Baloxvir derivative and application thereof

Abstract

The invention discloses a baloxvir derivative and application thereof, and belongs to the field of pharmacy, the baloxvir derivative, a stereoisomer thereof, a crystalline hydrate thereof, a deuterated compound thereof, a solvate thereof, a prodrug thereof and a pharmaceutically acceptable salt thereof, and the structural formula of the baloxvir derivative is shown as a formula I, and the sEH inhibitor is a residue after the esterification of the soluble epoxy hydrolase inhibitor. The application refers to application of the baloxvir derivative or the pharmaceutical composition thereof in preparation of anti-inflammatory and antiviral drugs. A series of sEH inhibitors and BAX are connected to synthesize twin drugs to simultaneously inhibit an NF-kappa B pathway and target virus cap-dependent endonuclease to play anti-inflammatory and antiviral dual roles, and new compounds with anti-inflammatory and antiviral activity are identified in vitro and in vivo. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of pharmacy and relates to a baloxavir derivative and a use thereof. Background Art

[0002] Seasonal influenza is an acute respiratory infectious disease caused by influenza virus. It is highly contagious and spreads rapidly, and is a major public health problem worldwide. Most hospitalizations and deaths are attributed to complications such as pulmonary fibrosis, bacterial pneumonia, acute respiratory distress syndrome, and congestive heart failure. In the early stages of influenza virus infection, influenza can induce upregulation of TLR3 protein levels, activate the NF-kB signaling pathway and mitochondria-related oxidative stress responses, and then cause the host's immune system to activate an inflammatory response to eliminate the virus. However, the production of inflammatory mediators does not decrease with the reduction of viral load, resulting in the accumulation of excessive inflammatory cytokines in the later stages of infection, which may cause further damage to the host. Currently approved anti-influenza drugs do not address the excessive inflammatory response of the host, highlighting the urgent need for strategies to alleviate this response during viral infection. High-dose glucocorticoid treatment has been shown to alleviate lung injury in patients with severe influenza pneumonia; however, it may also reduce viral clearance and prolong recovery time. The simultaneous use of antiviral and anti-inflammatory drugs has raised concerns about the metabolic side effects that may be caused by multiple drugs, including liver and kidney damage. In addition, when multiple drugs need to be taken, patients often show poor compliance. Therefore, Applicants have proposed a novel approach to combine antiviral and anti-inflammatory compounds into one entity to achieve two therapeutic effects in the treatment of influenza virus infection.

[0003] Baloxavir marboxil (MBAX) is currently the first single-dose oral antiviral drug approved in the European Union for the treatment of uncomplicated influenza and post-exposure prophylaxis in people aged 1 year and above. The active form, baloxavir acid, inhibits transcription of influenza viruses by blocking the endonuclease activity of the PA protein. The keto-enol structure of baloxavir (BAX) contributes to its hydrophilicity, resulting in a very low intestinal membrane transport rate. In order to improve the bioavailability of baloxavir, the hydroxyl group in the keto-enol structure was substituted to form baloxavir ester.

[0004] EETs, which are metabolized by arachidonic acid (AA) through the cytochrome P450s enzyme pathway, are closely related to inflammation. Studies have found that the inhibition of NF-κB activity is the key to the anti-inflammatory effect of EpFA, including EETs. EETs inhibit NF-κB activity in three ways: first, inhibiting the activation of NF-κB by tumor necrosis factor (TNF)-α by inhibiting IκB kinase; second, inhibiting the transcription of NF-κB and AP-1 by activating PPARγ; third, directly inhibiting the activity of PGE2, an inflammatory factor downstream of the NF-κB signaling pathway, which is an effect that NSAIDs and steroidal anti-inflammatory drugs do not have. EETs produced by AA through the CYP450 metabolic pathway have many physiological functions, such as anti-inflammation, analgesia, anti-ischemia, anti-angiogenesis, anti-proliferation, cardiovascular protection and regulation of metabolic diseases. EETs and inflammatory metabolites such as prostaglandins and leukotrienes in the COX and LOX metabolic pathways of AA form the body's inflammatory / anti-inflammatory balance and play a very important role in maintaining the inflammatory / anti-inflammatory balance in the body. sEH inhibitors can stabilize endogenous EpFAs (EETs). Therefore, sEH is a potential therapeutic target for pain and inflammatory diseases.

[0005] After searching, no research has been found so far on using sEH inhibitors to connect with BAX to synthesize twin drugs to simultaneously inhibit the NF-κB pathway and target viral cap-dependent endonucleases to exert dual anti-inflammatory and antiviral effects. Summary of the invention

[0006] The present invention synthesizes twin drugs by connecting a series of sEH inhibitors with BAX to simultaneously inhibit the NF-κB pathway and target viral cap-dependent endonucleases to exert dual anti-inflammatory and antiviral effects, and identifies new compounds with anti-inflammatory and antiviral activities in vitro and in vivo.

[0007] In order to achieve the above object, the present invention adopts the following technical solution.

[0008] In a first aspect, the present invention provides a baloxavir derivative, a stereoisomer thereof, a crystalline hydrate thereof, a deuterated product thereof, a solvate thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof,

[0009] The structural formula of the baloxavir derivative is shown in Formula I:

[0010]

[0011] Here, sEH inhibitor is the esterified residue of soluble epoxide hydrolase inhibitor.

[0012] Preferably, the soluble epoxide hydrolase inhibitor contains a substituted benzene ring or a substituted adamantane.

[0013] Preferably, the soluble epoxide hydrolase inhibitor is t-AUCB, 12-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}dodecanoic acid (AUDA), (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid, 7-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}heptanoic acid, 4-(((1r,4r)-4-(2-(3-fluoro-4-(trifluoromethoxy)phenyl)acetylamino)cyclohexyl)oxy)benzoic acid, t-TUCB, 12-({[(1,3-dimethyltricyclo[3.3.1.1 3,7 ]dec-7-yl)amino]carbonyl}amino)dodecanoic acid, 12-{[({4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid, 12-{[({3-fluoro-4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid, 11-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}undecanoic acid, 11-({[(3,5-dimethyltricyclo[3.3.1.1 3,7 ]dec-1-yl)amino]carbonyl}amino)undecanoic acid, 8-{[(tricyclo[3.3.1.1 3,7 ]dec-3-ylamino)carbonyl]amino}octanoic acid, 12-[({[1-(tricyclo[3.3.1.1 3,7 ]dec-1-yl)ethyl]amino}carbonyl)amino]dodecanoic acid, 12-[({[1-(tricyclo[3.3.1.1 3,7 At least one of {[(bicyclo[2.2.1]hept-2-en-6-ylmethyl)amino]carbonyl}amino)dodecanoic acid and 12-({[(bicyclo[2.2.1]hept-2-en-6-ylmethyl)amino]carbonyl}amino)dodecanoic acid.

[0014] In some embodiments, the compound represented by formula I is selected from the following compounds:

[0015]

[0016]

[0017] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned baloxavir derivative, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or a pharmaceutically acceptable salt thereof.

[0018] Preferably, the pharmaceutical composition further comprises traditional Chinese medicine ingredients and / or western medicine ingredients.

[0019] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0020] Preferably, the pharmaceutical composition is a tablet, a pill, a cream, an emulsion, an ointment, a suspension, a lyophilized agent, a capsule, a sustained-release agent, a granule, an elixir, an injection or a spray.

[0021] In a third aspect, the present invention provides the use of the above-mentioned baloxavir derivatives, their stereoisomers, their crystalline hydrates, their deuterated products, their solvates, their prodrugs, their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of anti-inflammatory and antiviral drugs.

[0022] Preferably, the virus is an influenza virus.

[0023] More preferably, the influenza virus is influenza A virus and / or influenza B virus.

[0024] Further preferably, the influenza virus is at least one of H1N1, H5N1, H7N9, H3N2 and influenza B virus.

[0025] More preferably, the influenza virus is at least one of H1N1, H3N2 or type B influenza virus.

[0026] In a fourth aspect, the present invention provides the use of the above-mentioned baloxavir derivative, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or its pharmaceutically acceptable salt or pharmaceutical composition in the preparation of an inhibitor of NF-κB signaling pathway.

[0027] The anti-inflammatory and antiviral drugs or NF-κB signaling pathway inhibitors described in the present invention are suitable for humans or animals.

[0028] Preferably, the animal comprises bovine, equine, ovine, porcine, canine, feline, lagomorph, avian, rodent or primate.

[0029] Beneficial Effects of the Invention

[0030] (1) The present invention synthesizes twin drugs by connecting a series of sEH inhibitors with BAX to simultaneously inhibit the NF-κB pathway and target the viral cap-dependent endonuclease to exert anti-inflammatory and antiviral dual effects, and identifies new compounds with anti-inflammatory and antiviral activities in vitro and in vivo.

[0031] (2) Compared with BAX and MBAX, the baloxavir derivatives of the present invention have better anti-influenza virus effects.

[0032] (3) Antiviral drugs widely used in clinic, such as oseltamivir and baloxavir, are recommended to be administered within 48 hours after infection to effectively protect influenza patients. However, the baloxavir derivatives of the present invention still have a good protective effect when administered within 48-96 hours after influenza virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The inhibitory effects of MBAX, ATV-114 and AUDA on H1N1 viral protein expression in cell lines with high and low AADAC enzyme expression.

[0034] Figure 2 ATV-114 exerts anti-inflammatory activity by inhibiting NF-κB signaling, wherein A shows the inhibitory effect of different concentrations of MBAX, ATV-114 and AUDA on LPS-induced inflammatory protein expression in RAW264.7 cells; B shows the inhibition of LPS-induced NF-κB transcriptional activation in RAW264.7 cells transfected with NF-κB luciferase reporter plasmid by different concentrations of MBAX, ATV-114 and AUDA.

[0035] Figure 3 ATV-114 prevents the nuclear export of RNP by inhibiting the nuclear translocation of NF-κB.

[0036] Figure 4 For anti-influenza virus effect, A is the weight change of mice infected with A / PR / 8 / 34 within 14 days after oral administration 12 hours, B is the survival of mice infected with A / PR / 8 / 34 within 14 days after oral administration 12 hours, C is the weight change of mice infected with A / PR / 8 / 34 within 14 days after oral administration at different time points of 48, 72, and 96 hours, D is the survival of mice infected with A / PR / 8 / 34 within 14 days after oral administration at different time points of 48, 72, and 96 hours, E is the weight change of mice infected with A / PR / 8 / 34with NA-H274Y strain within 14 days after oral administration 12 hours, and F is the weight change of mice infected with A / PR / 8 / 34withNA-H274Y strain within 14 days after oral administration 12 hours. DETAILED DESCRIPTION

[0037] Definition of terms

[0038] Unless otherwise indicated, the following terms and phrases as used herein have the following meanings:

[0039] In the present invention, "room temperature" refers to ambient temperature, which is from 10°C to 40°C. In some embodiments, "room temperature" refers to a temperature from 20°C to 30°C; in other embodiments, "room temperature" refers to a temperature from 25°C to 30°C; in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0040] The "esterified residue" in the present invention refers to the residual fragment after the carboxyl group of the soluble epoxide hydrolase inhibitor forms an ester bond with the hydroxyl group of baloxavir.

[0041] Baloxavir derivatives

[0042] "Stereoisomers" refer to compounds with the same chemical structure but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0043] The compound shown in Formula I of the present invention may have a chiral center, such as a chiral carbon. The compound therefore includes a racemic mixture of all stereoisomers, including enantiomers, diastereomers and atropisomers. In addition, the compound of the present invention includes an optical isomer enriched or split at any or all asymmetric chiral atoms. In other words, the chiral center similar to the description is provided in the form of a chiral isomer or a racemic mixture. A mixture of racemic and diastereoisomers, as well as a separate or synthesized, substantially free of its enantiomer or diastereoisomer partner, is within the scope of the present invention. The racemic mixture is separated into their separate, substantially optically pure isomers by known techniques, such as separation of diastereoisomer salts formed with optically active auxiliary agents (such as acids or bases), which are then converted back to optically active substances. In most cases, starting from the appropriate stereoisomer of the desired raw material, the desired optical isomer is synthesized by stereospecific reactions.

[0044] "Hydrate" refers to an association formed when the solvent molecule is water.

[0045] "Deuterated" means that one or more hydrogen atoms in a compound or group are replaced by deuterium. Deuterated can be monosubstituted, disubstituted, polysubstituted or fully substituted.

[0046] "Solvate" refers to an association formed between one or more solvent molecules and the compounds of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid and aminoethanol.

[0047] "Prodrug" is used in its broadest sense and includes those derivatives that are converted into the compounds of the present invention in vivo. These derivatives are obvious to those skilled in the art. When designing prodrugs, the most common form is to modify the structure of drugs containing carboxyl, hydroxyl, and amino groups to make prodrugs of the types of esters, carboxylates, amino acid esters, amides, and phosphates. In addition, azo-type prodrugs, nitric oxide-type prodrugs, and prodrugs of new structures such as open-loop and closed-loop structures can also be made.

[0048] Preferred prodrugs are those that increase the bioavailability of the compounds of the invention relative to the parent substance when these compounds are administered to a patient (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or enhance the delivery of the parent compound to a biological compartment (brain or lymphatic system).

[0049] The compounds of the present invention may be in crystalline form as a favorable compound or as a solvate, both of which are included within the scope of the present invention. Methods of solvation are well known in the art. Suitable solvates are pharmaceutically acceptable solvates.

[0050] "Pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to humans and generally do not produce allergic or similar untoward reactions, such as gastrointestinal discomfort, dizziness, etc. Preferably, the term "pharmaceutically acceptable" as used in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the Chinese Pharmacopoeia, the United States Pharmacopoeia, and other generally recognized pharmacopeias for use in animals, more particularly in humans.

[0051] Examples of "pharmaceutically acceptable salts" include salts derived from suitable bases such as alkali metals or alkaline earth metals (e.g., Na + , Li + , K + , Ca +2 and Mg +2) and ammonium. Physiologically acceptable salts of nitrogen atoms or amino groups include: (a) acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc.; (b) salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalene, etc. (c) salts with elemental anions, such as chlorine, bromine and iodine. Physiologically acceptable salts of hydroxy compounds include salts of the anions of the compounds with hydroxy groups, such as sodium + and NR4 + A combination of appropriate cations.

[0052] For therapeutic use, "acceptable" as used herein is physiologically acceptable, i.e., they are salts derived from physiologically acceptable acids or bases. However, salts that are not physiologically acceptable acids or bases may also be used, for example, to prepare or purify physiologically acceptable compounds. All salts, whether or not derived from physiologically acceptable acids or bases, are within the scope of the present invention.

[0053] The compound shown in formula I or its pharmaceutically acceptable salt can exist as different polymorphs or pseudopolymorphs. The crystal polymorphism used in the present invention refers to the ability of a crystalline compound to exist in different crystal structures. Crystal polymorphism can be derived from differences in crystal packing (stacking polymorphism) or packing differences between different conformers of the same molecule (conformational polymorphism). The crystal pseudopolymorphism used in the present invention refers to the ability of a hydrate or solvate of a compound to exist in different crystal structures. The pseudopolymorphs of the present invention can exist due to differences in crystal packing (stacking pseudopolymorphism) or due to packing differences between different conformers of the same molecule (conformational pseudopolymorphism). The present invention includes all polymorphs and pseudopolymorphs of compounds of formula I and their pharmaceutically acceptable salts.

[0054] The compound of formula I or its pharmaceutically acceptable salt can also exist as an amorphous solid. The amorphous solid used in the present invention is such a solid, wherein there is no long-range order in the positions of the atoms in the solid. This definition also applies when the crystal size is 2 nanometers or less. Additives including solvents can be used to establish the amorphous form of the present invention. The present invention includes all amorphous forms of the compound of formula I and their pharmaceutically acceptable salts.

[0055] The "residue after esterification" is the remaining carboxylic acid fragment after the carboxyl group of the soluble epoxide hydrolase inhibitor forms an ester bond with the hydroxyl group of baloxavir.

[0056] "Substituted" means that one or more hydrogen atoms in the benzene ring or adamantane are independently replaced by a non-hydrogen substituent.

[0057] Screening of active compositions with anti-inflammatory and antiviral properties

[0058] The baloxavir derivatives of the present invention are suitable for treating or preventing influenza virus infection in animals or humans. However, in the process of screening for anti-inflammatory and anti-influenza virus compounds, cell-based and animal (rodent) assays are the main screening tools.

[0059] The present compositions are screened for baloxavir derivatives having anti-inflammatory and anti-viral activity by any conventional technique for evaluating anti-inflammatory and anti-influenza virus activity. In the context of the present invention, typically, the compositions having anti-inflammatory and anti-viral activity are first screened, and then the compositions exhibiting anti-inflammatory and anti-viral activity are screened for in vivo activity. -6 M and preferably less than about 1×10 -7 Compositions with in vitro Ki (inhibition constant) values ​​of M are preferably used in vivo. Useful in vitro screens have been described in detail in the literature and will not be described in detail here. However, the Examples describe suitable in vitro assays.

[0060] Pharmaceutical composition

[0061] The pharmaceutical composition described in the present invention comprises the above-mentioned baloxavir derivative and a pharmaceutically acceptable excipient. In certain embodiments, the baloxavir derivative described in the present invention is provided in the pharmaceutical composition in an effective amount (eg, a therapeutically effective amount).

[0062] The pharmaceutical composition of the present invention is formulated with conventional carriers and excipients, which are selected according to conventional practice. Although the active ingredient can be used alone, it is preferred that they are made into pharmaceutical preparations. The preparation of the present invention, whether for veterinary or human use, comprises at least one active ingredient as defined above and one or more acceptable carriers therefor, and optionally comprises other therapeutic ingredients, especially those other therapeutic ingredients disclosed by the present invention. The carrier must be "acceptable", which means that it is compatible with the other components in the preparation and is physiologically harmless to its recipient.

[0063] The pharmaceutical compositions described herein can be prepared by any method known in pharmacy. In general, these preparation methods include associating the baloxavir derivative (i.e., the "active ingredient") with a carrier or excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0064] The pharmaceutical composition of the present invention can be prepared according to known methods, such as the methods described in the general rules for preparation of the Chinese Pharmacopoeia, the Japanese Pharmacopoeia 16th edition, the United States Pharmacopoeia, and the European Pharmacopoeia 9th edition. Depending on the dosage form, the pharmaceutical composition of the present invention can be appropriately administered to patients.

[0065] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to a subject and / or a convenient fraction of the dose (e.g., one-half or one-third of the dose).

[0066] The active ingredients, pharmaceutically acceptable excipients in the pharmaceutical compositions described herein will vary depending on the identity, size and / or condition of the subject being treated and further on the route of administration of the composition. The pharmaceutical composition may contain between 0.1% and 100% (w / w) active ingredients.

[0067] Pharmaceutically acceptable excipients used in the preparation of the provided pharmaceutical compositions include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients (e.g., cocoa butter and suppository wax), colorants, coating agents, sweeteners, flavoring agents, and aromatics may also be present in the pharmaceutical compositions.

[0068] The invention further provides veterinary compositions comprising at least one active ingredient as defined above together with a veterinary carrier therefor.

[0069] Veterinary carriers are substances useful for the purpose of the veterinary composition and may be solid, liquid or gaseous substances which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.

[0070] The term "treat", as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder, or one or more symptoms of such condition or disorder, to which the term applies. The term "treat", as used herein, refers to the act of treating, as "treat" is defined immediately above.

[0071] The "effective amount" of the compound described in the present invention refers to an amount sufficient to induce a desired biological response. The effective amount of the compound described in the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition to be treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. Alternatively, in a separate method or use, the present invention can be used as a preventive treatment when indicated and effective. In certain embodiments, the effective amount is the amount of the compound described herein in a single dose. In certain embodiments, the effective amount is the combined amount of the compound described herein in multiple doses.

[0072] As used herein, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount of a baloxavir derivative means an amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a disorder alone or in combination with other therapies. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids symptoms, signs or causes of a disorder, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any of the diseases or disorders described.

[0073] Route of administration

[0074] One or more compounds of the present invention (referred to herein as active ingredients) are administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including oral and sublingual) and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), etc. It should be understood that the preferred route may vary with, for example, the condition of the recipient. The benefits of the compounds of the present invention are that they are orally bioavailable and can be administered orally.

[0075] It should also be noted that the specific dosage and method of use of the baloxavir derivative for different patients are determined by many factors, including the patient's age, weight, sex, natural health, nutritional status, drug activity, time of administration, metabolic rate, severity of the disease, and the subjective judgment of the treating physician. The effective dose of the active ingredient depends at least on the nature of the disease to be treated, toxicity (whether the compound is used for prevention or against active viral infection), the method of delivery and the drug formulation, and will be determined by the clinician using conventional dose escalation studies. It can be expected that the dose is about 0.0001 to about 100 mg / kg body weight per day; typically, about 0.01 to about 10 mg / kg body weight per day; more typically, about 0.01 to about 5 mg / kg body weight per day; most typically, about 0.05 to about 0.5 mg / kg body weight per day. For example, for an adult with a body weight of about 70 kg, the daily candidate dose will be in the range of 1 mg to 1000 mg, preferably 5 mg to 500 mg, and can be in the form of a single dose or multiple doses.

[0076] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0077] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0078] Example 1: 4-[(4-{[(tricyclic[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexin-7-yl]oxy}methyl ester (ATV-103)

[0079]

[0080] 4-[(4-{[(tricyclo[3.3.1.1 3,7]Decan-1-ylamino)carbonyl]amino}cyclohexyl)oxy]benzoic acid t-AUCB (1.0g, 2.42mmol), sodium bicarbonate (1.01g, 12.1mmol) and tetrabutylammonium hydrogen sulfate (82.1mg, 0.242mmol) were dissolved in a mixture of 20ml of dichloromethane-water (1:1), and chloromethyl chlorosulfonate (0.97ml, 3.36mmol) was added dropwise. After the addition, it was stirred at room temperature for 8h. The organic layer was separated and the aqueous layer was extracted with 50ml of dichloromethane. The extract and the organic layer were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by rapid silica gel chromatography (0-50% ethyl acetate / petroleum ether) to obtain intermediate 1 (0.88g, yield 79.1%).

[0081] Baloxavir (0.5 g, 1.03 mmol), intermediate 1 (0.57 g, 1.24 mmol) and potassium carbonate (0.35 g, 2.57 mmol) were dissolved in 15 ml of acetonitrile and reacted at 80° C. for 8 h. After the reaction solution was cooled to room temperature, it was concentrated to obtain a crude product, which was purified by flash silica gel chromatography (0-80% ethyl acetate / petroleum ether) to obtain compound ATV-103 (0.56 g, yield 59.3%). 1 H NMR(600MHz,DMSO-d6)δ7.92-7.86(m,2H),7.43-7.35(m,2H),7.22(d,J= 7.8Hz,1H),7.18-7.14(m,1H),7.13-7.08(m,1H),7.08-7.05(m,2H),7.03 -7.00(m,1H),6.88-6.84(m,1H),6.00(d,J=6.5Hz,1H),5.94(d,J=6.5Hz ,1H),5.73(d,J=7.8Hz,1H),5.62(d,J=7.6Hz,1H),5.40(d,J=4.0Hz,1H), 4.48-4.42(m,1H),4.42-4.38(m,1H),4.27-4.20(m,1H),4.08-4.04(m,1 H),3.97(dd,J=10.9,3.1Hz,1H),3.49(dd,J=11.3,3.2Hz,1H),3.43-3.35 (m,2H),297-2.91(m,1H),2.88-2.81(m,1H),2.05-1.96(m,6H),1.85(d,J =2.9Hz,8H),1.59(q,J=3.1Hz,6H),1.49-1.41(m,2H),1.28-1.19(m,3H). 13C NMR (151MHz, DMSO-d6) δ173.6,170.8,165.3,162.2,156.9,156.9,154.5,151.9,150.4,150.3,150. 2,148.0,147.9,146.4,146.3,141.2,136.4,134.2,132.7,132.1,130.2,129.4,128.6,127.8,127.0 ,125.5,125.1,125.0,121.7,117.0,116.9,115.7,114.0,88.7,75.0,73.7,69.8,68.8,65.9,60.2,49.8,47.2,46.0,42.5,42.5,40.5,36.6,31.4,30.7,30.1,29.4,26.8,23.6,22.6,21.2,14.6,14.4.

[0082] Example 2: 12-{[(tricyclic[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexinano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-114)

[0083]

[0084] According to the method described in Example 1, 12-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}dodecanoic acid (AUDA) was used as raw material to synthesize compound ATV-114, a total of 0.3 g of white solid, and the total yield of two steps was 45.6%. 1H NMR (600MHz, DMSO-d6) δ7.43-7.3(m,2H),7.22(d,J=7.7Hz,1H),7.15(t,J=7.6Hz,1H),7.10(d,J=7.9Hz,1H),7.02(d,J=7.7H z,1H),6.85(t,J=7.4Hz,1H),5.73-5.69(m,3H),5.58(t,J=5.6Hz,1H),5.42(d,J=3.1Hz,1H),4.47-4.37(m,2H),4.06(d,J=1 4.3Hz,1H),3.99(dd,J=11.0,3.0Hz,1H),3.67(dd,J=11.7,3.3Hz,1H),3.47(t,J=10.4Hz,1H),3.32(s,1H),3.31-3.26(m,1H ),2.98-2.86(m,3H),2.31(t,J=7.4Hz,2H),2.00-1.95(m,3H),1.84(d,J=2.9Hz,6H),1.62-1.50(m,8H),1.34-1.16(m,16H). 13 C NMR(151MHz,DMSO-d6)δ173.7,172.9,157.5,154.6,150.6,150.3,141.2,13 6.4,134.2,132.7,130.2,129.3,128.6,127.8,127.0,125.4,125.2,125.1, 117.0,116.9,113.9,88.3,73.7,69.8,68.9,66.2,49.8,46.0,42.5,40.5,39.2,36.6,34.0,30.5,29.5,29.4,29.4,29.4,29.3,28.9,26.9,24.5,23.6.

[0085] Example 3: (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid (Intermediate 5)

[0086]

[0087] Add 4-(bromomethyl)-2-fluoro-1-nitrobenzene (3.0 g, 12.9 mmol) and dry acetonitrile (35 mL) to a single-mouth bottle. After 4-(bromomethyl)-2-fluoro-1-nitrobenzene is dissolved, add potassium carbonate (2.13 g, 15.4 mmol), potassium iodide (0.15 g, 1.29 mmol), and (S)-piperidine-3-carboxylic acid ethyl ester (2.03 g, 12.9 mmol) and reflux to react. After 6 hours, the reaction is complete as monitored by TLC. The acetonitrile was removed by concentration under reduced pressure, water (20 mL) was added, and ethyl acetate was extracted (30 mL×2). The organic layers were combined, washed with water (20 mL), and washed once with saturated brine (25 mL). The organic phase was concentrated under reduced pressure to obtain 5.2 g of yellow oil, which was loaded into a column with 4 times silica gel, mixed with 1.2 times silica gel, and eluent (EA:PE=1:40). The yellow oil was obtained by column chromatography, namely (S)-1-(3-fluoro-4-nitrobenzyl)piperidine-3-carboxylic acid ethyl ester (intermediate 2), with a yield of 4.4 g. The yield was 82%.

[0088] To a single-mouth bottle, add (S)-1-(3-fluoro-4-nitrobenzyl)piperidine-3-carboxylic acid ethyl ester (3.60 g, 11.6 mmol), 5% Pd-C (0.4 g) and anhydrous ethanol (50 mL), replace with argon three times, replace with hydrogen three times, heat to 60 ° C, stir and react for 12 h, and monitor the reaction completion by TLC; after the reaction solution is cooled to room temperature (25 ° C), filter it with suction, and concentrate the filtrate under reduced pressure to obtain a yellow oil, namely (S)-1-(4-amino-3-fluorobenzyl)piperidine-3-carboxylic acid ethyl ester (intermediate 3), with a yield of 2.86 g and a yield of 86%.

[0089] Solid phosgene (1.06 g, 3.8 mmol) and dry DCM (30 mL) were added to a three-necked flask, the temperature was cooled to below -78 °C, (S)-1-(4-amino-3-fluorobenzyl)piperidine-3-carboxylic acid ethyl ester (2.13 g, 7.6 mmol) and a dry dichloromethane (60 mL) solution of triethylamine (1.06 g, 3.980 mmol) were added dropwise, and the mixture was stirred at room temperature for 0.5 h, and then the reaction was stopped; the obtained reaction solution was concentrated to dryness under reduced pressure, and dry DCM (10 mL) was added to the residue to dissolve it to obtain an isocyanate solution for later use.

[0090] Memantine (1.48 g, 7.6 mmol), triethylamine (1.54 g, 15.2 mmol), and dry dichloromethane (25 mL) were added to a single-mouth bottle, and the above isocyanate solution was added dropwise. The reaction was allowed to react at room temperature for 0.5 h. TLC showed that the reaction was complete. The reaction solution was poured into water (40 mL), extracted with EA (40 mL × 3), and then washed with 1 mol / L HCl (40 mL × 2), water (40 mL × 2), saturated brine (40 mL) and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3.4 g of a light yellow oil, namely (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid ethyl ester (intermediate 4), which was directly used in the next step without purification.

[0091] To a single-necked bottle, add (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid ethyl ester (Intermediate 4) (3.4 g, 7.0 mmol), dissolve in tetrahydrofuran (20 mL), sodium hydroxide (0.36 g, 9.00 mmol), and water (10 mL), and reflux for reaction. After 12 h, TLC showed that the reaction was complete; the reaction solution was concentrated under reduced pressure to remove tetrahydrofuran, water (300 mL) was added to the residue, and the mixture was placed in a cold trap. The pH was adjusted to 3 with 6N hydrochloric acid (10 mL) to precipitate a light yellow solid, which was filtered off with suction. The filter cake was rinsed with water (20 mL) and dried to obtain 2.96 g of a crude product (dried in an oven at 60°C for 24 h). The crude product was purified by slurrying with petroleum ether and diethyl ether to obtain a light yellow solid, namely (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid (intermediate 5). The yield was 2.8 g, and the yield was 85.5%.

[0092] Example 4: (3S)-1-{[4-({[(1,3-dimethyltricyclo[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexinano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-116)

[0093]

[0094] According to the method described in Example 1, (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid (Intermediate 5) was used as raw material to synthesize compound ATV-116 as a white solid (0.41 g), with a total yield of 47.1% for two steps. 1 H NMR(600MHz,DMSO-d6)δ8.29(d,J=3.2Hz,1H),8.20(t,J=8.3Hz,1H),7.43 -7.35(m,2H),7.27-7.17(m,2H),7.17-7.07(m,3H),7.00(s,1H),6.83(s, 1H),6.57(s,1H),5.70(d,J=7.9Hz,2H),5.42(d,J=14.4Hz,1H),4.47-4.2 8(m,2H),4.06(d,J=14.3Hz,1H),4.00-3.94(m,1H),3.65(d,J=11.2Hz,1H ),3.47(d,J=10.4Hz,1H),3.31(s,2H),3.11-3.04(m,1H),2.91(s,1H),2. 62-2.56(m,1H),2.11-2.07(m,,1H),2.05-2.00(m,1H),1.76(d,J=3.2Hz, 2H),1.72-1.63(m,2H),1.58(s,4H),1.48(d,J=13.3Hz,1H),1.36-1.29(m ,3H),1.29-1.22(m,4H),1.18(t,J=7.1Hz,1H),1.12(s,2H),0.83(s,6H). 13 C NMR(151MHz,DMSO-d6)δ173.6,172.5,168.5,154.5,153.8,150.4,150.3,150.2,150.1,146.4,1 41.1,136.3,134.2,132.7,130.3,130.2,129.3,128.7,128.7,128.5,127.9,127.0,125.4,125.1 ,123.9,119.1,117.0,116.9,114.4,114.3,113.9,88.4,73.8,69.8,68.9,66.3,60.2,52.1,50.7,48.0,45.9,42.8,41.2,40.5,40.5,35.3,32.4,31.6,30.5,30.3,30.0,27.0,26.8,23.6,22.6.

[0095] Example 5: 7-{[(tricyclic[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}heptanoic acid

[0096]

[0097] Solid phosgene (1.06 g, 3.8 mmol) and dry DCM (30 mL) were added to a single-necked bottle, and the temperature was cooled to below -78 °C. A solution of adamantane amine (1.15 g, 7.6 mmol) and triethylamine (1.06 g, 3.980 mmol) in dry dichloromethane (60 mL) was added dropwise. After the addition was completed, the mixture was stirred at room temperature for 0.5 h, and then the reaction was stopped. The obtained reaction solution was concentrated to dryness under reduced pressure, and dry DCM (10 mL) was added to the residue to dissolve it to obtain an isocyanate solution for later use.

[0098] To a three-necked flask, methyl 7-aminoheptanoate (1.21 g, 7.6 mmol), triethylamine (1.54 g, 15.2 mmol), and dry dichloromethane (25 mL) were added, and the above isocyanate solution was added dropwise. The reaction was allowed to react at room temperature for 0.5 h. TLC showed that the reaction was complete. The reaction solution was poured into water (40 mL), extracted with EA (40 mL × 3), and then washed with 1 mol / L HCl (40 mL × 2), water (40 mL × 2), saturated brine (40 mL) and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 2.5 g of a light yellow oil, i.e., methyl 7-(3-(3s,5s,7s)-adamantan-1-yl)ureido)heptanoate (intermediate 6), which was directly used for the next step without purification.

[0099] Add 7-(3-(3s,5s,7s)-adamantan-1-yl)ureido)heptanoic acid methyl ester (intermediate 6) (3.4 g, 10.1 mmol) to a single-mouth bottle, dissolve in tetrahydrofuran (20 mL), add sodium hydroxide (0.48 g, 12.1 mmol), water (10 mL), and reflux for reaction. After 12 hours, TLC shows that the reaction is complete; the reaction solution is concentrated under reduced pressure to remove tetrahydrofuran, and water (300 mL) is added to the residue, which is placed in a cold trap and adjusted to pH 3 with 6N hydrochloric acid (10 mL). A light yellow solid is precipitated, which is filtered with suction, and the filter cake is rinsed with water (20 mL), which is 7-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}heptanoic acid (Intermediate 7) yield 2.8 g, yield 86.0%.

[0100] Example 6: 7-{[(tricyclic[3.3.1.1 3,7[(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazazepino[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-117)

[0101]

[0102] According to the method described in Example 1, 7-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}heptanoic acid (Intermediate 7) was used as the raw material to synthesize compound ATV-117, a total of 0.56 g of white solid, and the total yield of the two steps was 52.6%. 1 H NMR (600MHz, DMSO-d6) δ7.44-7.37(m,2H),7.22(d,J=7.8Hz,1H),7.18-7.13(m,1H),7.09(dd,J=8.0,1.3Hz,1H),7.02(dd, J=8.0,1.5Hz,1H),6.87-6.83(m,1H),5.73-5.68(m,4H),5.60(t,J=5.7Hz,1H),5.43(d,J=13.8Hz,2H),4.47-4.39(m,2H), 4.06(d,J=14.3Hz,1H),3.99(dd,J=10.9,3.1Hz,1H),3.68(dd,J=11.6,3.3Hz,1H),3.48(t,J=10.4Hz,1H),3.31-3.26(m,S 1H),2.98-2.88(m,3H),2.34-2.29(m,2H),1.99-1.94(m,3H),1.83(d,J=2.9Hz,6H),1.62-1.51(m,8H),1.34-1.22(m,6H). 13C NMR(151MHz,DMSO-d6)δ173.7,172.9,157.5,154.6,152.0,151.9,150.6,150.3,150 .2,148.0,147.9,146.4,141.2,136.4,134.2,132.7,132.7,130.2,129.3,128.5,12 7.8,127.0,127.0,125.4,125.2,125.1,117.1,116.9,113.9,88.3,73.8,69.8,68.9,66.2,49.8,46.0,42.5,40.5,39.2,36.6,33.9,30.4,29.4,28.7,26.7,24.5,23.6.

[0103] Example 7: 4-(((1r,4r)-4-(2-(3-fluoro-4-(trifluoromethoxy)phenyl)acetylamino)cyclohexyl)oxy)benzoic acid

[0104]

[0105] Add tert-butyl (1r, 4r)-4-hydroxycyclohexyl)carbamate (3.0 g, 13.95 mmol) and dry DMF (10 mL) to a single-mouth bottle, cool to below 0°C, add NaH (0.62 g, 15.5 mmol), move to room temperature for reaction for 0.5 h, then add 4-fluorobenzonitrile (1.69 g, 13.95 mmol), and after 6 h, TLC monitors the reaction to be complete. Pour the reaction solution into 200 mL of ice water, and a white solid precipitates. Filter and dry to obtain tert-butyl ((1r, 4r)-4-(4-cyanophenoxy)cyclohexyl)carbamate (intermediate 8), with a yield of 3.61 g and a yield of 82%.

[0106] To a single-necked bottle, intermediate 8 (3.61 g, 11.42 mmol), CF3COOH (3 mL) and DCM (10 mL) were added, and the reaction was stirred at room temperature for 6 h. The reaction was complete after monitoring by TLC. The solvent was removed under reduced pressure and the product was directly used for the next step without purification.

[0107] Solid phosgene (1.69 g, 5.71 mmol) and dry DCM (50 mL) were added to a three-necked flask, and the temperature was cooled to below -78 °C. A solution of 4-(((1r, 4r)-4-aminocyclohexyl)oxy)benzonitrile and triethylamine (6.92 g, 68.52 mmol) in dry dichloromethane (60 mL) was added dropwise. After the addition was completed, the mixture was stirred at room temperature for 0.5 h, and then the reaction was stopped. The obtained reaction solution was concentrated to dryness under reduced pressure, and dry DCM (10 mL) was added to the residue to dissolve it to obtain an isocyanate solution for later use.

[0108] 3-Fluoro-4-(trifluoromethoxy)aniline (2.22 g, 11.42 mmol), triethylamine (6.92 g, 68.52 mmol), and dry dichloromethane (50 mL) were added to a single-mouth bottle, and the above isocyanate solution was added dropwise. The reaction was allowed to react at room temperature for 0.5 h. TLC showed that the reaction was complete. The reaction solution was poured into water (40 mL), extracted with EA (40 mL × 3), and then washed with 1 mol / L HCl (40 mL × 2), water (40 mL × 2), saturated brine (40 mL) and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1-((1r, 4r)-4-(4-cyanophenoxy)cyclohexyl)-3-(3-fluoro-4-(trifluoromethoxy)phenyl)urea (intermediate 10), which was directly used in the next step without purification.

[0109] Add the intermediate 10 obtained in the previous step to a single-mouth bottle, dissolve it in tetrahydrofuran (30 mL), and add sodium hydroxide (0.55 g, 13.70 mmol) and water (10 mL) to reflux the reaction. After 12 hours, TLC shows that the reaction is complete; the reaction solution is concentrated under reduced pressure to remove tetrahydrofuran, and water (300 mL) is added to the residue, which is placed in a cold trap and the pH is adjusted to 3 with 6N hydrochloric acid (10 mL). A light yellow solid is precipitated, which is filtered and the filter cake is rinsed with water (20 mL), which is 4-(((1r,4r)-4-(2-(3-fluoro-4-(trifluoromethoxy)phenyl)acetylamino)cyclohexyl)oxy)benzoic acid (intermediate 11). The yield is 4.06 g, and the yield is 78.0%.

[0110] Example 8: Methyl ((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12-12a-hexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl)oxy)benzoate (ATV-123)

[0111]

[0112] According to the method described in Example 1, 4-(((1r,4r)-4-(2-(3-fluoro-4-(trifluoromethoxy)phenyl)acetylamino)cyclohexyl)oxy)benzoic acid (Intermediate 11) was used as a raw material to synthesize compound ATV-123 as a white solid (0.59 g), with a total yield of 54.7% in two steps. 1H NMR(600MHz,DMSO-d6)δ8.71(s,1H),7.91-7.85(m,2H),7.66(dd,J=13.4,2.5Hz,1H),7.38-7.35(m,2H),7.21(d,J=7.8Hz,1H),7.16-7.12(m,1H),7.10-7.04(m,4H),7.01(dd,J=8.0,1.5Hz,1H),6.86-6.82(m,1H),6.30(d,J=7.6Hz,1H),5.98(d,J=6.5Hz,1H),5.93(d,J=6.6Hz,1H),5.73-5.68(m,2H),5.39(dd,J=14.4,2.4Hz,1H),4.50-4.43(m,1H),4.39(dd,J=9.9,3.1Hz,1H),4.26-4.18(m,1H),4.04(d,J=14.3Hz,1H),3.95(dd,J=10.9,3.1Hz,1H),3.57-3.44(m,2H),3.38(t,J=10.4Hz,1H),3.31(s,1H),2.96-2.90(1H),2.86-2.80(m,1H),2.08-2.01(m,2H),1.95-1.88(m,2H),1.53-1.44(m,2H),1.41-1.33(m,2H). 13 C NMR(151MHz,DMSO-d6)δ173.6,165.3,162.1,155.0,154.6,154.5,153.3,151.9,151.8,150.4,150.3,150.2,147.9,146.4,146.3,141.9,141.8,141.2,136.4,134.2,132.7,132.1,130.2,129.4,128.8,128.8,128.6,127.8,127.0,127.0,125.5,125.1,125.0,124.7,123.2,121.8,121.5,119.8,117.0,116.9,115.7,114.0,114.0,114.0,106.1,106.0,88.7,74.8,73.7,69.8,68.8,65.9,60.2,47.7,46.0,40.5,30.3,30.0,23.6,21.2,14.5.

[0113] Example 9: (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4-c]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl)oxy)methyl 4-(((1r,4r)-4-(3-(4-(trifluoromethoxy))ATV-126)

[0114]

[0115] According to the method described in Example 1, t-TUCB was used as a raw material to synthesize compound ATV-126, a total of 0.43 g of white solid, with a two-step total yield of 46.7%. 1 H NMR(600MHz,DMSO-d6)δ8.51(s,1H),7.90(d,J=8.8Hz,2H),7.50-7.45(m,2H) ,7.39(dd,J=8.4,5.1Hz,2H),7.22(dd,J=8.1,2.8Hz,3H),7.18-7.14(m,1H),7 .12-7.06(m,3H),7.02(dd,J=7.9,1.5Hz,1H),6.88-6.83(m,1H),6.20(d,J=7. 6Hz,1H),6.00(d,J=6.6Hz,1H),5.95(d,J=6.6Hz,1H),5.76-5.69(m,2H),5.41 (dd,J=14.4,2.4Hz,1H),4.52-4.45(m,1H),4.41(dd,J=9.9,3.1Hz,1H),4.27 -4.20(m,1H),4.06(d,J=14.3Hz,1H),3.97(dd,J=10.9,3.1Hz,1H),3.58-3.47 (m,2H),3.40(t,J=10.4Hz,1H),2.98-2.91(m,1H),2.88-2.81(m,1H),2.10-2. 03(m,2H),1.94(dd,J=13.1,3.9Hz,2H),1.55-1.45(m,2H),1.43-1.32(m,2H). 13C NMR (151MHz, DMSO-d6) δ173.6,165.3,162.1,154.9,154.5,150.4,150.3,146.4,146. 3,142.4,141.2,140.3,136.4,134.2,132.7,132.7,132.1,130.2,129.4,128.6,127. 8,127.0,125.5,125.1,125.1,122.1,121.8,121.5,119.8,119.0,117.0,116.9,115.7,114.0,88.7,74.9,73.7,69.8,68.8,65.9,47.6,46.0,40.5,30.4,30.0,23.6,14.6.

[0116] Example 10: 12-({[(1,3-dimethyltricyclo[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexan-7-yl]oxy}methyl ester (ATV-140)

[0117]

[0118] According to the method described in Examples 1 and 5, 12-({[(1,3-dimethyltricyclo[3.3.1.1 3,7 ]dec-7-yl)amino]carbonyl}amino)dodecanoic acid was used as raw material to synthesize compound ATV-140, a total of 0.38 g of white solid, and the total yield of two steps was 41.9%. 1H NMR(600MHz, DMSO-d6)δ7.41(dd,J=8.2,5.6Hz,2H),7.22(d,J=7.8Hz,1H),7.18-7.12(m,1H),7.12-7.07(m,1H),7.04-7.00 (m,1H),6.89-6.83(m,1H),7.30-5.66(m,4H),5.57(t,J=5.6Hz,1H),5.43(d,J=12.9Hz,2H),4.47-4.38(m,2H),4.09-3.97(m ,2H),3.67(dd,J=11.6,3.4Hz,1H),3.48(t,J=10.4Hz,1H),3.31-3.26(m,1H),2.97-2.91(m,1H),2.89(q,J=6.5Hz,2H),2.31 (t,J=7.4Hz,2H),2.04-2.01(m,1H),1.67(d,J=3.2Hz,2H),1.57-1.45(m,6H),1.33-1.19(m,20H),1.07(s,2H),0.79(s,6H). 13 C NMR(151MHz,DMSO-d6)δ173.7,172.9,157.6,154.6,150.6,150.2,141.1,136.4,134.3, 132.7,130.2,129.3,128.6,127.8,127.0,125.4,125.2,125.1,117.0,116.9,113.9,88 .3,73.7,69.8,68.9,66.2,60.2,51.4,50.8,48.6,46.0,42.9,41.0,40.5,39.2,34.0,32.4,32.3,30.6,30.5,30.1,29.5,29.4,29.4,29.3,28.9,26.9,24.5,23.6,21.2,14.6.

[0119] Example 11: 12-{[({4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid-{[(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexinano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-148)

[0120]

[0121] According to the method described in Examples 1 and 5, 12-{[({4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid was used as the raw material to synthesize compound ATV-148 as a white solid (0.45 g), with a total yield of 39.9% in two steps. 1 H NMR (600MHz, DMSO-d6) δ8.59(s,1H),7.50-7.46(m,2H),7.40(d,J=6.8Hz,2H),7.21(t,J=8.0Hz,3H),7.17-7.13(m,1H),7.09(d, J=8.0Hz,1H),7.04-7.00(m,1H),6.15(t,J=5.7Hz,1H),5.71(dd,J=9.7,6.1Hz,4H),5.42(dd,J=14.3,2.3Hz,1H),4.47-4.39(m, 2H),4.05(d,J=14.3Hz,1H),3.99(dd,J=10.9,3.0Hz,1H),3.67(dd,J=11.6,3.3Hz,1H),3.47(t,J=10.4Hz,1H),3.31-3.25(m,1H ),3.06(q,J=6.5Hz,2H),2.96-2.90(m,1H),2.31(t,J=7.4Hz,2H),1.55-1.50(m,2H),1.42-1.37(m,2H),1.25(d,J=5.4Hz,15H). 13 C NMR(151MHz,DMSO-d6)δ173.7,172.9,155.5,154.6,152.0,151.9,150.6,150.3,150.2,148.0,147.9,146 .4,146.3,142.4,141.1,140.4,136.4,134.2,132.7,132.7,130.2,129.3,128.5,127.8,127.0,125.4,12 5.2,125.1,123.2,122.0,121.5,119.8,119.0,117.0,116.9,113.9,88.3,73.7,69.8,68.9,66.2,46.0,40.5,39.5,34.7,34.0,30.2,29.5,29.4,29.4,29.2,28.9,28.9,26.8,24.5,23.6,23.5,22.9,19.7,11.7.

[0122] Example 12: 12-{[({3-fluoro-4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid-{[(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazazepino[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-149)

[0123]

[0124] According to the method described in Examples 1 and 5, 12-{[({3-fluoro-4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid was used as the raw material to synthesize compound ATV-149 as a white solid (0.47 g), with a total yield of 45.9% for two steps. 1 HNMR(600MHz,DMSO-d6)δ8.82(s,1H),7.68(dd,J=13.5,2.5Hz,1H),7.42-7.35(m,3H),7.21(d,J=7.7Hz,1H),7.16-7.12(m,1H) ,7.12-7.08(m,2H),7.02-7.00(m,1H),6.86-6.83(m,1H),6.27(t,J=5.7Hz,1H),5.72-5.68(m,4H),5.42(dd,J=14.4,2.3Hz,1H ),4.47-4.38(m,2H),4.05(d,J=14.3Hz,1H),3.99-3.96(m,1H),3.68-3.64(m,1H),3.47(t,J=10.4Hz,1H),3.30-3.25(m,1H),3 .06(q,J=6.6Hz,2H),2.96-2.90(m,3.5Hz,1H),2.30(t,J=7.4Hz,2H),1.55-1.49(m,2H),1.42-1.37(m,2H),1.29-1.22(m,14H). 13C NMR(151MHz,DMSO-d6)δ173.7,172.9,155.2,154.9,154.6,153.3,150.6,150.3,150.2,146.4,146.3,142.1,142.0,14 1.1,136.4,134.2,132.7,130.2,129.3,128.8,128.7,128.5,127.8,127.0,125.4,125.2,125.1,124.6,121.5,119.8,1 17.0,116.9,114.0,114.0,113.9,106.1,105.9,88.3,73.7,69.8,68.9,66.2,46.0,41.3,40.5,39.5,35.9,34.7,34.4,34.0,30.1,29.5,29.4,29.4,29.2,29.2,28.9,28.9,27.6,26.8,25.2,24.5,23.6,22.9,21.1,20.3,19.7,19.1,11.7.

[0125] Example 13: 11-{[(tricyclic[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexinano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-150)

[0126]

[0127] According to the method described in Examples 1 and 5, 11-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}undecanoic acid was used as raw material to synthesize compound ATV-150, a total of 0.45 g of white solid, and the total yield of two steps was 49.9%. 1H NMR (600MHz, DMSO-d6) δ7.43-7.38(m,2H),7.21(d,J=7.7Hz,1H),7.15(t,J=7.5Hz,1H),7.09(d,J=7.9Hz,1H),7.02(d,J=7.7Hz,1H ),6.85(t,J=7.4Hz,1H),5.73-5.67(m,4H),5.57(t,J=5.6Hz,1H),5.41(s,1H),4.46-4.38(m,2H),4.06(d,J=14.4Hz,1H),3.99(dd ,J=10.9,3.0Hz,1H),3.67(dd,J=11.6,3.3Hz,1H),3.47(t,J=10.4Hz,1H),3.30-3.25(m,1H),2.96-2.91(m,1H),2.89(q,J=6.5Hz, 2H), 2.31 (t, J = 7.5Hz, 2H), 1.99-1.95 (m, 3H), 1.83 (d, J = 2.9Hz, 6H), 1.62-1.55 (m, 6H), 1.53 (q, J = 7.3Hz, 2H), 1.33-1.18 (m, 15H). 13 CNMR(151MHz,DMSO-d6)δ173.7,172.9,157.5,154.6,150.6,150.3,150.2,147.9 ,146.4,141.1,136.4,134.2,132.7,130.2,129.3,128.6,127.8,127.0,125.4,12 5.2,125.1,117.0,116.9,113.9,88.3,73.7,69.8,68.9,66.2,49.8,46.0,42.5,40.5,39.2,36.6,34.0,30.5,29.5,29.4,29.3,29.3,29.2,28.9,26.9,24.5,23.6.

[0128] Example 14: 11-({[(3,5-dimethyltricyclo[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazazepino[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-151)

[0129]

[0130] According to the method described in Examples 1 and 5, 11-({[(3,5-dimethyltricyclo[3.3.1.1 3,7 ]dec-1-yl)amino]carbonyl}amino)undecanoic acid was used as raw material to synthesize compound ATV-151, a total of 0.46 g of white solid, and the total yield of two steps was 41.3%. 1 H NMR (600MHz, DMSO-d6) δ7.40(dd,J=8.3,4.7Hz,2H),7.21(d,J=7.8Hz,1H),7.16-7.13(m,1H),7.09(dd,J=8.0,1.3Hz,1H),7.02(dd,J=7.9,1 .5Hz,1H),6.86-6.83(m,1H),5.73-5.68(m,4H),5.57(t,J=5.6Hz,1H),5.43(d,J=12.0Hz,2H),4.46-4.39(m,2H),4.06(d,J=14.3Hz,1H),3. 99(dd,J=10.8,3.1Hz,1H),3.67(dd,J=11.4,3.3Hz,1H),3.47(t,J=10.4Hz,1H),3.31-3.25(m,1H),2.96-2.91(m,1H),2.89(q,J=6.5Hz,2H) ,2.33-2.29(m,2H),2.04-2.01(m,1H),1.66(d,J=3.1Hz,2H),1.54(q,J=7.0Hz,2H),1.48(d,J=5.0Hz,4H),1.33-1.18(m,20H),0.79(s,6H). 13C NMR(151MHz,DMSO-d6)δ173.7,172.9,157.6,154.6,152.0,151.9,150.6,150.3,150.2,148.0,147.9,146.4,146.3, 141.1,136.4,134.2,132.7,130.2,129.3,128.6,127.8,127.0,125.4,125.2,125.1,117.0,116.9,113.9,88.3,73. 7,69.8,68.9,66.2,51.4,50.8,48.6,46.0,42.9,41.3,41.0,40.5,39.2,35.9,34.7,34.4,34.0,32.3,30.6,30.5,30.1,29.5,29.3,29.3,29.0,28.9,27.6,26.9,25.3,24.5,23.6,23.6,23.0,21.1,20.4,19.7,19.1,14.6,14.4,11.7.

[0131] Example 15: 8-{[(tricyclic[3.3.1.1 3,7 [(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexinano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-157)

[0132]

[0133] According to the method described in Examples 1 and 5, 8-{[(tricyclo[3.3.1.1 3,7 ]dec-3-ylamino)carbonyl]amino}octanoic acid was used as raw material to synthesize compound ATV-157, a total of 0.36 g of white solid, and the total yield of two steps was 47.3%. 1HNMR(600MHz, DMSO-d6) δ 7.41 (dd, J=9.5, 6.3Hz, 2H), 7.22 (d, J=7.7Hz, 1H), 7.16 (t, J=7.6Hz, 1H), 7.09 (d, J=8.0Hz, 1H), 7.02 (d, J=7.7Hz, 1H), 6.85 (t, J=7.5Hz, 1H), 5.73 - 5.66 (m, 4H), 5.59 (t, J=5.6Hz, 1H), 5.43 (d, J=17.1Hz, 2H), 4.47 - 4.38 (m, 2H), 4.06 (d, J=14.4Hz, 1H), 3.99 (dd, J=10.9, 3.1Hz, 1H), 3.68 (dd, J=11.5, 3.3Hz, 1H), 3.47 (t, J=10.4Hz, 1H), 3.31 - 3.25 (m, 1H), 2.98 - 2.87 (m, 3H), 2.31 (t, J=7.5Hz, 2H), 1.97 (s, 3H), 1.83 (d, J=3.0Hz, 6H), 1.61 - 1.56 (m, 6H), 1.56 - 1.52 (m, 2H), 1.33 - 1.27 (m, 4H), 1.24 (q, J=9.7Hz, 4H). 13 C NMR(151MHz, DMSO-d6) δ 173.7, 172.9, 157.5, 154.6, 152.0, 151.9, 150.6, 150.2, 148.0, 147.9, 146.4, 146.3, 141.1, 136.4, 134.2, 132.7, 132.7, 130.2, 129.3, 128.6, 127.8, 127.0, 127.0, 125.4, 125.2, 125.1, 117.1, 116.9, 113.9, 88.2, 73.7, 69.8, 68.9, 66.2, 49.8, 46.0, 42.5, 42.1, 40.5, 39.2, 36.6, 34.7, 33.9, 33.6, 30.5, 29.5, 29.4, 29.0, 28.9, 28.7, 26.8, 25.3, 24.4, 23.6.

[0134] Example 16: 12 - [({[1 - (Tricyclo[3.3.1.1 3,7[(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazacyclohexinano[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-158)

[0135]

[0136] According to the method described in Examples 1 and 5, 12-[({[1-(tricyclo[3.3.1.1 3,7 ]dec-1-yl)ethyl]amino}carbonyl)amino]dodecanoic acid was used as raw material to synthesize compound ATV-158, a total of 0.41 g of white solid, and the total yield of two steps was 44.3%. 1 HNMR (600MHz, DMSO-d6) δ7.40(t,J=6.5Hz,2H),7.22(d,J=7.7Hz,1H),7.15(t,J=7.7Hz,1H),7.09(d,J=7.9Hz,1H),7.02(d,J=7.7Hz,1H),6.85 (t,J=7.5Hz,1H),5.73-5.68(m,4H),5.65(t,J=5.6Hz,1H),5.50(d,J=9.5Hz,1H),5.43(dd,J=14.1,2.4Hz,1H),4.47-4.38(m,2H),4.06(d,J=1 4.3Hz,1H),3.99(dd,J=11.0,3.1Hz,1H),3.67(dd,J=11.6,3.3Hz,1H),3.47(t,J=10.4Hz,1H),3.31-3.24(m,2H),2.98-2.90(m,3H),2.31(t,J =7.4Hz,2H),1.94-1.89(m,3H),1.64(d,J=12.0Hz,3H),1.61-1.50(m,6 H),1.46(d,J=12.4Hz,4H),1.41(d,J=12.3Hz,3H),1.32-1.19(m,17H). 13C NMR (151MHz, DMSO-d6) δ173.7,172.9,158.3,154.6,152.0,151.9,150.6,150.3,150.2,148.0,147.9,146.4,146. 3,141.1,136.4,134.2,132.7,132.7,130.2,129.3,128.5,127.8,127.0,127.0,125.4,125.2,125.1,117.0,116. 9,113.8,88.3,73.7,69.8,68.9,66.2,53.0,46.0,41.3,40.5,38.5,37.2,36.1,35.9,34.7,34.0,30.5,29.5,29.4,29.4,29.3,29.3,29.0,28.9,28.3,26.9,25.3,24.5,23.6,23.6,22.9,21.1,20.4,19.7,19.1,15.6,14.6,11.7.

[0137] Example 17: (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4-c]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl)oxy)methyl 12-(3-(((3r,5r,7r)-adamantane (ATV-159)

[0138]

[0139] According to the method described in Examples 1 and 5, 12-[({[1-(tricyclo[3.3.1.1 3,7 ]dec-1-yl)ethyl]amino}carbonyl)amino]dodecanoic acid was used as raw material to synthesize compound ATV-159, a total of 0.41 g of white solid, and the total yield of two steps was 44.3%. 1HNMR(600MHz,DMSO-d6)δ7.40(t,J=6.6Hz,2H),7.22(d,J=7.7Hz,1H),7.15(t,J=7.6Hz,1H),7.09(d,J=7.9Hz,1H),7.02(d,J=7.7Hz,1H),6.85(t,J=7.5Hz,1H),5.73-5.65(m,6H),5.43(dd,J=14.4,2.4Hz,1H),4.47-4.38(m,2H),4.06(d,J=14.3Hz,1H),3.99(dd,J=10.9,3.1Hz,1H),3.67(dd,J=11.6,3.4Hz,1H),3.47(t,J=10.4Hz,1H),3.31-3.25(m,1H),2.98-2.90(m,3H),2.68(d,J=6.1Hz,2H),2.31(t,J=7.4Hz,2H),1.93-1.88(m,3H),1.65(d,J=12.1Hz,3H),1.59-1.50(m,6H),1.39(d,J=2.8Hz,6H),1.29-1.20(m,16H). 13 CNMR(151MHz,DMSO-d6)δ173.7,172.9,158.8,154.6,150.6,150.2,147.9,146.4,146.3,141.1,136.4,134.2,132.7,130.2,129.3,128.5,127.8,127.0,127.0,125.4,125.2,125.1,117.0,116.9,113.8,88.3,73.7,69.8,68.9,66.2,51.5,46.0,39.6,37.1,35.9,34.7,34.0,34.0,33.7,30.5,29.5,29.4,29.4,29.3,29.3,29.1,29.0,28.9,28.3,28.2,26.9,25.3,24.7,24.5,23.6,22.9,19.1,14.6,11.7.

[0140] Example 18: 12-({[(bicyclo[2.2.1]hept-2-en-6-ylmethyl)amino]carbonyl}amino)dodecanoic acid-{[(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[1,2-e:1',2'-b]thiepin-11-yl]-6,8-dioxylidene-1,3,4,6,12,12a-hexahydro[1,4]oxazazepino[3,4-c]pyrido[2,1-f][1,2,4]triazacyclohexin-7-yl]oxy}methyl ester (ATV-160)

[0141]

[0142] According to the method described in Example 1, 0.49 g of white solid compound ATV-160 was synthesized using 12-({[(bicyclo[2.2.1]hept-2-en-6-ylmethyl)amino]carbonyl}amino)dodecanoic acid as raw material, and the total yield of two steps was 47.1%. 1 H NMR (600MHz, DMSO-d6) δ7.40(d,J=6.4Hz,2H),7.22(d,J=7.8Hz,1H),7.15(t,J=7.6Hz,1H),7.09(d,J=7.9Hz,1H),7.04-7.00(m,1H),6 .87-6.83(m,1H),6.13(dd,J=5.8,3.0Hz,1H),5.93(dd,J=5.8,2.8Hz,1H),5.77-5.66(m,6H),5.43(dd,J=14.4,2.4Hz,1H),4.47-4.38( m,2H),4.06(d,J=14.3Hz,1H),3.99(dd,J=10.9,3.0Hz,1H),3.67(dd,J=11.5,3.3Hz,1H),3.47(t,J=10.4Hz,1H),3.31-3.25(m,1H),2. 94-2.90(m,3H),2.80-2.72(m,3H),2.31(t,J=7.4Hz,2H),2.13-2.06(m,1H),1.76-1.70(m,1H),1.55-1.49(m,2H),1.36-1.17(m,20H). 13C NMR (151MHz, DMSO-d6) δ173.7,172.9,158.5,158.4,154.6,152.0,151.9,150.6,150.3,150.2,148.0,147.9,146.4,146.3,141. 1,137.3,136.9,136.9,136.4,134.2,132.8,132.7,132.7,130.2,129.3,128.5,127.8,127.0,125.4,125.2,125.1,117.0,116.9 ,113.9,88.3,73.7,69.8,68.9,66.2,49.4,46.0,45.1,44.9,44.1,43.9,42.3,41.6,41.3,40.5,39.6,35.9,34.7,34.4,34.0,30.8,30.5,30.2,29.5,29.4,29.4,29.3,29.3,29.0,28.9,26.9,25.3,24.5,23.6,23.0,21.1,20.4,19.7,19.1,14.6,14.3,11.7.

[0143] Example 19: In vitro biological activity studies and cytotoxicity studies

[0144] Compounds to be tested: Compounds of the present invention and control compound baloxavir. In vitro biological activity study test method: MDCK cells were seeded into 96-well cell culture plates at a density of 2,0000 cells per well, and then placed in a 37°C, 5% CO2 incubator for overnight culture. The next day, influenza virus A / WSN / 1933 (H1N1) strain was inoculated at 2*TCID 90 Add to the cell culture wells, dilute the compound and add it to the cell wells (2-fold dilution, 8 test concentration points), the final DMSO concentration in the culture medium is 0.5%. The cell plate is placed in a 37°C, 5% CO2 incubator for 2 days. After 2 days of culture, the cell activity is detected using the cell viability detection kit CCK8. The raw data is analyzed by nonlinear fitting of the inhibition rate of the compound using GraphPadPrism9.0 software to obtain the EC 50 The results are shown in Table 1.

[0145] Cytotoxicity study method: The cytotoxicity assay and antiviral activity assay of the compound were carried out in parallel. Except for the absence of virus, the other experimental conditions were the same as those of the antiviral activity assay. After 2 days of culture, the cell viability was detected using the cell viability detection kit CCK8. The raw data were used for compound cytotoxicity (CCK8) 50 ) were calculated, and the results are shown in Table 1.

[0146] Table 1 Inhibitory activity and toxicity of compounds against influenza virus A / WSN / 1933 (H1N1)

[0147]

[0148]

[0149] As shown in Table 1, the EC values ​​of BAX and MBAX for H1N1 50 The values ​​were 2.19nM and 1.68nM, respectively, which are very close to the values ​​previously reported in the literature. In contrast, most of the twin drugs showed strong inhibitory effects on H1N1. Specifically, ATV-114, ATV-148, ATV-149, ATV-150, ATV-151, and ATV-158 showed better anti-influenza virus effects than MBAX, especially ATV-114 had an EC of 0.10 for H1N1. 50 is 0.51nM. ATV-114 is synthesized by connecting the carboxyl structure of the sEH inhibitor AUDA to the enol structure of BAX through a methylene group. Therefore, the inventors evaluated the inhibitory activity of compounds ATV-114 and MBAX against various clinical isolates of influenza A and influenza B viruses, including the oseltamivir-resistant strain A / PR / 8 / 1934 (NA-H274Y). It is worth noting that the inhibitory activity of ATV-114 against all test strains exceeded that of MBAX. In particular, for the A / PR / 8 / 1934 (H1N1) virus, the activity of ATV114 was almost four times that of MBAX, and the results are shown in Table 2.

[0150] Table 2 Inhibitory activity of ATV-114 and MBAX against different influenza virus strains

[0151]

[0152] Example 20: ATV-114 is more effective than MBAX in inhibiting viral proteins in both cells with high and low AADAC enzyme expression

[0153] In order to exert its antiviral activity, baloxavir must be converted to baloxavir acid, a process that relies on arylacetamide deacetylase (AADAC). AADAC is primarily expressed in the liver, intestine, pancreas, and adrenal glands, with lower levels detected in other tissues. In contrast, carboxylases that hydrolyze ATV-114 into active metabolites AUDA and BAX are widely distributed throughout the body. In the experiments of the present invention, cells were infected with the H1N1 virus and treated with MBAX, ATV-114, and AUDA under conditions of high and low AADAC expression. Figure 1As shown in Figures AF, AC represents the inhibitory effect of different concentrations of MBAX, ATV-114 and AUDA on the expression of H1N1 virus proteins in cell lines with high AADAC expression (i.e., Caco-2, HEPG-2 and MDCK cells); DF represents the inhibitory effect of different concentrations of MBAX, ATV-114 and AUDA on the expression of H1N1 virus proteins in cell lines with low AADAC expression (A549, Beas-2B and HaCat cells).

[0154] Figure 1 MBAX significantly inhibited the expression of influenza virus nucleoprotein (NP) and polymerase acidic (PA) proteins, and enhanced efficacy was observed in cells showing high AADAC expression. In cells with high AADAC expression, 10nM MBAX almost completely inhibited influenza virus protein expression, while ATV-114 had a more significant inhibitory effect on viral protein expression in cells with high and low AADAC expression, achieving complete inhibition of viral protein expression at a concentration of 5nM.

[0155] Example 21: ATV-114 exerts anti-inflammatory activity by inhibiting NF-κB signaling

[0156] In this study, the inventors used the LPS-induced RAW264.7 inflammation model to evaluate the anti-inflammatory effect of AUDA in vitro and determined that ATV-114 could successfully hydrolyze AUDA to obtain similar anti-inflammatory effects. Figure 2 As shown in A, ATV-114 and AUDA inhibited LPS-induced upregulation of inflammatory proteins and activation of the NF-κB pathway, while MBAX had no significant effect. Subsequently, the inventors transfected RAW264.7 cells with NF-κB luciferase reporter plasmid, and the expression of luciferase represented NF-κB transcriptional activity. Figure 2 As shown in Figure B, the NF-κB inhibitor BAY11-7085 almost completely inhibited LPS-induced NF-κB transcriptional activation, and both AUDA and ATV-114 inhibited NF-κB transcription in a concentration-dependent manner, indicating that NF-κB is indeed the target of AUDA and ATV-114 inhibits NF-κB by releasing AUDA molecules in cells.

[0157] Example 22: Confocal experiment to detect the inhibitory effect of twin drug ATV-114 on RNP

[0158] Six hours after influenza virus infected A549 cells, the viral nucleic acid nucleoprotein complex RNP was released from the cell nucleus for a new round of infection and replication; after cells were infected with influenza virus, the NF-κB signaling pathway was activated, NF-κB phosphorylated and entered the cell nucleus, promoting RNP to exit the nucleus.

[0159] A549 cells were infected with A / WSN / 1933 virus for 6 hours before indirect immunofluorescence assay using NF-κB and NP antibodies. DAPI was used to indicate nuclear localization. Scale bar: 10 μm. Figure 3 As shown, compound ATV-114 inhibits RNP nuclear export by reducing NF-κB nuclear import.

[0160] Example 23: ATV-114 effectively protects mice from influenza virus infection and death

[0161] Evaluate the efficacy of ATV-114 in the influenza A virus H1N1 mouse infection model. Mice were infected with influenza A virus A / PR / 8 / 1934 or A / PR / 8 / 34with NA-H274Y via intranasal drops and treated with the compound at 12, 48, 72 and 96 hours after infection, respectively. MBAX, ATV-114 and HEAUDA were administered once, and oseltamivir phosphate was administered once a day for three consecutive days. The anti-influenza virus H1N1 effect of the compound in this model was evaluated by observing the changes in mouse body weight and survival rate, such as Figure 4 As shown. The experiment used ATVF-level BALB / c mice (Experimental Animal Management Center of Southern Medical University), 6-7 weeks old, half male and half female. The experiment began after the mice arrived at the BSL-2 animal room and adapted for at least 3 days, and the day of infection was set as day 0 of the experiment.

[0162] Currently, widely used antiviral drugs in clinical practice, such as oseltamivir and baloxavir, are recommended to be administered within 48 hours after infection to effectively protect influenza patients. In the inventors' study, BALB / c mice were infected with the A / PR / 8 / 1934 virus and orally administered MBAX or ATV-114 at 48, 72, and 96 hours after infection. The survival rate of ATV-114 treatment at 72 hours and 96 hours after infection was still 37.5%. In contrast, oseltamivir had no protective effect when administered at 72 hours and 96 hours after infection, and MBAX only provided a survival rate of 12.5% ​​when administered at 72 hours after infection. These findings highlight the great potential of AUDA's potent anti-inflammatory effect.

[0163] After influenza virus infection, in addition to direct damage caused by the virus, the inflammatory cytokine storm also poses a serious threat to lung cells. Therefore, although antiviral treatment started 72 hours after infection can inhibit viral replication, high viral loads coupled with uncontrolled inflammatory responses will hinder survival. Due to the effects of cytokine storm and pulmonary fibrosis, surviving mice often recover slowly, resulting in irreversible lung damage.

[0164] The results of the study showed that ATV-114 released AUDA in the middle stage of viral infection to inhibit the over-activation of inflammatory pathways, thereby alleviating the impact of cytokine storms. At the same time, ATV-114 released BAX, which rapidly inhibited viral replication and prevented the production of new progeny viruses, reducing viral load from the source. The dual anti-inflammatory and antiviral effects of ATV-114 provided crucial treatment for infected mice during this critical period.

[0165] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A baloxavir derivative, a stereoisomer thereof, a crystalline hydrate thereof, a deuterated product thereof, a solvate thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the baloxavir derivative is shown in Formula I: Here, sEH inhibitor is the esterified residue of soluble epoxide hydrolase inhibitor.

2. The baloxavir derivative, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or its pharmaceutically acceptable salt according to claim 1, characterized in that: The soluble epoxide hydrolase inhibitor contains a substituted benzene ring or a substituted adamantane.

3. The baloxavir derivative, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or its pharmaceutically acceptable salt according to claim 1, characterized in that: The soluble epoxide hydrolase inhibitor is t-AUCB, 12-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}dodecanoic acid (AUDA), (S)-1-(4-(3-(1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)piperidine-3-carboxylic acid, 7-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}heptanoic acid, 4-(((1r,4r)-4-(2-(3-fluoro-4-(trifluoromethoxy)phenyl)acetylamino)cyclohexyl)oxy)benzoic acid, t-TUCB, 12-({[(1,3-dimethyltricyclo[3.3.1.1 3,7 ]dec-7-yl)amino]carbonyl}amino)dodecanoic acid, 12-{[({4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid, 12-{[({3-fluoro-4-[(trifluoromethyl)oxy]phenyl}amino)carbonyl]amino}dodecanoic acid, 11-{[(tricyclo[3.3.1.1 3,7 ]dec-1-ylamino)carbonyl]amino}undecanoic acid, 11-({[(3,5-dimethyltricyclo[3.3.1.1 3,7 ]dec-1-yl)amino]carbonyl}amino)undecanoic acid, 8-{[(tricyclo[3.3.1.1 3,7 ]dec-3-ylamino)carbonyl]amino}octanoic acid, 12-[({[1-(tricyclo[3.3.1.1 3,7 ]dec-1-yl)ethyl]amino}carbonyl)amino]dodecanoic acid, 12-[({[1-(tricyclo[3.3.1.1 3,7 At least one of {[(bicyclo[2.2.1]hept-2-en-6-ylmethyl)amino]carbonyl}amino)dodecanoic acid and 12-({[(bicyclo[2.2.1]hept-2-en-6-ylmethyl)amino]carbonyl}amino)dodecanoic acid.

4. The baloxavir derivative, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or its pharmaceutically acceptable salt according to claim 1, characterized in that: The compound represented by formula I is selected from the following compounds:

5. A pharmaceutical composition, characterized in that The invention comprises the baloxavir derivative according to any one of claims 1 to 4, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or its pharmaceutically acceptable salt.

6. The pharmaceutical composition according to claim 5, characterized in that Includes a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of tablets, pills, creams, emulsions, ointments, suspensions, lyophilized preparations, capsules, sustained-release preparations, granules, granules, injections or sprays.

8. Use of the baloxavir derivative according to any one of claims 1 to 4, its stereoisomer, its crystalline hydrate, its deuterated product, its solvate, its prodrug or its pharmaceutically acceptable salt, or the pharmaceutical composition according to any one of claims 5 to 7 in the preparation of anti-inflammatory and antiviral drugs.

9. The use according to claim 8, characterized in that The virus is an influenza virus.

10. The use according to claim 9, characterized in that The influenza virus is influenza A virus and / or influenza B virus.

11. The use according to claim 9, characterized in that The influenza virus is at least one of H1N1, H5N1, H7N9, H3N2 and influenza B viruses.

12. The use according to claim 11, characterized in that The influenza virus is at least one of H1N1, H3N2 or B influenza virus.

13. Use of the baloxavir derivative, stereoisomer thereof, crystalline hydrate thereof, deuterated product thereof, solvate thereof, prodrug thereof or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 7 in the preparation of an inhibitor of NF-κB signaling pathway.

14. The use according to any one of claims 8 to 13, characterized in that: The anti-inflammatory and antiviral drugs or the NF-κB signaling pathway inhibitor are suitable for humans or animals.

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