature, and the system was allowed to react at room temperature for 3 hours. Concentrating to dryness, purifying with silica gel column to obtain 0.968g compound N13-2.1H-NMR(CDCl3,300MHz)σ:0.08(s,6H),0.42-0.58(m,4H),0.93(s,9H),2.76(brs,1H),3.44-3.60(s,2H),3.62-3.65(s,2H).
Synthesis of Compound N13-3
To the reaction flask, 0.91g of Compound N13-2 and 40ml of methylene chloride were added, and 1.96g of dess-Martin reagent (DMP) was added to the system at 0℃to carry out a reaction at 0℃for 30 minutes. The system was purified by silica gel column to give 0.72g of a compound N13-3.1H-NMR(CDCl3,300MHz)σ:0.05(s,6H),0.87(s,9H),1.12(s,4H),3.87-3.95(s,2H),8.97-9.04(s,1H).
Synthesis of Compound N13-4
Under nitrogen protection, 1.2g of triphenylphosphine bromomethane and 20ml of anhydrous tetrahydrofuran were added to the reaction flask. The temperature of the system was lowered to 0℃and 1.5ml (2.5M) of n-butyllithium was added thereto to react for 10 minutes. 10ml of an anhydrous tetrahydrofuran solution of Compound N13-3 (0.66 g) was added thereto, and the mixture was reacted at room temperature for 2 hours. Quenching the reaction with water, concentrating the system to dryness, extracting with dichloromethane and water, concentrating the organic phase to dryness, purifying with silica gel column to obtain 0.53g compound N13-4.1H-NMR(CDCl3,300MHz)σ:0.05(s,6H),0.50-0.75(m,4H),0.85-0.92(s,9H),3.62-3.66(s,2H),4.90-5.05(m,2H),5.65-5.75(m,1H).
Synthesis of Compound N13-5
To the reaction flask was added 0.52g of Compound N13-4, followed by 7.5ml of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (1M TBAF in THF). The reaction is carried out at room temperature. Concentrating the system to dryness, extracting with dichloromethane, and purifying with silica gel column to obtain 25mg of compound N13-5.1H-NMR(CDCl3,300MHz)σ:0.50-0.77(m,4H),3.60-3.66(s,2H),5.01-5.20(m,2H),5.61-5.75(m,1H).
Synthesis of Compound N13-6
Under nitrogen, 22mg of chloromethyl chloroformate was dissolved in anhydrous methylene chloride, 24mg of pyridine was added at 0℃and then 15mg of a methylene chloride solution of compound N13-5 was added dropwise. The system was then transferred to room temperature for 3 hours, and concentrated to dryness to give crude compound N13-6, which was used directly in the next step without work-up.
Synthesis of Compound N13
30Mg of compound M04 was added to 1ml of DMA (N, N-dimethylacetamide) at room temperature, followed by 2mg of potassium iodide and 16.6mg of potassium carbonate. The DMA solution of the compound N13-6 is added into the system and reacted for 5 hours at 50 ℃. 18.45mg of compound N13 was obtained by reverse phase column chromatography in a yield of 47%, ESI-MS (+): m/z 655.1[ M+H ].
The following examples were synthesized in the same manner as in the above examples, using commercially available compounds or intermediate compounds appropriately synthesized from the commercially available compounds:
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Or a hydrate, solvate, optical isomer, polymorph, isotopic derivative, pharmaceutically acceptable salt thereof.
Example nine: cytopathic degree (CPE) assay
MDCK cells are inoculated into 96-well culture plates and cultured at 37 ℃ with 5% CO 2. During the cell index growth period, maintaining liquid containing different dilutions of sample and positive reference medicine is added, and 3 compound wells are set in each concentration, and normal cell reference wells are set simultaneously. After the sample was added, the sample was cultured for 72 hours, and the cytotoxicity test of the sample was performed by the CPE method. MDCK cells were also inoculated into 96-well plates and incubated at 37 ℃ with 5% co 2. After 24 hours, influenza virus (A/han-defenses/359/95 (H3N 2)) was infected, adsorbed for 2 hours, the virus solution was discarded, a maintenance solution containing samples of different dilutions and positive control was added, 3 duplicate wells were set for each concentration, and cell control wells and virus control wells were set simultaneously, 5% CO 2, and incubated at 37 ℃. The antiviral test of the tested sample is carried out by a CPE method, and when the pathological change degree (CPE) of the virus control group reaches 4+, the cytopathic change degree (CPE) of each group is observed. The half toxic concentration of the sample on cells (TC 50) and the drug effective concentration (EC 50) to inhibit 50% of the cytopathic effect were calculated using the Reed-Muench method, respectively, as shown in table 1:
table 1: cytotoxicity of the compound and inhibitory Activity against influenza Virus
Sample description: in the experiments, N01, N02, N11 and N12 are compounds disclosed in the prior literature and are used as comparative examples in the invention.
Experiments show that the compound has better anti-influenza virus activity. Wherein, compared with the comparative example compounds N01, N02 and Ballon Sha Wei, the cell activities of M01 to M08 (all are non-esterified structures) in the compound of the invention are obviously better; compared with the comparative example compounds N11, N12 and Ballon Sha Weizhi, the compound also has better anti-influenza virus activity; and none of the compounds showed significant cytotoxicity.
Example ten: in vivo anti-influenza virus activity
BALB/c mice 30, females, 6-8 weeks old. The randomization was divided into 6 groups: vehicle group, positive control group, test group. Mice were vaccinated nasally on day 0 with virus (A/PR/8/34 (H1N 1)) at 1500p.f.u./mouse. The vehicle or positive control or test sample is continuously treated for 7 days from day 1 to day 7, 2 times daily, by gastric lavage, administration volume of 10mL/kg, and first administration time of 24 hours after virus inoculation. Animals were observed continuously from day 0 to day 14 and weight, health and survival were recorded.
As can be seen from fig. 2: vehicle group, group 3 (M19-1 low dose group) and group 4 (M19-1 high dose group) mice underwent a significant decrease in body weight starting on day 3, followed by a continued decrease until either died or euthanized; the mice in the baluo Sha Wei (5 mpk) group showed a significant decrease in body weight starting on day 3, with the body weight at day 3 falling to a minimum of 9.59% and then returning to normal levels starting on day 4; group 5 (M19 low dose group) mice showed a significant decrease in body weight starting on day 3, a minimum decrease in body weight on day 6 of 15.51% maximum, and a recovery starting on day 7 to normal levels. Group 6 (M19 high dose group) mice showed a significant decrease in body weight starting on day 3, with the 3 rd day body weight decreasing to the lowest point, the maximum decrease being 5% and the 5 th day starting to recover to normal levels.
As can be seen from fig. 3: mice in vehicle group died from day 6 and all died on day 9 with a final survival of 0%; group 3 (M19-1 low dose group) mice died from day 6 and all died on day 8 with a final survival of 0%; group 4 (M19-1 high dose group) mice died from day 7 and all died on day 8 with a final survival of 0%; no death occurred in both the baluo Sha Weizu and M19 high/low dose groups, and the survival rates were 100%.
The results show that: the mice in the vehicle group show infection symptoms after virus inoculation and finally die completely, the median survival time is 7.5 days, and the final survival rate is 0%; under the set experimental conditions, the baluo Sha Wei can relieve the weight loss of mice caused by virus infection, protect the mice from death, and show the expected in vivo anti-influenza virus drug effect. The results reach the selection standard, meet the expected model, prove experimental science and credibility, and provide references and windows for drug effect evaluation of tested compounds. Unexpectedly, the test compound M19-1, which performed better in the cell activity test, failed to alleviate the weight loss of mice due to infection under the set experimental conditions, indicating no in vivo anti-influenza virus efficacy. And the other test compound M19 with slightly poorer performance in the cell activity test starts to be administered after 24 hours of virus inoculation, can relieve the weight reduction of mice caused by virus infection, and protect the mice from death, and has good in vivo anti-influenza A virus drug effect.
Example eleven: virus titre test and histopathological examination
C57BL/6J mice 20 (female, 8 weeks, 18-20 g) were divided into 4 groups of 5. The test drug was administered by gavage twice daily after nasal drip inoculation with a semi-lethal dose of A/PR/8/34 (H1N 1) virus. Lung tissue samples were harvested 5 days post infection, examined for lung pathology, and right lung homogenates were assayed for TCID 50. The results of viral titers are shown in figure 4.
The pulmonary virus titer test results show that the pulmonary tissue virus titers of the compounds M19, M20 and the comparative compound N12 of the invention are lower compared with the vehicle group; the compounds M19 and M20 of the present invention showed lower viral titers and better anti-influenza virus effects at the same dose as compared to the comparative example compound N12. At the same time, the results of the lung histopathological examination also show that the lung diseases of the mice treated by the compounds M19 and M20 of the invention are slightly changed compared with the comparative example compound N12.
Embodiment twelve: oral pharmacokinetic test in rats
20 SD rats, male, 180g-220g, were cannulated for jugular vein before the start of the experiment, and after three days of adaptation (free feeding of drinking water, room temperature: 20-26 ℃ C.; humidity: 40-70%; light illumination: dark = 12h:12 h) the experiment was started. The experimental animals were divided into 6 groups of 3 animals each, A/B/C/D/E/F. The suspensions of the test substances (M04, M19, M24, M25, M26, and N13, each of which was suspended with 0.5% sodium carboxymethyl cellulose) were administered orally by gavage, respectively, at a dose of 2.25mg/kg in terms of M19, and equimolar. Fasted food begins at 5 pm on the day before administration but water is not forbidden, and fasted for 16-17h. Animals fed after 4h of administration, and water is not forbidden in the whole process.
15Min, 30min, 1h, 2h, 4h, 6h, 8h, 10h, 24h before and after administration. About 0.25mL of whole blood was taken through the jugular vein in heparin sodium anticoagulants, respectively. After taking the blood, the anticoagulation tube containing the blood sample is immediately reversed for 5-10 times and temporarily stored in an ice bath. The blood samples were centrifuged at 3000rpm at 4℃for 5 minutes within 1 hour after collection. Transferring the centrifugally collected plasma to a new labeled centrifuge tube, temporarily storing in a refrigerator at-20 ℃, and after all samples are collected, delivering the collected plasma to a biological sample manager and storing in the refrigerator at-80 ℃. The biological sample is treated and then tested for substance (M04 as the tested substance) by LC-MS/MS. The essential pharmacokinetic parameters were calculated according to the non-compartmental model method using winnonlin 7.0. The main pharmacokinetic parameters after intragastric administration of each set of samples are shown in table 2:
Table 2: main pharmacokinetic parameters of test sample in rat after equimolar gastric lavage administration
M04 M19 M24 M25 M26 N13
T1/2(h) 5.41 4.33 4.27 5.15 4.22 4.53
Tmax(h) 2 1 1 1 1 2
Cmax(ng/ml) 4.39 34.5 22.9 5.36 19.7 2.89
AUC(h*ng/ml) 66.974 289.914 147.285 81.360 145.629 40.970
In pharmacokinetic studies of intragastric administration in rats, the inventors selected several different ester compounds that are relatively close in structure and all of which are precedent for patent medicine, and the results showed that: the compounds of the invention have shorter peak time (Tmax) after oral gavage administration in rats compared to M04, indicating that the compounds of the invention can act rapidly; in addition, the plasma exposure (AUC) of compound M19 was significantly increased and the exposure of M24 and M26 was also increased to different extents, but the exposure of M25 and M13 was even reduced, compared to M04, indicating that in the pharmacokinetic experiments of rats, different ester-forming patterns affected the in vivo exposure of gavage administration, presumably due to different degrees of absorption or metabolism of the different ester-forming compounds.
Embodiment thirteen: mouse tissue distribution test
45 DBA/1J mice (6 to 8 week old, females) were weighed one day prior to dosing to calculate dosing. Randomly, 3 groups of 15 were divided, and 1mg/kg of M19, M20 and N12 (N12 was used as a comparative compound in this experiment) was administered by oral gavage. Mice were sampled at the following 5 time points: 1 hour, 2 hours, 4 hours, 8 hours and 24 hours (3 mice per time point). Mice were euthanized by CO 2 at each time point. Collecting organs (lung, liver, kidney, spleen and heart) from euthanized mice, cleaning the surface with physiological saline, sucking water with filter paper, weighing, placing into a glass homogenizing tube, adding 3 times of physiological saline, and grinding to obtain homogenate of each tissue. The homogenate is preserved at-80 ℃. The biological samples were processed and analyzed by LC-MS/MS. The results are shown in FIG. 5.
As can be seen from fig. 5: within 1-4 hours after administration, the three compounds reached the highest concentrations in all tissues, with the highest concentrations in lung tissue all reaching around 2 hours. The tissue concentration of compound N12 then decreased rapidly, whereas the decrease in concentration of compounds M19 and M20 of the invention was slower, was still detectable in lung tissue 24 hours after administration, but compound N12 was below the limit of detection 24 hours after administration, indicating that compounds M19 and M20 of the invention had a low rate of elimination in lung tissue and a long half-life.
The present application has been described in terms of several embodiments, but the description is intended to be illustrative and not limiting and many more embodiments and implementations are possible within the scope of the described embodiments.

Claims (5)

1. A compound of formula (I-0), or an isotopically derivative, pharmaceutically acceptable salt thereof:
in the formula (I-0), n is 0,1, 2,3 and 4;
R 0 is fluorine, chlorine, bromine, iodine, trifluoromethyl or cyano;
R a、Rb and R c are each independently selected from hydrogen, deuterium or methyl;
Q is hydrogen.
2. The compound of any one of claims 1, selected from the structures:
or an isotopically derivative, a pharmaceutically acceptable salt thereof.
3. A pharmaceutical composition comprising a compound of any one of claims 1-2 or an isotopic derivative thereof, a pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.
4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is in the form of tablet, capsule, powder, granule, pill, suspension, syrup, injection.
5. Use of a compound according to any one of claims 1-2, including isotopic derivatives, pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to claim 3, for the preparation of an anti-influenza virus medicament.
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