CN115960148B - Novel cytidine derivative, and pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a novel cytidine derivative, a pharmaceutical composition and application thereof, wherein the cytidine derivative is shown as a formula (I) ₀ ) Shown; the compound can be used for preparing medicines for resisting viral infection.

Description

A novel cytidine derivative and its pharmaceutical composition and use

Technical Field

The present invention relates to the field of pharmaceutical chemistry technology, but is not limited thereto, and in particular to a novel cytidine derivative and a pharmaceutical composition and use thereof.

Background Art

Influenza is an acute respiratory infection caused by influenza virus infection. It is a particularly important disease for high-risk groups such as infants and the elderly. Among the elderly, the incidence of pneumonia is high.

Since influenza viruses are highly variable, there is a long way to go in the development of anti-influenza virus drugs due to concerns about the emergence of drug-resistant strains or side effects, as well as the spread of pathogenic or lethal new influenza viruses. Therefore, the field is still eager to develop antiviral drugs with new structures.

Summary of the invention

The present inventors have developed a novel cytidine derivative having antiviral effects and low cytotoxicity.

In one aspect, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof as shown in (I ₀ ):

In formula (I ₀ ),

_R1 and _R2 are independently selected from hydrogen, Alternatively, R ₁ and R ₂ form an acetal or ketal with their adjacent oxygen;

wherein n ₁ is selected from 0, 1, 2 or 3;

n ₂ is selected from 1, 2 or 3;

_Ra and _Rb are independently selected from hydroxyl, the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, C2-C8 alkenyl, C3-C8 cycloalkyl, C6-C18 aryl, aryloxy, arylalkyl, alkylaryl;

R _c and R _d are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by one or more groups A;

_R3 and _R4 are the same or different and are independently selected from hydrogen or _R3 and _R4 cannot both be hydrogen;

wherein _na is selected from 0, 1, 2, 3, 4, or 5;

n _b is selected from 1, 2, 3, 4, or 5;

n ₃ is selected from 0, 1, 2, 3, 4, or 5;

n ₄ is selected from 0, 1, 2, 3, or 4;

R ₅ and R ₆ are the same or different and are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by one or more groups A; or R ₅ , R ₆ and the carbon to which they are connected form a cycloalkyl group;

_R7 is hydrogen, halogen, amino, C1-C8 alkyl which is substituted or unsubstituted by one or more groups A;

Z is selected from

wherein n ₅ is independently 0, 1, 2, 3, 4, or 5;

R ₈ is selected from H, hydroxy, nitro, halogen, the following groups which are substituted or unsubstituted by one or more groups A: amino, C1-C8 alkyl, C6-C18 aryl, C1-C8 alkoxy, aminoalkyl, C1-C8 alkylaryl, arylcarbonyl, C1-C8 alkylcarbonyloxy;

R _e and R _f are independently selected from hydrogen, the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C3-C8 cycloalkyl, heterocycloalkyl, C6-C18 aryl, heteroaryl, non-aromatic heterocyclic group;

_RX1 , _RX2 , _RX3 , _RX4 , _RX5 , _RX6 , _RX7 and _RX8 are independently selected from hydrogen and deuterium;

The group A is: hydroxyl, carboxyl, amino, halogen, cyano, aldehyde, nitro, trifluoromethyl, C3-C8 cycloalkyl, C1-C8 alkoxy, chlorophenylcarbonyl.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof as shown in formula (I ₀ -1):

The substituents in formula (I ₀ -1) are as defined above.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof as shown in formula (I ₀ -2):

The substituents in formula (I ₀ -2) are as defined above.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -3):

The definitions of the substituents in formula (I ₀ -3) are as described above. In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -4):

The substituents in formula (I ₀ -4) are as defined above.

In one aspect, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, and pharmaceutically acceptable salt thereof as shown in (I ₀ -1):

In formula (I ₀ -1),

_R1 and _R2 are the same or different and are independently selected from hydrogen, Alternatively, R ₁ and R ₂ together form an acetal or ketal with their adjacent oxygen;

wherein _n1 and _n2 are selected from 0, 1, 2 or 3;

_Ra and _Rb are independently selected from hydroxyl, alkyl substituted or unsubstituted by group A, alkoxy substituted or unsubstituted by group A, alkenyl substituted or unsubstituted by group A, cycloalkyl substituted or unsubstituted by group A, aryl substituted or unsubstituted by group A, aryloxy substituted or unsubstituted by group A, arylalkyl substituted or unsubstituted by group A, alkylaryl substituted or unsubstituted by group A;

R _c and R _d are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by group A;

_R3 and _R4 are the same or different and are independently selected from hydrogen and _R3 and _R4 cannot both be hydrogen;

wherein n _a , n _b and n ₃ are independently selected from 0, or 1, or 2, or 3, or 4, or 5;

n ₄ is selected from 0, 1, 2, 3 or 4;

R ₅ and R ₆ are the same or different and are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by group A; or C cycloalkyl to which R ₅ , R ₆ are connected;

R ₇ is selected from hydrogen, halogen, amino, C1-C8 alkyl substituted or unsubstituted by group A;

Z is selected from

wherein n ₅ is independently 0, or 1, or 2, or 3, or 4, or 5;

R ₈ is selected from H, hydroxyl, halogen, amino substituted or unsubstituted by group A, C1-C8 alkyl substituted or unsubstituted by group A, aryl substituted or unsubstituted by group A, C1-C8 alkyloxy substituted or unsubstituted by group A, aminoalkyl substituted or unsubstituted by group A, C1-C8 alkylaryl substituted or unsubstituted by group A, arylcarbonyl substituted or unsubstituted by group A, C1-C8 alkylcarbonyloxy substituted or unsubstituted by group A;

The group A is: hydroxyl, carboxyl, amino, halogen, cyano, aldehyde, nitro, trifluoromethyl, C3-C8 cycloalkyl, C1-C8 alkoxy, chlorophenylcarbonyl.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -1-1):

The substituents in formula (I ₀ -1-1) are as defined above.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -1-2):

The substituents in formula (I ₀ -1-2) are as defined above.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -1-3):

The substituents in formula (I ₀ -1-3) are as defined above.

In one aspect, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, and pharmaceutically acceptable salt thereof as shown in (I ₀ -1):

In formula (I ₀ -1),

_R1 and _R2 are independently selected from hydrogen, Alternatively, R ₁ and R ₂ together form an acetal or ketal with their adjacent oxygen;

wherein _n1 and _n2 are selected from 0, 1, 2 or 3;

_Ra and _Rb are independently selected from hydroxyl, alkyl substituted or unsubstituted by group A, alkoxy substituted or unsubstituted by group A, alkenyl substituted or unsubstituted by group A, cycloalkyl substituted or unsubstituted by group A, aryl substituted or unsubstituted by group A, aryloxy substituted or unsubstituted by group A, arylalkyl substituted or unsubstituted by group A, alkylaryl substituted or unsubstituted by group A;

R _c and R _d are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by group A;

_R3 and _R4 are the same or different and are independently selected from hydrogen, or _R3 and _R4 cannot be hydrogen at the same time;

Wherein, n _a and n _b are independently selected from 1, or 2, or 3, or 4, or 5;

n ₃ is selected from 0, or 1, or 2, or 3, or 4, or 5;

n ₄ is selected from 0, 1, 2, 3 or 4;

R ₅ and R ₆ are the same or different and are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by group A; or R ₅ , R ₆ and the carbon to which they are connected form a cycloalkyl group;

_R7 is hydrogen, halogen, amino, C1-C8 alkyl which is substituted or unsubstituted by group A;

Z is selected from in,

n ₅ are independently 0, 1, 2, 3, 4, or 5;

R ₈ is selected from H, hydroxyl, halogen, amino substituted or unsubstituted by group A, C1-C8 alkyl substituted or unsubstituted by group A, aryl substituted or unsubstituted by group A, C1-C8 alkyloxy substituted or unsubstituted by group A, aminoalkyl substituted or unsubstituted by group A, C1-C8 alkylaryl substituted or unsubstituted by group A, arylcarbonyl substituted or unsubstituted by group A, C1-C8 alkylcarbonyloxy substituted or unsubstituted by group A;

_Re and _Rf are independently selected from hydrogen, the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C3-C8 cycloalkyl, heterocycloalkyl, C6-C18 aryl, heteroaryl, non-aromatic heterocyclic group; wherein _Re and _Rf cannot be hydrogen at the same time;

The group A is: hydroxyl, carboxyl, amino, halogen, cyano, aldehyde, nitro, trifluoromethyl, C3-C8 cycloalkyl, C1-C8 alkoxy, chlorophenylcarbonyl.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -1-1):

The substituents in formula (I ₀ -1-1) are as defined above.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salts thereof of formula (I ₀ -1-2):

The substituents in formula (I ₀ -1-2) are as defined above.

In some embodiments, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof of formula (I ₀ -1-3):

The substituents in formula (I ₀ -1-3) are as defined above.

In one aspect, the present invention provides a novel cytidine derivative, tautomer, stereoisomer, and pharmaceutically acceptable salt thereof as shown in (I ₀ -5):

In formula (I ₀ -5),

_R1 and _R2 are independently selected from hydroxyl, In particular, when R _x6 and R _x7 are both hydrogen or deuterium, and R ₁ and R ₂ are independently selected from C1-8 alkoxy, R ₁ and R ₂ may form an acetal or ketal together with the carbon atom to which they are attached;

Wherein, the above _n1 and _n2 are independently selected from 0, 1, 2 or 3;

_Ra and _Rb are independently selected from hydroxyl, substituted or unsubstituted by one or more groups A: alkyl, alkoxy, alkenyl, cycloalkyl, aryl, aryloxy, arylalkyl, alkylaryl;

R _c and R _d are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by one or more groups A;

_R3 and _R4 are the same or different and are independently selected from hydrogen, or _R3 and _R4 cannot be hydrogen at the same time;

wherein n _a , n _b and n ₃ are independently selected from 0, 1, 2, 3, 4 or 5;

n ₄ is selected from 0, 1, 2, 3 or 4;

R ₅ and R ₆ are the same or different and are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by one or more groups A; or R ₅ , R ₆ and the carbon to which they are connected form a cycloalkyl group;

_R7 is hydrogen, halogen, amino, C1-C8 alkyl which is substituted or unsubstituted by one or more groups A;

Z is selected from in,

n ₅ are independently 0, 1, 2, 3, 4, or 5;

R ₈ is selected from H, hydroxyl, halogen, the following groups substituted or unsubstituted by one or more groups A: amino, C1-C8 alkyl, aryl, alkyloxy, aminoalkyl substituted or unsubstituted by one or more groups A, C1-C8 alkylaryl, arylcarbonyl, C1-C8 alkylcarbonyloxy;

R _e and R _f are independently selected from hydrogen, the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C3-C8 cycloalkyl, heterocycloalkyl, C6-C18 aryl, heteroaryl, non-aromatic heterocyclic group;

_RX1 , _RX2 , _RX3 , _RX4 , _{RX5, RX6, RX7 and RX8 are independently selected from hydrogen and deuterium. In particular, RX1, RX2, RX3, RX4, RX5 , RX6 , RX7 and RX8 cannot be hydrogen at the} same time.

The group A is: hydroxyl, carboxyl, amino, halogen, cyano, aldehyde, nitro, trifluoromethyl, C3-C8 cycloalkyl, C1-C8 alkoxy, chlorophenylcarbonyl.

In some embodiments, in the above formula (I ₀ ) and/or (I ₀ -2)-(I ₀ -4), _RX1 , _RX2 , _RX3 , _RX4 are independently selected from hydrogen and deuterium;

In some embodiments, in the above formula (I ₀ -1), _RX1 , _RX2 , _RX3 , _RX4 , _RX5 , _RX6 , _RX7 and _RX8 are all hydrogen.

In some embodiments, in the above formulae (I ₀ -2)-(I ₀ -4), _RX5 , _RX6 , _RX7 and _RX8 are all hydrogen.

In some embodiments, in the above formula (I ₀ )-(I ₀ -4), R ₁ and R ₂ are both hydrogen;

In some embodiments, in the above formula (I ₀ )-(I ₀ -1), R ₁ is hydrogen, and R ₂ is selected from wherein n ₁ is selected from 0, 1, 2 or 3;

In some more specific embodiments, n ₁ is selected from 0 or 1;

n ₂ is selected from 1, 2 or 3;

In some more specific embodiments, _n2 is 1;

_Ra and _Rb are independently selected from hydroxyl, the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkoxy, aryloxy, alkylaryl;

In some more specific embodiments, the above-mentioned _Ra and _Rb are independently selected from hydroxyl, the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C1-C8 alkyloxy;

In some embodiments, in the above formula (I ₀ )-(I ₀ -1), R ₂ is hydrogen, and R ₁ is selected from wherein n ₁ , n ₂ , _Ra and R _b are as defined above;

In some embodiments, in the above formula (I ₀ )-(I ₀ -1), R ₁ and R ₂ are not hydrogen, and are independently selected from wherein n ₁ , n ₂ , _Ra and R _b are as defined above respectively.

In some embodiments, in the above formula (I ₀ )-(I ₀ -2), R ₄ is hydrogen, R ₃ is

In some embodiments, _na is selected from 0, 1, 2, or 3;

In some more specific embodiments, _na is 0;

In some more specific embodiments, _na is 1;

In some embodiments, n _b is selected from 0, 1, 2, or 3;

In some more specific embodiments, n _b is 1;

In some embodiments, n ₃ is selected from 0, 1, 2, 3;

In some more specific embodiments, n ₃ is 0;

In some more specific embodiments, _n3 is 2;

In some more specific embodiments, _n3 is 3;

In some embodiments, n ₄ is selected from 0, 1, 2;

In some more specific embodiments, n ₄ is 0;

In some more specific embodiments, n ₄ is 2;

In some embodiments, the above-mentioned R _e is hydrogen, and R _f is the following groups which are substituted or unsubstituted by one or more groups A: C1-C8 alkyl, C3-C8 cycloalkyl, heterocycloalkyl, C6-C18 aryl, heteroaryl, non-aromatic heterocyclic group;

In some more specific embodiments, the above-mentioned _Re is hydrogen, and _Rf is methyl;

In some embodiments, the above-mentioned _Re and _Rf are both C1-C8 alkyl groups which are substituted or unsubstituted by group A;

In some more specific embodiments, the above-mentioned _Re and _Rf are both C1-C8 alkyl;

In some embodiments, the above R ₅ and R ₆ are the same or different, and are independently selected from hydrogen, C1-C8 alkyl substituted or unsubstituted by one or more groups A; or C cycloalkyl to which R ₅ , R ₆ are connected;

In some more specific embodiments, the above R ₅ and R ₆ are both C1-C4 alkyl; preferably, R ₅ and R ₆ are both methyl;

In some embodiments, the above R ₅ is methyl, and R ₆ is selected from C1-C4 alkyl substituted or unsubstituted by one or more groups A; preferably, R ₅ is methyl, and R ₆ is ethyl;

In some embodiments, the above R ₇ is hydrogen, halogen, amino, C1-C8 alkyl substituted or unsubstituted by one or more groups A;

In some more specific embodiments, the above R ₇ is selected from hydrogen, or methyl;

In some embodiments, the above Z is

In some embodiments, the above n ₅ is selected from 0, 1, 2, 3, 4, or 5;

In some more specific embodiments, the above n ₅ is preferably selected from 0, 1, or 2;

In some embodiments, the above R ₈ is selected from hydrogen, hydroxyl, nitro, halogen, the following groups which are substituted or unsubstituted by one or more groups A: amino, C1-C8 alkyl, C6-C18 aryl, C1-C8 alkoxy, C1-C8 alkylaryl, C1-C8 alkylcarbonyloxy;

In some more specific embodiments, the above R ₈ is preferably selected from hydrogen, nitro, chlorine, bromine, the following groups which are substituted or unsubstituted by one or more groups A: amino, C1-C3 alkyl, C6-C18 aryl, C1-C3 alkoxy, C1-C3 alkylcarbonyloxy;

In some more specific embodiments, the above Z is selected from 4-chlorophenylcarbonyl, hydrogen, chlorine, 4-chlorobenzyl,

The group A is: hydroxyl, carboxyl, amino, halogen, cyano, aldehyde, nitro, trifluoromethyl, C3-C8 cycloalkyl, C1-C8 alkoxy, chlorophenylcarbonyl.

In some embodiments, in the above formula (I ₀ )-(I ₀ -1) and/or (I ₀ -3), R ₃ and R ₄ are wherein Z, _R5 , _R6 , _R7 , _Re , _Rf , _na , _nb , _n3 and _n4 are as defined above.

In some embodiments, in the above formula (I ₀ )-(I ₀ -1) and/or (I ₀ -4), R ₃ is hydrogen, R ₄ is wherein Z, _R5 , _R6 , _R7 , _Re , _Rf , _na , _nb , _n3 and _n4 are as defined above.

In some embodiments, the novel cytidine derivatives, tautomers, stereoisomers, isotope derivatives and pharmaceutically acceptable salts thereof provided by the present invention are selected from the following compounds:

On the other hand, in some embodiments, the present invention provides a pharmaceutical composition comprising the above-mentioned novel cytidine derivatives, tautomers, stereoisomers, isotopic derivatives and pharmaceutically acceptable salts thereof.

In some embodiments, the present invention discloses a pharmaceutical composition, which comprises the compound, isomer or pharmaceutically acceptable salt thereof described in the present invention as an active ingredient or a main active ingredient, and is supplemented with a pharmaceutically acceptable carrier.

On the other hand, in some embodiments, the present invention provides the above-mentioned novel cytidine derivatives, tautomers, stereoisomers, isotope derivatives and pharmaceutically acceptable salts thereof which can be used to treat and/or prevent diseases related to antiviral disorders.

In some embodiments, the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of antiviral drugs; wherein the above-mentioned viruses include but are not limited to: Arenaviridae, Filoviridae and Coronaviridae viruses, etc., including but not limited to adenovirus, rhinovirus, influenza virus, Lassa virus, respiratory syncytial virus, severe acute respiratory syndrome virus, parainfluenza virus, coronavirus, etc.

In some embodiments, the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of antiviral drugs; wherein the above-mentioned influenza viruses and coronaviruses include but are not limited to: influenza A virus, influenza B virus, SARS virus, MERS virus, COVID-19 virus, etc.

In some embodiments, the novel cytidine derivatives of the present invention can be formulated into pharmaceutical compositions and administered to patients according to a variety of suitable administration routes, including systemic administration such as oral or parenteral, intravenous, intramuscular, transdermal or subcutaneous administration.

Compared with compound A disclosed in CN111372592A, the compound disclosed in the present invention has better stability.

Compared with compound A disclosed in CN111372592A, the compound disclosed in the present invention has better anti-influenza virus activity, lower cytotoxicity and higher selectivity index.

Compared with compound A, the compound disclosed in the present invention has better anti-new coronavirus activity.

The bioavailability of the compound disclosed in the present invention is nearly 1.4 times that of compound A, and has a better safety range.

Compared with the combination of compound A and compound C, the compounds disclosed in the present invention have more excellent therapeutic and preventive effects in terms of anti-influenza virus. According to the excellent therapeutic and preventive effects, after the compounds of the present invention enter the body, the two metabolic components play a certain synergistic role. The compounds of the present invention can be used as antiviral drugs with new structures.

The structure of compound A is as follows:

The structure of compound C is as follows:

definition:

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

Certain compounds of the present invention may exist in unsolvated or solvated forms, such as hydrates and ethanolates. Generally speaking, the solvated forms are equivalent to the unsolvated forms and are all included in the scope of the present invention.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents discovered by the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include aluminum, sodium, potassium, calcium, manganese, iron, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, etc.; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight chain and branched groups. Alkyl groups may be substituted or unsubstituted. When substituted alkyl groups, the substituents are preferably one or more, more preferably 1-3, and most preferably 1 or 2 substituents.

The term "alkenyl" refers to an aliphatic hydrocarbon group containing an unsaturated carbon-carbon double bond, including straight-chain and branched groups. The alkyl group may be substituted or unsubstituted. The carbon-carbon double bond may be one or more.

The term "cycloalkyl" refers to a monocyclic or fused ring ("fused" ring means that each ring in the system shares a pair of adjacent carbon atoms with other rings in the system) group that is all carbon, wherein one or more rings do not have a completely connected π electron system, examples of cycloalkyl (not limited to) are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and cycloheptatriene. Cycloalkyl groups may be substituted and unsubstituted.

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 1 to 12 carbon atoms with a completely conjugated π electron system. Non-limiting examples of aryl groups include phenyl, naphthyl and anthracenyl. Aryl groups may be substituted or unsubstituted. When substituted, the substituents are preferably one or more, more preferably one, two or three, and even more preferably one or two.

The term "arylalkyl" refers to an alkyl group substituted with an aryl group.

The term "heteroaryl" refers to a monocyclic or fused ring group of multiple atoms containing one, two, three or four ring heteroatoms selected from N, O or S, the remaining ring atoms being C, and having a completely conjugated π electron system. Non-limiting examples of unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine and carbazole.

The term "alkoxy" refers to a group in which an alkyl group is linked to an oxygen group, where the alkyl group may be a straight chain, branched chain or cyclic alkyl group.

The term "hydroxy" refers to the -OH group.

The term "amino" refers to a _-NH2 group.

The term "carboxy" refers to a -COOH group.

The term "halogen" denotes fluorine, chlorine, bromine or iodine.

The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc.

The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

Numeric ranges mentioned in this application, such as "C1-C8", mean that the group may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 8 carbon atoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the therapeutic administration trial against influenza virus (H1N1).

FIG. 2 shows the results of the preventive administration test against influenza virus (H1N1).

DETAILED DESCRIPTION

Many exemplary preparation methods of the compounds of the present invention are provided in the following examples. The present invention is described in detail below by way of example, but it is not intended to limit the present invention in any adverse way. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed, and it will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. Certain compounds of the present invention can be used as intermediates for preparing other compounds of the present invention, and the structures of all compounds are determined by LC-MS or NMR.

The raw materials in the examples of this application were purchased through commercial channels.

Example 1: Synthesis of compound PY-01

Reaction:

Preparation method:

Step 1: Preparation of compound PY-0102:

Tetrahydrofuran (300 ml), PY-01-SM2 (31.8 g, 100 mmol), 4-dimethylaminopyridine (DMAP, 18.33 g, 150 mmol) were added to the reaction flask in sequence, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 18.63 g, 120 mmol) and compound PY-01-SM1 (28.43 g, 100 mmol) were added after dissolution, the temperature was raised to 70°C, the reaction was stirred, TLC was monitored for completion of the reaction, the system was cooled, evaporated to dryness, 200 ml of ethyl acetate and 100 ml of water were added, the organic phase was separated, washed twice with water again, dried, evaporated to dryness under reduced pressure, and the residue was purified by column to obtain the product PY-0102 (44.34 g) with a yield of 75.8%. ESI-MS (+): m/z=585.16.

Step 2: Preparation of compound PY-0101:

Compound PY-0102 (44.00 g, 75.2 mmol), N,N-dimethylformamide (250 ml), DIPEA (19.44 g, 150.4 mmol) were added to the reaction flask, and PyBroP (38.55 g, 82.7 mmol) was added after dissolution. The system was stirred at room temperature for 30 min, and then hydroxylamine hydrochloride (62.71 g, 90.24 mmol) was added. The reaction was carried out at 40-50°C for 4-6 h. The reaction was completed by TLC detection, and the temperature was lowered, water was added, and ethyl acetate was extracted three times. The organic phases were combined, washed with water twice, evaporated to dryness under reduced pressure, and the residue was recrystallized from methyl tert-butyl ether/n-heptane to obtain product PY-0101 (39.00 g), with a yield of 86.4%. ESI-MS (+): m/z = 600.17.

Step 3: Preparation of compound PY-01:

PY-0101 (38.00 g, 63.3 mmol) and formic acid (500 ml) were added to the reaction flask, and the system was reacted at room temperature for 20 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was recrystallized from isopropanol/methyl tert-butyl ether to obtain compound PY-01 (29.21) with a yield of 82.4%. ESI-MS(+): m/z=560.2. 1H NMR (DM SO-D6, 500MHz): δ10.02(s,1H),9.49(s,1H),7.72-7.75(m,4H),7.63-7.64(d,2H),6.94-6.96(d,2H),6.78-6.80(d,1H) ),5.71-5.72(d,1H),5.46-5.48(d,1H),5.32-5.33(d,1H), 5.20-5.21(d,1H),4.28-4.36(m,2H),3.97(s,1H),3.89-3.92(m,1H),3.78-3.79(d,1H ),1.64-1.66(d,6H).

Example 2: Synthesis of Compound PY-02

Reaction:

Preparation method:

Step 1: Preparation of compound PY-0201

Referring to the preparation of step 1 of Example 1, compound PY-0101 was used to replace compound PY-01-SM1 to prepare compound PY-0201 (13.21 g) with a yield of 73.5%. ESI-MS (+): m/z = 900.22.

Step 2: Preparation of compound PY-02

Referring to the preparation of step 3 of Example 1, compound PY-0201 was used to replace compound PY-0101 to prepare compound PY-02 (10.14 g) with a yield of 83.2%. ESI-MS (+): m/z = 860.19.

Example 3: Synthesis of Compound PY-03

Reaction:

Preparation method:

Step 1: Preparation of compound PY-0303

Under nitrogen protection, compound PY-03-SM3 (25.0 g, 102.0 mmol) and 500 ml of dichloromethane were added to a three-necked flask. The resulting solution was cooled to 0°C, and DMAP (1.3 g, 10.6 mmol) and imidazole (27.9 g, 409.0 mmol) were added in sequence. TBSCl (61.7 g, 40.0 mmol) was added within 10 minutes, and the resulting mixture was warmed to ambient temperature and stirred for 18 hours. 300 mL of water was added to the system, stirred at room temperature for 2 hours, separated, the aqueous phase was extracted 3 times with dichloromethane, the combined organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by column to obtain compound PY-0303 (43.4 g) with a yield of 72.5%. ESI-MS (+): m/z = 587.33.

Step 2: Preparation of compound PY-0302

Compound PY-0303 (28.0 g, 47.7 mol) and dichloromethane (700 mL) were placed in a 1 L round bottom flask. The solution was cooled to 0 ° C using an ice bath; DMAP (0.583 g, 4.77 mmol) and N, N-diisopropylethylamine (30.9 g, 239 mmol) were added in sequence. 2,4,6-triisopropylphenyl-1-sulfonyl chloride (28.9 g, 95 mmol) was slowly added to the flask. After the addition, the flask was warmed to ambient temperature and stirred for 18 hours. The system was cooled to 0 ° C, N, N-diisopropylethylamine (24.6 g, 191 mol) was added dropwise, and then solid hydroxylamine hydrochloride (13.26 g, 191 mmol) was immediately added. The mixture was warmed to room temperature and stirred for 3 hours. The reaction was quenched with water (200 mL) and the resulting layers were separated. The aqueous layer was extracted with dichloromethane (200 mL), and the combined organic matter was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column to obtain compound PY-0302 (20.0 g) with a yield of 69.5%. ESI-MS (+): m/z = 602.34.

Step 3: Preparation of compound PY-0301

Referring to the preparation of step 1 of Example 1, compound PY-0302 was used to replace compound PY-01-SM1 to prepare compound PY-0301 (18.6 g) with a yield of 76.2%. ESI-MS (+): m/z = 902.40.

Step 4: Preparation of compound PY-03

Compound PY-0301 (17.5 g, 19.4 mol) and tetrahydrofuran (50 mL) were added to a three-necked flask, followed by triethylamine trihydrofluoride (3.1 g, 19.4 mmol), and the mixture was stirred at ambient temperature for 18 hours. The mixture was concentrated under reduced pressure, and the residue was dissolved in a minimum amount of MeOH, and the solution was slowly added to a conical flask containing rapidly stirred dichloromethane (50 mL), and the mixture was stirred at room temperature for 15 minutes. Filtered and recrystallized from dichloromethane and petroleum ether to obtain the title compound PY-03 (7.65 g) with a yield of 70.5%. ESI-MS(+):m/z＝560.25。1H NMR(DMSO-D6,400MHz):δ11.34-11.35(s,1H),10.79-10.80(s,1H),7.70-7.74(m,4H),7.61-7. 63(d,2H),7.48-7.51(d,1H),6.94-6.96(d,2H),5.72-5.78(m,2H),5.32-5.34(d,1H),5.02-5.08(d,2H),3.96-4.04(m,2H),3.83-3.84(m,1H),3.55(s,2H),1.76(s,3H),1.69(s,3H)。

Example 4: Synthesis of Compound ZJT1

Reaction:

Preparation method:

Step 1: Preparation of compound ZJT1-03

In a three-necked flask, 500 ml of 80% methylamine solution, anisole (36.4 g, 0.34 mol), and cuprous chloride (35.6 g, 0.36 mol) were added. The solution temperature was controlled at 35°C. p-Bromobenzamide (101.9 g, 0.35 mol) was added dropwise. After the addition, the solution temperature was raised to 45°C. The stirring speed was maintained to react for 7 hours. The solution temperature was lowered to 30°C. The reaction solution was poured into a 30% sodium bisulfite solution. The solution temperature was maintained at 0-5°C. The organic layer was separated, and the aqueous layer was extracted 6 times with methylamine solution. After the organic layers were combined, methylamine was evaporated to obtain compound ZJT1-03 for later use.

Step 2: Preparation of compound ZJT1-02

Add 300 ml of hexane to the above-mentioned compound ZJT1-03 spare product, heat to 60°C, add cuprous chloride (59.4 g, 0.6 mol), reflux for 2 h, evaporate the hexane, lower the solution temperature to 5°C, add 200 ml of 60% potassium bicarbonate solution, stir for 2 h, filter, wash with potassium sulfate solution, and obtain compound ZJT1-02 (79.2 g), with a yield of 84.1%. ESI-MS (+): m/z = 276.98.

Step 3: Preparation of compound ZJT1

Acetone (120 g, 2.07 mol) and compound ZJT1-02 (59.6 g, 0.21 mol) were added to a three-necked flask and stirred for about 10 minutes. Sodium hydroxide (60.0 g, 1.5 mol) was added and chloroform (45 ml) was added dropwise at 20-30°C with stirring. After the addition of chloroform was completed, the mixture was kept at 20-30°C for 1.5 h, and then heated to reflux for 3.5 h. The organic solvent was evaporated under reduced pressure, and 100 ml of water and 130 ml of toluene were added to the residue. 36% hydrochloric acid (about 60 g) was added dropwise to acidify the liquid to p=3.5-4.5, and then 100 ml was added. The system was cooled to room temperature and stirred for 2 h, filtered, and dried to obtain compound ZJT1 (66.5 g) with a yield of 87.2%. ESI-MS (-): m/z=361.01.

Example 5: Synthesis of Compound ZJT2

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 4, the starting material was replaced by p-nitrobenzamide to obtain compound ZJT2 (55.2 g) with a yield of 85.8%. ESI-MS (-): m/z = 328.09.

Example 6: Synthesis of Compound ZJT3

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 4, the starting material was replaced by p-aminobenzamide to obtain compound ZJT3 (51.9 g) with a yield of 86.1%. ESI-MS (-): m/z = 298.12.

Example 7: Synthesis of Compound ZJT4

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 4, the starting material was replaced by 3-chlorobenzamide to obtain compound ZJT4 (57.6 g) with a yield of 82.9%. ESI-MS (-): m/z = 317.07.

Example 8: Synthesis of Compound ZJT5

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 4, the starting material was replaced with 3,4-dichlorobenzamide to obtain compound ZJT5 (46.1 g) with a yield of 79.6%. ESI-MS (-): m/z = 351.03.

Example 9: Synthesis of Compound ZJT6

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 4, the starting material was replaced by p-methoxybenzamide to obtain compound ZJT6 (31.3 g) with a yield of 87.1%. ESI-MS (-): m/z = 313.12.

Example 10: Synthesis of Compound ZJT7

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 4, the starting material was replaced with 3,4,5-trimethoxybenzamide to obtain compound ZJT7 (32.6 g) with a yield of 84.3%. ESI-MS (-): m/z = 373.14.

Example 11: Synthesis of compound PY-ZJT8

Reaction:

Preparation method:

Referring to the operating procedures of each step of Example 4, the starting material was replaced by 3-(trifluoromethyl)benzamide to obtain compound ZJT8 (36.8 g) with a yield of 81.1%. ESI-MS (-): m/z = 351.09.

Example 12: Synthesis of compound PY-ZJT9

Reaction:

Preparation method:

Compound PY-01-SM2 (21.5 g, 67.5 mmol), water (300 mL), dichloromethane (300 mL), tetrabutylammonium hydrogen sulfate (2.3 g, 6.75 mmol) and sodium bicarbonate (22.7 g, 270 mmol) were placed in a three-necked flask. Chloromethyl chlorosulfonate (13.5 g, 81.7 mmol) was added dropwise at room temperature, and the reaction was stirred at room temperature overnight. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted with dichloromethane (300 mL). The organic phases were combined, dried, concentrated, and purified by column (0 to 5% ethyl acetate/hexane gradient elution) to obtain compound ZJT9 (18.7 g) with a yield of 75.4%.

Example 13: Synthesis of Compound PY-04

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 1, compound ZJT1 was used to replace compound PY-01-SM2 as the starting material to obtain compound PY-04 (10.8 g) with a total yield of 51.1%. ESI-MS (+): m/z = 604.09.

Example 14: Synthesis of Compound PY-05

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 2, compound ZJT1 was used to replace compound PY-01-SM2 as the starting material to obtain compound PY-05 (12.3 g) with a total yield of 57.6%. ESI-MS (+): m/z = 948.09.

Example 15: Synthesis of Compound PY-06

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 1, compound ZJT2 was used to replace compound PY-01-SM2 as the starting material to obtain compound PY-06 (14.1 g) with a total yield of 46.9%. ESI-MS (+): m/z = 571.16.

Example 16: Synthesis of Compound PY-07

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 1, compound ZJT3 was used to replace compound PY-01-SM2 as the starting material to obtain compound PY-07 (13.6 g) with a total yield of 48.5%. ESI-MS (+): m/z = 541.19.

Example 17: Synthesis of Compound PY-08

Reaction:

Preparation method:

Step 1: Preparation of compound PY-0804:

PY-01-SM2 (1.91 g, 6 mmol) was added to dichloromethane (20 mL) under nitrogen protection, cooled to 0°C, and thionyl chloride (0.86 g, 7.2 mmol) was slowly added. After the addition was completed, the mixture was stirred at room temperature for 2.0 h. The system was concentrated to dryness, and the residue was removed from the remaining thionyl chloride with toluene (10 mL × 3). The residue was compound PY-0804, which was not treated and was set aside;

Step 2: Preparation of compound PY-0803:

Dichloromethane (25 mL) was added to the residue obtained in step 1 (compound PY-0804, 6 mmol), and then the above mixture was slowly added to a dichloromethane solution (20 mL) containing aluminum chloride (0.48 g, 3.6 mmol). After the addition was completed, the mixture was stirred at room temperature for 20 minutes. The system was cooled to 0°C, and acetaldehyde (0.27 g, 6 mmol) was added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 1 hour, and the system was gradually added to the vigorously stirred ice-water slurry. It was extracted with dichloromethane three times, the organic phases were combined, washed with ice water twice, the organic phases were separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound PY-0803 (1.11 g) with a yield of 48.7%. ESI-MS (+): m/z = 381.06.

Step 3: Preparation of compound PY-0802:

Compound PY-01-SM1 (0.56 g, 2.0 mmol), triethylamine (0.31 g, 3 mmol) and compound PY-0803 (0.92 g, 2.4 mmol) were added to the reaction flask, and N,N-dimethylformamide (50 mL) was added, and the system was heated to 50°C and stirred for 24 hours. The mixture was cooled to room temperature, stirred with water, and filtered to obtain a crude product, which was purified by silica gel column to obtain compound PY-0802 (0.96 g) with a yield of 76.4%, ESI-MS (+): m/z = 628.20.

Step 4: Preparation of compound PY-0801:

Compound PY-0802 (0.47 g, 0.75 mmol), N,N-dimethylformamide (25 ml), DIPEA (0.20 g, 1.50 mmol) were added to the reaction flask, and PyBroP (0.39 g, 0.83 mmol) was added after dissolution. The system was stirred at room temperature for 30 min, and then hydroxylamine hydrochloride (0.63 g, 0.90 mmol) was added. The reaction was carried out at 40-50°C for 4-6 h. The reaction was completed by TLC detection, and the temperature was lowered, water was added, and ethyl acetate was extracted 3 times. The organic phases were combined, washed with water twice, evaporated to dryness under reduced pressure, and the residue was purified by column to obtain the product compound PY-0801 (0.36 g) with a yield of 74.5%. ESI-MS (+): m/z = 644.19.

Step 5: Preparation of compound PY-08:

Compound PY-0801 (0.36 g, 0.56 mmol) and formic acid (10 mL) were added to the reaction flask, and the system was reacted at room temperature for 20 hours. After the reaction was completed, the product was concentrated under reduced pressure and the residue was purified by column chromatography to obtain compound PY-08 (0.23 g) with a yield of 68.7%. ESI-MS (+): m/z = 604.16. 1H NMR (DMSO-D6, 400 MHz): δ11.32-11.33 (s, 1H), 10.77-10.78 (s, 1H), 7.71-7.76 (m, 4H), 7.59-7.61 (d, 2H), 7.45-7.49 (d, 1H), 6.91-6.94 (d, 2H), 6.61 (m, 4H), 5.70-5.75 (m, 2H), 5.29-5.31 (d, 1H), 5.01 -5.06(d,2H),3.94-4.03(m,2H),3.81-3.83(m,1H),3.53(s,2H),1.72(d,3H),1.70(s,6H).

Example 18: Synthesis of Compound PY-09

Reaction:

Preparation method:

Referring to the operating procedures of step 3 and step 4 in Example 3, compound PY-01-SM2 was replaced by compound ZJT5 to obtain compound PY-09 (9.3 g) with a total yield of 50.1%. ESI-MS (+): m/z = 592.12.

Example 19: Synthesis of Compound PY-10

Reaction:

Preparation method:

Referring to the operating procedures of step 3 and step 4 in Example 3, compound PY-01-SM2 was replaced by compound ZJT6 to obtain compound PY-10 (19.4 g) with a total yield of 55.3%. ESI-MS (+): m/z = 554.21.

Example 20: Synthesis of Compound PY-11

Reaction:

Preparation method:

Referring to the operation procedures of step 1 and step 2 in Example 1, compound PY-1102 was prepared by replacing compound PY-01-SM2 with compound ZJT7;

Referring to the operation procedures of each step of Example 2, compound PY-01-SM2 was replaced by compound ZJT7, and compound PY-0101 was replaced by compound PY-1102 to prepare compound PY-11 (5.7 g) with a total yield of 35.3%. ESI-MS (+): m/z = 972.33.

Example 21: Synthesis of Compound PY-24

Reaction:

Preparation method:

Referring to the operation procedures of each step of Example 1, compound ZJT8 was used to replace compound PY-01-SM2 as the starting material to obtain compound PY-24 (5.1 g) with a total yield of 45.9%. ESI-MS (+): m/z = 594.16.

Example 22: Synthesis of Compound PY-26

Reaction:

Preparation method:

Step 1: Synthesis of compound PY-2602

Compound PY-2602 (6.1 g) was prepared by referring to the operation procedure of step 3 in Example 1 with a yield of 78.3%. ESI-MS (+): m/z = 545.12.

Step 2: Compound PY-2601

Compound PY-2602 (5.6 g, 10.24 mol) and ethyl acetate (120 mL) were added to the three-necked flask. Triethylamine (5.2 g, 51.12 mol) and DMAP (0.063 g, 5.12 mmol) were added to the above system under stirring. The temperature was lowered to below 10 °C, and trifluoroacetic anhydride (6.45 g, 30.72 mmol) was slowly added to the above system within 5 minutes. The system exothermic during the addition. After the addition was completed, the system was stirred at room temperature for 1 h, and the reaction was completed by TLC detection. The system was quenched with 40 mL of water and stirred at room temperature for 20 minutes. The layers were separated, and the organic matter was washed with water (2×50 mL), saturated bicarbonate aqueous solution (50 mL×2), water (50 mL), and brine (50 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was compound PY-2601. It was used in the next step without further purification. ESI-MS (+): m/z = 737.09.

Step 3: Preparation of compound PY-26

Compound PY-26 (2.2 g) was prepared by referring to the operation procedure of step 2 in Example 1 with a yield of 83.6%. ESI-MS (+): m/z = 754.12.

Example 23: Synthesis of Compound PY-27

Reaction:

Preparation method:

Step 1: Synthesis of compound PY-2702

Compound PY-01-SM1 (14.2 g, 50.0 mmol), triethylamine (75.9 g, 75.0 mmol) and ZJT9 (36.7 g, 100.0 mmol) were added to the reaction flask, and 500 ml of N,N-dimethylformamide was added, and the system was heated to 50°C and stirred for 24 hours. The mixture was cooled to room temperature, stirred with water, and filtered to obtain a crude product, which was purified by silica gel column to obtain compound PY-2702 (17.2 g), with a yield of 55.8%, ESI-MS (+): m/z = 615.17.

Step 2: Compound PY-2701

Referring to the operation procedure of step 2 in Example 1, compound PY-2701 (10.1 g) was prepared with a yield of 82.3%. ESI-MS (+): m/z = 630.18.

Step 3: Preparation of compound PY-27

Compound PY-27 (5.3 g) was prepared by referring to the operation procedure of step 3 in Example 1 with a yield of 79.6%. ESI-MS (+): m/z = 590.15.

Example 24: Synthesis of Compound PY-28

Reaction:

Preparation method:

Step 1: Synthesis of compound PY-2801

Referring to the operating procedures of step 1 in Example 23, compound PY-2801 (12.2 g) was prepared with a yield of 51.4%, ESI-MS (+): m/z = 932.41.

Step 2: Compound PY-28

Referring to the operation procedure of step 4 in Example 3, compound PY-28 (5.2 g) was prepared with a yield of 68.3%. ESI-MS (+): m/z = 590.15.

Example 25: Synthesis of Compound PY-34

Reaction:

Preparation method:

Step 1: Preparation of compound PY-3404:

PY-01-SM2 (1.91 g, 6 mmol) was added to dichloromethane (20 mL) under nitrogen protection, cooled to 0°C, and thionyl chloride (0.86 g, 7.2 mmol) was slowly added. After the addition was completed, the mixture was stirred at room temperature for 2.0 h. The system was concentrated to dryness, and the residue was removed from the remaining thionyl chloride with toluene (10 mL × 3). The residue was compound PY-3404, which was not treated and was set aside.

Step 2: Preparation of compound PY-3403:

Dichloromethane (25 mL) was added to the residue obtained in step 1 (compound PY-3404, 6 mmol), and then the mixture was slowly added to a dichloromethane solution (20 mL) containing aluminum chloride (0.48 g, 3.6 mmol). After the addition was completed, the mixture was stirred at room temperature for 20 minutes. The system was cooled to 0°C, and acetaldehyde (0.27 g, 6 mmol) was added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 1 hour, and the system was gradually added to the vigorously stirred ice-water slurry. It was extracted with dichloromethane three times, the organic phases were combined, washed with ice water twice, the organic phases were separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound PY-3403 (1.11 g) with a yield of 48.7%. ESI-MS (+): m/z = 381.06.

Step 3: Preparation of compound PY-3402:

Compound PY-01-SM1 (0.56 g, 2.0 mmol), triethylamine (0.31 g, 3 mmol) and compound PY-3403 (0.92 g, 2.4 mmol) were added to the reaction flask, and N,N-dimethylformamide (50 mL) was added, and the system was heated to 50°C and stirred for 24 hours. The mixture was cooled to room temperature, stirred with water, and filtered to obtain a crude product, which was purified by silica gel column to obtain compound PY-3402 (0.96 g) with a yield of 76.4%, ESI-MS (+): m/z = 628.20.

Step 4: Preparation of compound PY-3401:

Compound PY-3402 (0.47 g, 0.75 mmol), N,N-dimethylformamide (25 ml), DIPEA (0.20 g, 1.50 mmol) were added to the reaction flask, and tripyrrolidinophosphonium bromide hexafluorophosphate (PyBroP, 0.39 g, 0.83 mmol) was added after dissolution. The system was stirred at room temperature for 30 min, and then hydroxylamine hydrochloride (0.63 g, 0.90 mmol) was added. The reaction was carried out at 40-50°C for 4-6 h. After TLC detection, the reaction was completed, the temperature was lowered, water was added, and ethyl acetate was extracted 3 times. The organic phases were combined, washed with water twice, evaporated to dryness under reduced pressure, and the residue was purified by column to obtain the product compound PY-3401 (0.36 g) with a yield of 74.5%. ESI-MS (+): m/z = 644.19.

Step 5: Preparation of compound PY-34:

Compound PY-3401 (0.36 g, 0.56 mmol) and formic acid (10 mL) were added to the reaction flask, and the system was reacted at room temperature for 20 hours. After the reaction was completed, the product was concentrated under reduced pressure and the residue was purified by column chromatography to obtain compound PY-34 (0.23 g) with a yield of 68.7%. ESI-MS (+): m/z = 604.16. 1H NMR (DMSO-D6, 400 MHz): δ11.32-11.33 (s, 1H), 10.77-10.78 (s, 1H), 7.71-7.76 (m, 4H), 7.59-7.61 (d, 2H), 7.45-7.49 (d, 1H), 6.91-6.94 (d, 2H), 6.61 (m, 4H), 5.70-5.75 (m, 2H), 5.29-5.31 (d, 1H), 5.01 -5.06(d,2H),3.94-4.03(m,2H),3.81-3.83(m,1H),3.53(s,2H),1.72(d,3H),1.70(s,6H).

Example 26: Synthesis of Compound PY-35

Reaction:

Preparation method:

Step 1: Preparation of compound PY-3501

Referring to the preparation of step 3 of Example 25, compound PY-3401 was used to replace compound PY-01-SM1 to prepare compound PY-3501 (1.26 g) with a yield of 69.5%. ESI-MS (+): m/z = 988.27.

Step 2: Preparation of compound PY-35

Referring to the preparation of step 5 of Example 25, compound PY-3501 was used to replace compound PY-3401 to prepare compound PY-35 (0.19 g) with a yield of 63.2%. ESI-MS (+): m/z = 948.24.

Example 27: Synthesis of Compound PY-36

Reaction:

Preparation method:

Step 1: Preparation of compound PY-3603

Under nitrogen protection, compound PY-03-SM3 (12.5 g, 51.0 mmol) and dichloromethane (250 mL) were added to a three-necked flask. The resulting solution was cooled to 0°C, and 4-dimethylaminopyridine (DMAP, 0.70 g, 5.7 mmol) and imidazole (14.0 g, 205.6 mmol) were added in sequence. Tert-butyldimethylsilyl chloride (TBSC1, 30.9 g, 205.0 mmol) was added within 10 minutes, and the resulting mixture was warmed to ambient temperature and stirred for 18 hours. Water was added to the system, stirred at room temperature for 2 hours, separated, the aqueous phase was extracted 3 times with dichloromethane, the combined organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by column to obtain compound PY-3603 (21.1 g) with a yield of 70.6%. ESI-MS (+): m/z = 587.33.

Step 2: Preparation of compound PY-3602

Compound PY-3603 (14.0 g, 23.9 mmol) and dichloromethane (350 mL) were added to a three-necked flask. The solution was cooled to 0°C using an ice bath. DMAP (0.29 g, 0.24 mmol) and N,N-diisopropylethylamine (15.5 g, 120 mmol) were added in sequence. 2,4,6-triisopropylbenzene-1-sulfonyl chloride (14.5 g, 47.5 mmol) was slowly added to the flask, and after the addition, the flask was warmed to ambient temperature and stirred for 18 hours. The system was cooled to 0°C, N,N-diisopropylethylamine (12.3 g, 95.5 mmol) was added dropwise, and then solid hydroxylamine hydrochloride (6.63 g, 95.5 mmol) was immediately added. The mixture was warmed to room temperature and stirred for 3 hours. The reaction was quenched with water and the resulting layers were separated. The aqueous layer was extracted with dichloromethane, and the combined organic matter was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column to obtain compound PY-3602 (9.41 g) with a yield of 65.4%. ESI-MS (+): m/z = 602.34.

Step 3: Preparation of compound PY-3601

Referring to the preparation of step 3 of Example 25, compound PY-3602 was used to replace compound PY-01-SM1 to prepare compound PY-3601 (10.1 g) with a yield of 71.3%. ESI-MS (+): m/z = 946.42.

Step 4: Preparation of compound PY-36

Compound PY-0301 (9.5 g, 1.0 mmol) and tetrahydrofuran (100 mL) were added to a three-necked flask, followed by triethylamine trihydrofluoride (1.61 g, 1.0 mmol), and the mixture was stirred at ambient temperature for 18 hours. The mixture was concentrated under reduced pressure, and the residue was dissolved in a minimum amount of methanol, the solution was slowly added to dichloromethane containing rapid stirring, and the mixture was stirred at room temperature for 15 minutes. Filtered and recrystallized from dichloromethane and petroleum ether to obtain the title compound PY-36 (4.05 g) with a yield of 67.1%. ESI-MS (+): m/z = 604.16.

Example 28: Synthesis of Compound ZJT10

Reaction:

Preparation method:

Step 1: Preparation of compound ZJT10-02:

Under nitrogen protection, pyrimidine-5-D (11.31 g, 0.1 mol), BSA (40.7 g, 0.2 mol) and anhydrous acetonitrile (250 mL) were added to a reaction bottle, heated to 80°C, and stirred for 30 min; cooled to room temperature, tetraacetyl ribose (63.3 g, 0.2 mol) was added, TMSOTf (44.5 g, 0.2 mol) was added dropwise, and after the addition was completed, the temperature was raised to 80°C, reacted for 2 hours, cooled to room temperature, and acetonitrile was evaporated under reduced pressure; ethyl acetate was added to the residue, washed twice with saturated sodium bicarbonate aqueous solution, the organic phase was separated, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain compound ZJT10-02 (31.6 g) with a yield of 85.2%. ESI-MS (+): m/z=372.13.

Step 2: Preparation of compound ZJT10-01:

Compound ZJT10-02 (31.0 g, 0.084 mol) and 7M ammonia/methanol solution (500 mL, 3.5 mol) were added to the reaction flask, stirred at room temperature for 36 hours, the solvent was evaporated, and the residue was subjected to column chromatography to obtain compound ZJT10-01 (15.5 g), with a yield of 75.2%. ESI-MS (+): m/z = 246.10.

Step 3: Preparation of compound ZJT10

Compound ZJT10-01 (15.0 g, 0.061 mol) was dissolved in acetone (300 mL), 2, 2-dimethoxypropane (31.8 g, 0.305 mol) was slowly added, and after the addition was completed, concentrated sulfuric acid (2.0 g, 0.020 mol) was slowly added, and stirred for 30 minutes; saturated sodium bicarbonate was added to the system to quench, the solvent was evaporated under reduced pressure, and the residue was subjected to column chromatography to obtain compound ZJT10 (11.5 g), with a yield of 66.1%. ESI-MS (+): m/z = 286.20.

Example 29: Synthesis of Compound ZJT11

Reaction:

Preparation method:

Referring to the operation procedure of Example 1, uracil-2D was used to replace pyrimidine-5-D as the starting material to obtain compound ZJT11 (10.2 g) with a total yield of 40.1%. ESI-MS (+): m/z = 287.22.

Example 30: Synthesis of Compound ZJT12

Reaction:

Preparation method:

Step 1: Preparation of compound ZJT12-01

Under nitrogen protection, PY-01-SM1 (6.3 g, 22.0 mmol) and dichloromethane (200 mL) were added to the reaction flask at room temperature. Then, pyridinium dichromate (16.6 g, 44.1 mmol), acetic anhydride (22.5 g, 220 mmol) and tert-butyl alcohol (16.3 g, 220 mmol) were added to the system in sequence under stirring. The above system was stirred at room temperature for 24 hours, washed with water, the aqueous phase was separated, extracted twice with dichloromethane, the organic phases were combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was subjected to column chromatography to obtain compound ZJT12-01 (5.5 g) with a yield of 70.5%. ESI-MS (+): m/z = 355.15.

Step 2: Preparation of compound ZJT12

Under nitrogen protection, compound ZJT12-01 (5.4 g, 15.2 mmol) was added to ethanol-D1 (200 mL) at room temperature, and sodium borodeuteride-D4 (2.5 g, 60.8 mmol) was added at once under stirring. After the addition was completed, the mixture was stirred at room temperature for 1 hour, heated to 55°C for 7 hours, and then stirred at room temperature overnight. The system was cooled to 0°C, quenched with acetic acid-D1, evaporated to dryness under reduced pressure, and the residue was subjected to column chromatography to obtain compound ZJT12 (3.0 g) with a yield of 68.2%. ESI-MS (+): m/z = 287.15

Example 31: Synthesis of Compound PY-49

Reaction:

Preparation method:

Step 1: Preparation of compound PY-4902

Tetrahydrofuran (30 mL), PY-01-SM2 (3.18 g, 10 mmol), 4-dimethylaminopyridine (DMAP, 1.83 g, 15 mmol) were added to the reaction bottle in sequence, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 1.86 g, 12 mmol) and ZJT10 (2.85 g, 10 mmol) were added after dissolution, and the temperature was raised to 70°C, and the reaction was stirred. After the reaction was completed under TLC monitoring, the system was cooled and evaporated to dryness. Ethyl acetate and water were added to the residue, and the organic phase was separated, washed twice with water, dried, and evaporated to dryness under reduced pressure. The residue was purified by column to obtain the product PY-4902 (4.2 g) with a yield of 71.7%. ESI-MS (+): m/z=586.20.

Step 2: Preparation of compound PY-4901

Compound PY-4902 (4.1 g, 7.0 mmol), N,N-dimethylformamide (25 mL), N,N-diisopropylethylamine (DIPEA, 1.81 g, 14.0 mmol) were added to the reaction flask, and tripyrrolidinylphosphonium bromide hexafluorophosphate (PyBroP, 3.59 g, 7.7 mmol) was added after dissolution. The system was stirred at room temperature for 30 min, and then hydroxylamine hydrochloride (0.59 g, 8.4 mmol) was added. The reaction was carried out at 40-50°C for 4-6 h. The reaction was completed by TLC detection, and the temperature was lowered, water was added, and ethyl acetate was extracted 3 times. The organic phases were combined, washed with water twice, evaporated to dryness under reduced pressure, and the residue was subjected to column chromatography to obtain the product PY-4901 (2.93 g) with a yield of 69.6%. ESI-MS (+): m/z = 601.17.

Step 6: Preparation of compound PY-49

PY-4901 (2.9 g, 4.83 mmol) and formic acid (50 mL) were added to the reaction flask, and the system was reacted at room temperature for 20 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was recrystallized from isopropanol/methyl tert-butyl ether to obtain compound PY-49 (2.17 g), with a yield of 80.1%, ESI-MS (+): m/z = 561.14. 1H NMR (DM SO-D6, 500 MHz): δ10.05 (s, 1H), 9.44 (s, 1H), 7.74-7.77 (m, 4H), 7.65-7.66 (d, 2H), 6.96-6.98 (d, 2H), 6.79-6.81 (d, 1H), 5.73-5.75 (d, 1H), 5.33-5.34 (d, 1H), 5.22-5.23 (d, 1H), 4.29-4.37(m,2H),3.98(s,1H),3.91-3.95(m,1H),3.79-3.80(d,1H),1.65-1.67(d,6H).

Example 32: Preparation of Compound PY-50

Reaction:

Preparation method:

Referring to the operation procedure of Example 31, ZJT12 was used instead of ZJT10 as the starting material to obtain compound PY-50 (1.21) with a total yield of 42.2%. ESI-MS (+): m/z = 562.18.

Example 33: Preparation of Compound PY-51

Reaction:

Preparation method:

Referring to the operation procedure of Example 31, ZJT12 was used instead of ZJT10 as the starting material to obtain compound PY-51 (1.52) with a total yield of 38.5%. ESI-MS (+): m/z = 562.20.

Example 34: Preparation of Compound PY-52

Reaction:

Preparation method:

Step 1: Preparation of compound PY-5203

Under nitrogen protection, compound ZJT10-01 (2.5 g, 10.2 mmol) and dichloromethane (50 mL) were added to the reaction flask and cooled to 0°C; then DMAP (0.13 g, 1.02 mmol) and imidazole (2.8 g, 40.9 mmol) were added in sequence. Tert-butyldimethylsilyl chloride (TBSCl, 6.17 g, 4.0 mmol) was added within 10 minutes, and the resulting mixture was warmed to ambient temperature and stirred for 18 hours. Water (30 mL) was added to the system, stirred at room temperature for 2 hours, separated, the aqueous phase was extracted 3 times with dichloromethane, the combined organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure, and the residue was purified by column to obtain compound PY-5203 (4.14 g) with a yield of 69.0%. ESI-MS (+): m/z = 588.33.

Step 2: Preparation of compound PY-5202

Compound PY-5203 (4.00 g, 6.8 mmol) and dichloromethane (100 mL) were added to the reaction flask, stirred and cooled to 0 ° C, and then DMAP (0.083 g, 0.68 mmol) and N, N-diisopropylethylamine (4.41 g, 34.1 mmol) were added to the above system in sequence. Under stirring, 2,4,6-triisopropylbenzene-1-sulfonyl chloride (4.13 g, 13.6 mmol) was slowly added to the flask. After the addition, it was stirred at room temperature for 18 hours. The system was cooled to 0 ° C again, N, N-diisopropylethylamine (3.51 g, 27.2 mmol) was added dropwise, and then solid hydroxylamine hydrochloride (1.9 g, 27.3 mmol) was added immediately. The mixture was warmed to room temperature and stirred for 3 hours. The reaction was quenched with water, separated, the aqueous layer was extracted twice with dichloromethane, the organic phases were combined, washed with brine, dried over sodium sulfate, concentrated under reduced pressure, and the residue was purified by column to obtain compound PY-5202 (2.70 g), with a yield of 65.9%. ESI-MS (+): m/z = 603.34.

Step 3: Preparation of compound PY-5201

Referring to the preparation of step 1 of Example 31, compound PY-5202 was used to replace compound ZJT10 to prepare compound PY-5201 (2.22 g) with a yield of 66.5%. ESI-MS (+): m/z = 903.43.

Step 4: Preparation of compound PY-52

Compound PY-5201 (1.75 g, 1.94 mol) and tetrahydrofuran (20 mL) were added to a three-necked flask, followed by triethylamine trihydrofluoride (0.31 g, 1.94 mmol), and the mixture was stirred at ambient temperature for 18 hours. The mixture was concentrated under reduced pressure, and the residue was subjected to column chromatography to obtain compound PY-52 (0.53 g) with a yield of 48.7%. ESI-MS (+): m/z = 561.16.

Example 35: Preparation of Compound PY-54

Reaction:

Preparation method:

Referring to the operating procedure of Example 34, ZJT11-01 was used instead of ZJT10-01 as the starting material to obtain compound PY-54 (0.46) with a total yield of 16.2%. ESI-MS (+): m/z = 562.20.

Example 36: Preparation of Compound PY-55

Reaction:

Preparation method:

Referring to the operation procedures of step 1 and step 3 of Example 31, compound PY-55 (0.25 g) was prepared using compound PY-4901 as the starting material with an overall yield of 50.2%. ESI-MS (+): m/z = 861.22.

Example 37: Preparation of Compound PY-56

Reaction:

Preparation method:

Step 1: Synthesis of compound PY-5604

PY-01-SM2 (3.82 g, 12 mmol) was added to dichloromethane (50 mL) under nitrogen protection, cooled to 0°C, and thionyl chloride (1.72 g, 14.4 mmol) was slowly added. After the addition was completed, the mixture was stirred at room temperature for 2.0 h. The system was concentrated to dryness, and the residue was removed from the remaining thionyl chloride by toluene three times. The residue was compound PY-5604, which was not treated and was set aside.

Step 2: Synthesis of compound PY-5603

Dichloromethane (50 mL) was added to the residue obtained in step 1 (compound PY-5604, 12 mmol), and then the above mixture was slowly added to a dichloromethane solution (40 mL) containing aluminum chloride (0.96 g, 7.2 mmol). After the addition was completed, the mixture was stirred at room temperature for 20 minutes. The system was cooled to 0°C, and acetaldehyde (0.54 g, 12 mmol) was added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 1 hour, and the system was gradually added to the vigorously stirred ice-water slurry. It was extracted with dichloromethane three times, the organic phases were combined, washed with ice water twice, the organic phases were separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound PY-5603 (2.07 g) with a yield of 45.2%. ESI-MS (+): m/z = 381.06.

Step 3-Step 5: Synthesis of Compound PY-56

Referring to the operation procedures of each step of Example 31, PY-5603 was used to replace PY-01-SM2 as the starting material to obtain compound PY-56 (1.12), with a total yield of 35.3% in three steps. ESI-MS (+): m/z = 605.20.

The following example compounds were synthesized by the same method as in the above examples using commercially available compounds or intermediate compounds appropriately synthesized from commercially available compounds.

Example 38: Stability Test

Compounds PY-01, PY-02, PY-03, PY-12, PY-15, PY-18, PY-21, PY-34, PY-49, and compound A were prepared with 0.5% sodium carboxymethylcellulose solution and 1.0% methylcellulose solution, respectively. The samples (sealed at the mouth) were placed in a stability box at 25.0°C, 37% humidity, and protected from light. HPLC (with DAD detector) was used to detect the stability on day 0, day 3, and day 7. The results are shown in Table 1.

Table 1 Stability test results

The results show that the stability of the series of compounds of the present invention is better than that of the reference compound A.

Example 39: In vitro anti-influenza virus activity and cytotoxicity assay

Toxicity determination of the test compound on MDCK:

MDCK cells in logarithmic growth phase were inoculated into 24-well cell culture plates, cultured in an incubator at 37°C and 5% CO ₂ for 24 hours, and then treated with drugs at different dilution concentrations for 2 hours, and then washed 3 times with Hanks solution. After drying, 1 ml of DMEM containing 2% newborn calf serum was added to each well to maintain growth, and cultured in an incubator at 37°C and 5% CO _2. The degree of cytopathic effect (CPE) was observed within 7 days, and the half toxic concentration (TC ₅₀ ) of the samples to cells was calculated by the Reed-Muench method.

Determination of anti-influenza virus activity of test compounds:

MDCK cells in the logarithmic growth phase were inoculated into 24-well plates, each well containing about 1×10 ⁴ cells, and cultured in a 37°C, 5% CO ₂ incubator. After 24 hours, the cells were infected with virus solution (H1N1, A/WSN/33), washed twice with Hanks solution, then acted on the cells with drug diluent, washed three times with Hanks solution, and finally cultured in a 37°C, 5% CO ₂ incubator with DMEM maintenance solution containing 2% newborn calf serum. The cytopathic effect (CPE) was observed under an inverted microscope at 1, 2, 3, 4, 5, 6, and 7 days after culture, and the half inhibitory concentration (IC ₅₀ ) of the sample on the virus on the cell was calculated by the Reed-Muench method, and then the selection index (SI) was calculated. The calculation method of SI is SI=TC ₅₀ /IC ₅₀ . The results are shown in Table 2.

Table 2 In vitro anti-influenza virus activity data

The experimental results show that the tested compound samples of the present invention have better inhibitory activity against influenza virus (H1N1), lower cytotoxicity, and higher selectivity index compared with the disclosed compound A.

Example 40: In vitro anti-novel coronavirus activity (EC ₅₀ ) assay

Vero E6 cells were seeded into microplates at a certain density and cultured overnight in a 5% CO ₂ , 37°C incubator. On the second day, doubly diluted compounds (8 concentration points, triplicate wells) and SARS-CoV-2 virus (B.1.1.7 (Alpha)) were added. Cell controls (cells, no compound treatment or virus infection) and virus controls (cells infected with virus, no compound treatment) were set up. Cells were cultured in an incubator for 3 or 4 days.

The antiviral activity of the compound is represented by the inhibition rate (%) of the compound at different concentrations on the cytopathic effect caused by the virus. The inhibition rate of the compound was analyzed by nonlinear fitting using GraphPad Prism to calculate the EC ₅₀ of the compound. The results are shown in Table 3.

Table 3 In vitro anti-novel coronavirus activity data

Sample No. EC ₅₀ (μmol) Compound A 1.62 PY-01 0.35 PY-02 0.58 PY-03 0.66 PY-12 0.79 PY-15 0.57 PY-18 0.66 PY-21 0.77 PY-34 0.82 PY-49 0.33

The experimental results show that the tested compound samples of the present invention have better inhibitory activity against SARS-CoV-2 virus (B.1.1.7 (Alpha)) compared with the disclosed compound A.

Example 41: Evaluation of the pharmacokinetic characteristics of compound PY-01 in rats

12 SD rats, male, 180g-220g. The feeding conditions were room temperature: 20-26℃, humidity: 40-70%, light illumination: dark = 12h:12h; the rats were adaptively fed for 3 days, during which they were free to eat and drink. They were randomly divided into 4 groups, 3 rats in each group. The two test products (PY-01 and compound A) were administered by single equimolar oral gavage and equimolar tail vein injection, respectively. The rats were fasted for 16-17h starting at 5 pm the day before the administration, and the animals were fed 4 hours after the administration, and water was not forbidden during the whole process.

Blood was collected before and 0.25h, 0.5h, 1h, 2h, 3h, 4h, 8h, and 24h after a single oral gavage administration, or before and 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h, and 24h after a single intravenous injection.

At each blood sampling time point, about 300 μL of venous blood was collected from the rat eyeball, added to a centrifuge tube with sodium heparin precooled in ice water, and placed in an ice bath. After standing, centrifugation (4000 rpm, 10 min) was performed, and the blood was divided into 50 μL units, placed in sterile EP tubes, and stored at -80 ° C for later use. The concentration of compound B in plasma was determined as soon as possible. HPLC (High Performance Liquid Chromatography) and LC/MS were used to determine the drug concentration in plasma, and the area under the concentration-time curve (AUC) in plasma was calculated using a nonlinear least squares program to calculate the bioavailability of the drug in rats.

Absolute bioavailability: F = AUClast-po/AUClast-iv x 100%. The results are shown in Table 4.

The structure of compound B is as follows:

Table 4 Pharmacokinetic results

The results of the pharmacokinetic experiment in SD rats showed that compound PY-01 had a longer half-life, a greater in vivo exposure after oral administration, and its bioavailability was nearly 1.4 times that of compound A. The Cmax of compound B after administration of compound PY-01 was lower than that of compound A, indicating that the compound of the present invention had a better safety margin.

Example 42: In vivo anti-influenza virus therapeutic administration trial

Thirty C57BL/6J mice, 6-8 weeks old, SPF grade, female, were randomly divided into 6 groups, numbered as group 1, group 2, group 3, group 4, group 5 and group 6, with 5 mice in each group. 24 hours after intranasal inoculation of a half-lethal dose of 100 pfu influenza virus (H1N1, A/WSN/33), group 1 (vehicle group) was gavaged with 0.5% sodium carboxymethyl cellulose solution; group 2 was gavaged with control drugs (equimolar, 17.33 mg/kg of compound A and 19.01 mg/kg of compound C, combined use); groups 3-5 were gavaged with test drugs (29.46 mg/kg of PY-01, 29.46 mg/kg of PY-03, 29.46 mg/kg of PY-49); group 6 was given 29.46 mg/kg of PY-01 starting 48 hours after intranasal inoculation of the virus. Each group was given equimolar doses twice a day, with groups 1 to 5 given drugs or vehicles for 4 days, and group 6 given drugs for 3 days. The animals were killed on the 5th day after virus inoculation, and lung tissue samples were collected and homogenized for virus titer determination. The results are shown in Figure 1.

The results showed that compound PY-01, compound PY-03 and compound PY-49 all showed excellent anti-influenza virus (H1N1) effects when administered 24 hours after virus infection, showing stronger anti-influenza virus (H1N1) effects than combined administration. Compound PY-01 was also able to significantly reduce the virus titer 48 hours after virus infection.

Example 43: In vivo prophylactic administration trial against influenza virus (H1N1)

Thirty BALB/c mice, 6-8 weeks old, SPF grade, female, were randomly divided into 6 groups, numbered as group 1, group 2, group 3, group 4, group 5 and group 6, with 5 mice in each group. Each group started to be administered on day -1, and 100 PFU of influenza virus (H1N1, A/Puerto Rico/8/1934) was inoculated intranasally on day 0, and the virus was inoculated 2 hours after administration on the day of virus inoculation. Among them, group 1 (solvent group) was gavaged with 0.5% sodium carboxymethyl cellulose solution; group 2 was gavaged with control drugs (17.33 mg/kg of compound A and 19.01 mg/kg of compound C, combined use); groups 3-5 were gavaged with different doses of test drugs (high, medium and low dose groups of PY-01: 88.37 mg/kg, 29.46 mg/kg, 9.82 mg/kg); group 6 was gavaged with a high dose of the test drug PY-01 (88.37 mg/kg). Each group was administered with equimolar dosage, once a day, groups 1-5 were administered or solvent for 5 days, and group 6 was administered for 2 days. The animals were killed on the 4th day after virus inoculation, lung tissue samples were collected, and the virus titer was determined after homogenization. The results are shown in Figure 2.

The results showed that compared with the vehicle group, all experimental groups showed lower virus titers; at the same number of administration days, the virus titers of the PY-01 high-dose group and the medium-dose group were lower than those of the combined administration group; compared with group 3 (high-dose PY-01 was administered 2 days before virus inoculation and 3 days after virus inoculation), the virus titer of group 6 (high-dose PY-01 was administered 2 days before virus inoculation and not administered after virus inoculation) was slightly larger, but still lower than that of other groups. This shows that the compound of the present invention has a significant preventive effect on influenza virus.

Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims (3)

1. A new type of cytidine derivative, tautomer, stereoisomer, isotope derivative and pharmaceutically acceptable salt thereof, selected from the following compounds: 2. Pharmaceutical compositions comprising novel cytidine derivatives, tautomers, stereoisomers, isotope derivatives and pharmaceutically acceptable salts thereof as claimed in claim 1. 3. The novel cytidine derivatives, tautomers, stereoisomers, isotope derivatives and pharmaceutically acceptable salts thereof described in claim 1, or the pharmaceutical composition described in claim 2 during preparation Application of anti-influenza A virus drugs.