Deuterated nucleoside compound and its use

Abstract

The present invention discloses a series of deuterated nucleoside compounds and their uses, specifically, compounds represented by formula (IV), their stereoisomers or their pharmaceutically acceptable salts. JPEG2025517042000037.jpg7084

Description

Technical Field

[0001] The present invention claims the following priorities. Application No.: CN202210548373.7, Filing Date: May 17, 2022; Application No.: CN202210695557.6, Filing Date: June 15, 2022; Application No.: CN202211002496.7, Filing Date: August 19, 2022; Application No.: CN202310213459.9, Filing Date: March 7, 2023. The present invention relates to the technical field of pharmaceutical chemistry, and particularly to a series of deuterated nucleoside compounds and their uses. Specifically, it relates to a compound represented by formula (IV), its stereoisomers or its pharmaceutically acceptable salts.

Background Art

[0002] Currently, seven types of coronaviruses that can infect humans are known, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (SARS), MERS-CoV (MERS), and SARS-CoV-2 (COVID-2019) that emerged at the end of 2019. Among them, the latter three can cause severe symptoms and even death when infecting humans. Coronavirus infections, especially the new COVID-2019 and its variants, spread very rapidly, and some patients may require a long period to recover, seriously affecting people's work and life.

Summary of the Invention

Problems to be Solved by the Invention

[0003] There is an urgent need for more effective drugs and methods for treating such infectious diseases.

Means for Solving the Problems

[0004] In one aspect, the present invention provides a compound represented by formula (IV), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0005]

Chemical formula

[0006] However, R 4 is selected from hydrogen and R 1 -C(=O)-, R 1 is C 1-6 alkyl, C 1-4 alkoxy, and phenyl, and the C 1-6 alkyl, C 1-4 alkoxy, and phenyl are each independently optionally substituted with one, two, or three Rs, R 2 and R 3 are each independently selected from hydrogen, C 1-6 alkyl-C(=O)-, C 1-4 alkoxy-C(=O)-, and phenyl-C(=O)-, and the C 1-6 alkyl, C 1-4 alkoxy, and phenyl are each independently optionally substituted with one, two, or three Rs, Each R is independently selected from hydroxyl, halogen, amino, and cyano.

[0007] In another aspect, the present invention provides a compound represented by formula (IV), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from the structures represented by formulas (VI-1) and (VI-2).

[0008]

Chemical formula

[0009] However, R 2 and R 3 are each independently hydrogen, C1-6 alkyl-C(=O)-, C 1-4 alkoxy-C(=O)- and phenyl-C(=O)-, and said C 1-6 alkyl, C 1-4 alkoxy and phenyl are each independently optionally substituted by one, two or three Rs, R 5 is selected from phenyl, and said phenyl is optionally substituted by one, two or three Rs, R 6 is C 1-6 alkyl and C 1-4 alkoxy, and said C 1-6 alkyl and C 1-4 alkoxy are each independently optionally substituted by one, two or three Rs, each R is independently selected from hydroxyl, halogen, amino and cyano. In some embodiments of the present invention, among the compounds represented by the above formula (IV), their stereoisomers or their pharmaceutically acceptable salts, each R is independently selected from hydroxyl, and the other variables are as defined in the present invention.

[0010] In some embodiments of the present invention, among the compounds represented by the above formula (IV), their stereoisomers or their pharmaceutically acceptable salts, R 1 is selected from isopropyl, methoxy, tert-butoxy and phenyl, and said isopropyl, methoxy, tert-butoxy and phenyl are each independently optionally substituted by one, two or three Rs, and the other variables are as defined in the present invention.

[0011] In some embodiments of the present invention, among the compounds represented by the above formula (IV), their stereoisomers or their pharmaceutically acceptable salts, R 1 is selected from isopropyl, methoxy, tert-butoxy and

[0012]

Chemical formula

[0013] selected from, and other variables are as defined in the present invention.

[0014] In some embodiments of the present invention, among the compounds represented by the above formula (IV), their stereoisomers or their pharmaceutically acceptable salts, R 4 is selected from hydrogen, isopropyl-C(=O)-, methoxy-C(=O)-, tert-butoxy-C(=O)- and phenyl-C(=O)-, and the isopropyl, methoxy, tert-butoxy and phenyl are each independently optionally substituted by one, two or three Rs, and other variables are as defined in the present invention.

[0015] In some embodiments of the present invention, among the compounds represented by the above formula (IV), their stereoisomers or their pharmaceutically acceptable salts, R 4 is hydrogen, isopropyl-C(=O)-, methoxy-C(=O)-, tert-butoxy-C(=O)- and

[0016]

Chemical formula

[0017] selected from, and other variables are as defined in the present invention.

[0018] In some embodiments of the present invention, among the compounds represented by the above formula (VI-1), their stereoisomers or their pharmaceutically acceptable salts, R 5 is

[0019]

Chemical formula

[0020] selected from, and other variables are as defined in the present invention.

[0021] In some embodiments of the present invention, among the compounds represented by the above formula (VI-2), their stereoisomers or their pharmaceutically acceptable salts, R 6 is selected from isopropyl, methoxy and tert-butoxy, and the isopropyl, methoxy and tert-butoxy are each independently optionally substituted by one, two or three Rs, and the other variables are as defined in the present invention.

[0022] In some embodiments of the present invention, among the compounds represented by the above formula (VI-2), their stereoisomers or their pharmaceutically acceptable salts, R 6 is selected from isopropyl, methoxy and tert-butoxy, and the other variables are as defined in the present invention.

[0023] In some embodiments of the present invention, among the compounds represented by the above formula (VI-2), their stereoisomers or their pharmaceutically acceptable salts, R 6 is selected from isopropyl, and the other variables are as defined in the present invention.

[0024] In some embodiments of the present invention, the above R 2 and R 3 are each independently selected from hydrogen, isopropyl-C(=O)- and phenyl-C(=O)-, and the isopropyl and phenyl are each independently optionally substituted by one, two or three Rs, and the other variables are as defined in the present invention.

[0025] In some embodiments of the present invention, R 2 and R 3 are each independently selected from hydrogen, isopropyl-C(=O)- and

[0026]

Chemical formula

[0027] selected from, and the other variables are as defined in the present invention.

[0028] In some embodiments of the present invention, R 2 and R 3 are each independently selected from hydrogen and isopropyl-C(=O)-, and the other variables are as defined in the present invention.

[0029] In some embodiments of the present invention, R 2 and R 3 are each independently selected from hydrogen and

[0030]

Chemical formula

[0031] and the other variables are as defined in the present invention.

[0032] The present invention also provides a compound represented by formula (IV) or a pharmaceutically acceptable salt thereof.

[0033]

Chemical formula

[0034] provided that R 4 is selected from hydrogen and R 1 -C(=O)-, R 1 is selected from C 1-6 alkyl, C 1-4 alkoxy and phenyl, and the phenyl is optionally substituted with one, two or three Rs, R 2 and R 3 are each independently selected from hydrogen, C 1-6 alkyl-C(=O)-, C 1-4 alkoxy-C(=O)- and phenyl-C(=O)-, and the phenyl-C(=O)- is optionally substituted with one, two or three Rs, R is selected from hydroxyl, halogen, amino and cyano. In some embodiments of the present invention, the above R is selected from hydroxyl, and the other variables are as defined in the present invention.

[0035] In another part of the embodiments of the present invention, the above R 1 is selected from isopropyl, methoxy, tert-butoxy and

[0036]

Chemical formula

[0037] and the other variables are as defined in the present invention.

[0038] In some embodiments of the present invention, the above R 4 is hydrogen, isopropyl-C(=O)-, methoxy-C(=O)-, tert-butoxy-C(=O)- and

[0039]

Chemical formula

[0040] and the other variables are as defined in the present invention.

[0041] The present invention also provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0042]

Chemical formula

[0043] However, R 1 is selected from C 1-6 alkyl, C 1-4 alkoxy and phenyl, and the phenyl is optionally substituted by one, two or three Rs, R 2 and R 3 are each independently hydrogen, C 1-6Alkyl-C(=O)-, C 1-4 selected from alkoxy-C(=O)- and phenyl-C(=O)-, wherein said phenyl-C(=O)- is optionally substituted with one, two or three Rs, R is selected from hydroxyl, halogen, amino and cyano.

[0044] In some embodiments of the present invention, the above R is selected from hydroxyl.

[0045] In some embodiments of the present invention, the above R 1 is isopropyl, methoxy, tert-butoxy and

[0046]

Chemical formula

[0047] selected from.

[0048] Some forms of the present invention are further formed by any combination of the above variables.

[0049] The present invention also provides a compound selected from the following formulas or a pharmaceutically acceptable salt thereof.

[0050]

Chemical formula

[0051] In some embodiments of the present invention, there is provided the use of the above compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease associated with an RNA-dependent RNA polymerase inhibitor.

[0052] In some embodiments of the present invention, the above use is characterized in that the disease associated with the RNA-dependent RNA polymerase inhibitor is a viral infection such as viral cold, viral pneumonia.

[0053] In some embodiments of the present invention, the above use is characterized in that the disease associated with the RNA-dependent RNA polymerase inhibitor is viral pneumonia.

[0054] The present invention also provides the following synthetic route.

[0055] [Chemical Formula] [Advantages of the Invention]

[0056] The compounds of the present invention have strong in vitro anti-coronavirus activity. After administering the compounds of the present invention to mice, they are rapidly metabolized into corresponding active metabolites in vivo to exert medicinal effects. The drug concentration of the compounds of the present invention lasts longer, the maintained concentration is also higher, which is advantageous for continuous virus inhibition and the exertion of medicinal effects. [Modes for Carrying Out the Invention]

[0057] [Definitions and Explanations]

[0058] Unless otherwise explained, the following terms and collocations used in this specification include the following meanings. If a particular term or collocation is not specially defined, it should not be uncertain or unclear and should be understood as an ordinary definition. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0059] As used in this specification, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms, which are within the scope of reliable medical judgment, suitable for contact with human and animal tissues, and have few toxicities, irritations, allergic reactions or other problems or complications, and meet a reasonable benefit / risk ratio.

[0060] Unless otherwise explained, the term "treatment" refers to all processes that may slow down, interrupt, prevent or stop the progression of a disease, but does not necessarily mean that all symptoms disappear.

[0061] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, which are prepared with acids or bases that are relatively non-toxic compared to compounds having the specific substituents discovered in the present invention. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting these compounds in their neutral form with a sufficient amount of base in a single solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine 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 these compounds in their neutral form with a sufficient amount of acid in a single solution or in a suitable inert solvent. Some specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into any base addition salt or acid addition salt.

[0062] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid or basic groups by conventional methods. Usually, the methods for producing such salts are carried out by reacting these compounds in the form of free acids or bases with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

[0063] Unless otherwise specified, the term "isomer" refers to including geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, enantiomers and tautomers.

[0064] The compounds of the present invention may exist in the form of specific geometric or stereoisomers. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as mixtures rich in enantiomers or non-enantiomers, and all these mixtures are included within the scope of the present invention. Other asymmetric carbon atoms may be present in substituents such as alkyl. All these isomers and mixtures thereof are included within the scope of the present invention.

[0065] Unless otherwise indicated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0066] Unless otherwise indicated, the terms "cis-trans isomer" or "geometric isomer" are due to the fact that the double bond or single bond of the ring-forming carbon atoms cannot rotate freely.

[0067] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers in which a molecule has two or more chiral centers and the molecules are in a non-mirror image relationship with each other.

[0068] Unless otherwise indicated, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic.

[0069]

Chem.

[0070] Unless otherwise specified, when a group has one or more bondable sites, any one or more sites of the group can be bonded to other groups by chemical bonds. When the bonding mode of the chemical bond is delocalized and there are H atoms at the bondable sites, when the chemical bond is formed, the number of H atoms at the site decreases to a group with a corresponding valence according to the number of the formed chemical bonds. The chemical bond by which the site is bonded to another group is

[0071]

Chem.

[0072] For example, the straight solid line bond of -OCH 3 means that it is bonded to another group through the oxygen atom of the group.

[0073]

Chem.

[0074] The dashed line connection of the straight line in the formula means that both ends of the nitrogen atom in the group are bonded to other groups.

[0075] [Chemical formula]

[0076] The dotted line connection in the formula means that it can be bonded to other groups by one chemical bond through any bondable site of the phenyl group.

[0077] Specific compounds of the present invention may exist. Unless otherwise explained, the terms "tautomer" or "tautomeric form" refer to isomers of different functional groups being in dynamic equilibrium and being able to rapidly convert into each other at room temperature. Tautomers can reach the chemical equilibrium of tautomers if possible (for example, in solution). For example, proton tautomers (also called prototropic tautomers) include interconversions via the transfer of protons, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by the rearrangement of some bonding electrons. Among them, a specific example of keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxy-3-penten-2-one.

[0078] Unless otherwise indicated, the terms "enriched in one isomer", "isomer-enriched", "enriched in one enantiomer", or "enantiomer-enriched" mean that the content of one isomer or enantiomer is less than 100% and the content of this isomer or enantiomer is 60% or more, or 70% or more, or 80% or more, or 90% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.6% or more, or 99.7% or more, or 99.8% or more, or 99.9% or more.

[0079] Unless otherwise indicated, the terms "isomer excess" or "enantiomer excess" mean the difference between the relative percentages of two isomers or two enantiomers. For example, when the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80%.

[0080] Optically active (R)- and (S)-isomers as well as D and L isomers can be produced using asymmetric synthesis or chiral reagents or other conventional techniques. To obtain one enantiomer of a compound of the present invention, it can be produced by asymmetric synthesis or by induction with a chiral auxiliary, where the resulting mixture of diastereomers is separated and the auxiliary group is decomposed to provide the desired enantiomeric purity. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), a diastereomeric salt is formed with an appropriate optically active acid or base, and after separating the diastereomers by a conventional method known in the art, the recovered and isolated enantiomer is obtained. Also, the separation of enantiomers and diastereomers is usually carried out by chromatography, where the chromatography uses a chiral stationary phase and may be combined with any chemical derivatization method (e.g., generating a carbamate from an amine).

[0081] The compounds of the present invention may contain non-natural atomic isotopes in one or more of the atoms constituting the compounds. For example, tritium (3 H), iodine-125( 125 I) or C-14( 14 C) and other radioisotopes can be used to label compounds. Also, for example, deuterium can be substituted for hydrogen to form deuterated drugs. The bond formed between deuterium and carbon is stronger than the bond formed between normal hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have the advantages of reduced toxic side effects, increased drug stability, improved therapeutic effects, and extended biological half-lives of the drugs. The conversion of the isotope composition of the compounds of the present invention is included within the scope of the present invention, whether radioactive or not.

[0082] "Optional" or "optionally" means that it is possible depending on the matters or circumstances described below but does not necessarily occur, and the description includes the case where the matters or circumstances described therein do not occur when such matters or circumstances occur.

[0083] The term "substituted" means that any one or more hydrogen atoms at a specific atom are substituted with substituents, and may include deuterium and hydrogen variants as long as the specific valence state is normal and the compound after substitution is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are substituted. Keto group substitution does not occur in aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise defined, the type and number of substituents are arbitrary as long as they can be realized chemically stably.

[0084] If any of the variables (e.g., R) appears one or more times in the composition or structure of the compound, its definition is independent in any case. Therefore, for example, when one group is substituted with 0 to 2 R's, the above group is optionally substituted with 2 or fewer R's, and in any case, R has independent options. Also, combinations of substituents and / or their variants are only permitted if such combinations result in stable compounds.

[0085] Unless otherwise defined, the term "C 1―6"Alkyl" represents a saturated hydrocarbon group composed of 1 to 6 straight-chain or branched carbon atoms. The above C 1-6 alkyl includes C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4 、C 6 、and C 5 alkyl etc., and it may be monovalent (e.g., methyl), divalent (e.g., methylene) or polyvalent (e.g., methine). Examples of C 1-6 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl, t-butyl), pentyl (including n-pentyl, isopentyl, neopentyl), hexyl, etc.

[0086] Unless otherwise defined, the term "C 1―4 alkoxy" represents an alkyl group containing 1 to 4 carbon atoms linked to the rest of the molecule through an oxygen atom. The above C 1-4 alkoxy includes C 1-3 、C 1-2 、C 2-4 、C 4 and C 3 alkoxy etc. Examples of C 1-6 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy and t-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy and neopentyloxy), hexyloxy, etc.

[0087] Unless otherwise defined, the term "halogen" or "halo" means a fluorine, chlorine, bromine or iodine atom, either by itself or as part of another substituent.

[0088] The compounds of the present invention can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthesis methods, and equivalent alternative methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0089] The structure of the compounds of the present invention can be confirmed by conventional methods well-known to those skilled in the art. When the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by the conventional technical means of those skilled in the art. For example, single crystal X-ray diffraction (SXRD), cultured single crystals are collected by a Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning. After collecting the relevant data, the direct method (Shelxs97) can be used for crystal structure analysis to confirm the absolute configuration.

[0090] All solvents used in the present invention can be obtained from commercially available products. The present invention uses the following abbreviations. aq represents water; eq represents equivalent; M represents mol / L; DCM represents dichloromethane; PE represents petroleum ether; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOAc represents ethyl acetate; EtOH represents ethanol; MeOH represents methanol; THF represents tetrahydrofuran; HCOOH represents formic acid; TFA represents trifluoroacetic acid; Py represents pyridine; DIPEA represents diisopropylethylamine; DMP represents Dess-Martin reagent; NaBD 4 represents sodium borohydride-d; MeOD represents methanol-d; TBAF represents tetrabutylammonium fluoride; ACN represents acetonitrile; mp represents melting point; RdRp represents RNA-dependent RNA polymerase; room temperature represents 25 °C.

[0091] Compounds are named according to the normal naming principles in this field or by ChemDraw (R) software, and the names of commercially available compounds are those used in the manufacturer's catalog.

Examples

[0092] The present invention will be specifically described below by way of examples, which do not mean any restrictive limitation of the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. It is obvious to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0093] Example 1

[0094]

Chemical formula

[0095] Synthesis scheme:

[0096]

Chemical formula

[0097] Step 1: Synthesis of compound 1-2 Compound 1-1 (3.0 g) and dichloromethane (20 mL) were added to a reaction flask, and pyridine (29.40 g) and compound 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (3.90 g) were sequentially added thereto. The reaction system was stirred at room temperature for 14 hours. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO (R) ; 40 g of SepaFlash (R) Silica Flash Column, mobile phase: 0-50% EtOAc / PE, flow rate: 50 mL / min) to obtain compound 1-2 (purity: 84.0%).

[0098] MS m / z (ESI): [M+H] + = 534.6.

[0099] Step 2: Synthesis of compound 1-3 Compound 1-2 (1.0 g, purity: 84%) and dichloromethane (50 mL) were added to a reaction flask, and Dess-Martin reagent (1.19 g) was further added. The reaction system was stirred at room temperature for 4 hours. After filtering the reaction solution, it was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO (R) ; 12 g of SepaFlash (R) Silica Flash Column, mobile phase: 0 - 33% EtOAc / PE, flow rate: 35 mL / min) to obtain compound 1-3.

[0100] MS m / z (ESI): [M+H] + = 532.2.

[0101] Step 3: Synthesis of compound 1-4 Compound 1-3 (200 mg) and deuterated methanol (10 mL) were added to a reaction flask, and sodium borohydride-d (28.46 mg) was further added. The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO (R) ; 12 g of SepaFlash (R) Silica Flash Column, mobile phase: 0 - 30% EtOAc / PE, flow rate: 30 mL / min) to obtain compound 1-4.

[0102] MS m / z (ESI): [M+H] + = 535.2.

[0103] Step 4: Synthesis of compound 1-5 Compound 1-4 (110 mg) and tetrahydrofuran (4 mL) were added to a reaction flask, and a tetrahydrofuran solution of tetrabutylammonium fluoride (1 M, 411.39 μL) was further added. The reaction system was stirred at room temperature for 0.5 hour. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. Compound 1-5 was obtained.

[0104] MS m / z (ESI): [M+H] + = 293.2. 1 1H NMR (400 MHz, DMSO-d6) δ = 7.88 (s, 1H), 6.91 - 6.94 (m, 2H), 3.95 (d, J = 5.3 Hz, 1H), 3.65 (br d, J = 3.5 Hz, 1H), 3.62 (br d, J = 3.3 Hz, 2H), 3.52 (br d, J = 4.5 Hz, 2H).

[0105] Step 5: Synthesis of Compound 1-6 Compound 1-5 (100 mg) and acetone (40 mL) were added to a reaction flask, and then sulfuric acid (50.34 mg) and compound acetonylidene (178.17 mg) were sequentially added. The reaction system was stirred at 45 °C for 0.5 h. Water (50 mL) was added to the reaction system, and the reaction solution was adjusted to neutral with a sodium bicarbonate solution (15%). It was extracted with EtOAc (50 mL × 3), and after combining the organic phases, it was further washed successively with saturated brine (50 mL) and water (50 mL). After separation, the organic phase was dried over anhydrous Na 2 SO 4 and finally the organic phase was directly concentrated under reduced pressure to obtain Compound 1-6.

[0106] MS m / z (ESI): [M + H] + = 333.2.

[0107] Step 6: Synthesis of Compound 1-7 Compound 1-6 (130 mg) and EtOAc (5 mL) were added to a reaction flask, and then isobutyric anhydride (123.77 mg), triethylamine (118.75 mg) and N,N-lutidine (9.56 mg) were sequentially added. The reaction system was stirred at room temperature for 1 h. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (ISCO (R) ; 12 g of SepaFlash (R) Silica Flash Column, mobile phase: 0 - 33% ethyl acetate / petroleum ether, flow rate: 30 mL / min) to obtain Compound 1-7.

[0108] MS m / z (ESI): [M + H] += 403.2。

[0109] Step 7: Synthesis of Compound 1 Compound 1-7 (100 mg) and H 2 O (1 mL) were added to the reaction flask, and further formic acid (11.94 mg) was added. The reaction system was stirred at room temperature for 1 hour. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (eluent: petroleum ether: ethyl acetate = 1:0 to 0:1) to obtain Compound 1.

[0110] MS m / z (ESI): [M+H] + = 534.6。 1 H NMR (400 MHz, CD 3 OD) δ = 7.88 (s, 1H), 6.91 - 6.94 (m, 2H), 4.63 - 4.44 (m, 1H), 4.30 - 4.38 (m, 2H), 4.15 - 4.16 (m, 1H), 2.55 - 2.57 (m, 1H), 1.88 - 1.91 (m, 6H).

[0111] Example 2

[0112]

Chemical formula

[0113] Synthesis scheme:

[0114]

Chemical formula

[0115] Step 1: Synthesis of Compound 2-2 Compound 1-6 (300 mg), salicylic acid (311.71 mg) and dichloromethane (3 mL) were added to a reaction flask, and 4-dimethylaminopyridine (275.71 mg) and N,N'-dicyclohexylcarbodiimide (465.63 mg) were sequentially added thereto. The reaction system was stirred at 35 °C for 12 hours. Water (30 mL) was added to the reaction system for quenching, and the mixture was extracted with EtOAc (10 mL × 2). After combining the organic phases, the combined organic phases were further washed with saturated brine (10 mL), and then dried over anhydrous Na 2 SO 4 and filtered. After filtration, the organic phase was concentrated under reduced pressure. The crude product was separated by flash column chromatography (ISCO (R) ; 4 g SepaFlash (R) Silica Flash Column, mobile phase: 0 - 80% ethyl acetate / petroleum ether, flow rate: 18 mL / min) to obtain Compound 2-2.

[0116] MS m / z (ESI): [M+H] + = 453.0.

[0117] Step 2: Synthesis of Compound 2 Compound 2-2 (275 mg) and water (3 mL) were added to a reaction flask, and formic acid (15 mL) was further added thereto. The reaction system was stirred at room temperature for 4 hours. The reaction solution was directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by prep-HPLC (column type: O-Welch C18 150×30 mm×5 μm; mobile phase: [H 2 O(FA)-ACN]; ACN%: 18% - 58%, 10 min) to obtain Compound 2.

[0118] MS m / z (ESI): [M+H] + = 413.3. 1 H NMR: (400 MHz, DMSO-d 6) δ = 10.43 (s, 1H), 7.98 - 7.81 (m, 2H), 7.64 (d, J=6.8 Hz, 1H), 7.53 (t, J=7.7 Hz, 1H), 6.98 (d, J=8.3 Hz, 1H), 6.91 (t, J=7.7 Hz, 1H), 6.87 (d, J=4.5 Hz, 1H), 6.80 (d, J=4.5 Hz, 1H), 6.34 (s, 1H), 5.46 (br s, 1H), 4.63 (dd, J=2.6, 12.2 Hz, 1H), 4.48 - 4.43 (m, 1H), 4.41 - 4.36 (m, 1H), 4.13 (br d, J=6.3 Hz, 1H).

[0119] Example 3

[0120]

Chem.

[0121] Synthesis scheme:

[0122]

Chem.

[0123] Step 1: Synthesis of Compound 3 Compound 1-5 (1 g) and ethyl acetate (5 mL) were added to a reaction flask, and isobutyric anhydride (1.62 g), 4-dimethylaminopyridine (83.60 mg), and triethylamine (1.04 g) were sequentially added. The reaction system was stirred at room temperature for 4 hours. The reaction solution was quenched with water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with saturated brine (20 mL × 2), further washed with anhydrous sodium sulfate, dried, and directly concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash column chromatography (ISCO (R) ; 20 g of SepaFlash (R)Silica Flash Column, mobile phase: 0 - 80% ethyl acetate / petroleum ether, flow rate: @35 mL / min), after separation, further purified by prep-HPLC (column: Welch Xtimate C18 150×30 mm×5 μm; mobile phase: [H 2 O(FA)-ACN]; ACN%: 38% - 78%, 8 min) to obtain Compound 3.

[0124] MS m / z (ESI): [M + H] + = 503.1. 1 H NMR: (400 MHz, DMSO-d 6 ) δ = 8.08 - 7.93 (m, 2H), 6.93 (d, J = 4.5 Hz, 1H), 6.74 (d, J = 4.5 Hz, 1H), 5.43 (d, J = 3.5 Hz, 1H), 4.82 - 4.82 (m, 1H), 4.62 (br d, J = 3.3 Hz, 1H), 4.68 - 4.55 (m, 1H), 2.69 - 2.55 (m, 2H), 2.48 - 2.41 (m, 1H), 1.15 (dd, J = 7.0, 10.3 Hz, 6H), 1.09 (d, J = 7.0 Hz, 6H), 1.02 (dd, J = 7.0, 13.3 Hz, 6H).

[0125] Biological tests: Test Example 1 In vitro antiviral activity test against coronavirus Research objective To detect the in vitro anti-human coronavirus (HCoV) 229E activity of the compound by the cytopathic effect (CPE) experiment and simultaneously detect the toxicity of the compound to cells.

[0126] 1. Experimental materials 1.1 Compounds The test compound was prepared as a 20 mM stock solution using DMSO solution. The starting test concentration of the test compound was 50 μM, which was tested at eight concentrations, diluted in a four-fold gradient, and a double-well experiment was set up. The control compound, Remdesivir, was provided by WuXi AppTec. Remdesivir was tested at eight concentrations, diluted in a three-fold gradient, and a double-well experiment was set up.

[0127] 1.2 Cells and Viruses MRC5 cells and coronavirus HCoV 229E were purchased from ATCC. MRC5 cells were cultured in EMEM (Sigma) medium supplemented with 10% fetal bovine serum (Excell), 1% double antibody (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acids (Gibco). EMEM (Sigma) medium supplemented with 5% fetal bovine serum (Excell), 1% double antibody (Hyclone), 1% L-glutamine (Gibco), and 1% non-essential amino acids (Gibco) was used as the experimental medium.

[0128] 2. Experimental Methods

[0129]

Table 1

[0130] Cells were seeded at a predetermined density in a 96-well microplate (Table 1) and cultured overnight in an incubator at 5% CO 2 , 37 °C. The next day, the diluted compound (eight concentration points, double-well) was added at 50 μL / well. Next, the diluted virus was added to the cells at 200 TCID 50 / well, 50 μL / well. Cell controls (cells without compound treatment or virus infection), virus controls (cells infected with virus without compound treatment), and medium controls (medium only) were set up. The final volume of the experimental medium was 200 μL, and the final concentration of DMSO in the medium was 0.5% respectively. The cells were incubated at 5% CO 2, They were cultured in an incubator at 35 °C for 3 days. Cell viability was detected using the CellTiter Glo cell viability detection kit (Promega). The cytotoxicity experiment was performed under the same conditions as the antiviral experiment, but without virus infection.

[0131] The antiviral activity and cytotoxicity of the compound are represented by the inhibition rate (%) of the cytopathic effect of the virus caused by the compound and the cell viability (%) at different concentrations, respectively. The calculation formulas are as follows: Inhibition rate (%) = (test well reading - virus control average value) / (cell control average value - virus control average value) × 100 Cell viability (%) = (test well reading - medium control average value) / (cell control average value - medium control average value) × 100 Using GraphPad Prism, a non-linear fitting analysis of the inhibition rate and cell viability of the compound was performed to calculate the values of the half-maximal effective concentration (EC 50 ) and the half-cytotoxic concentration (CC 50 ) of the compound.

[0132] The experimental results are as shown in Table 2.

[0133]

Table 2

[0134] Conclusion: The compounds of the present invention have strong anti-coronavirus activity in vitro.

[0135] Test Example 2: Pharmacokinetic evaluation of the compound Experimental purpose: To test the pharmacokinetics of the compound in CD-1 mice. Experimental materials: CD-1 mice (male, Beijing Vital River Laboratory Animal Technology Co., Ltd.)

[0136] Experimental operation: Using the standard protocol, the pharmacokinetic properties of the compound after intragastric administration in rodents were tested. In the experiment, the candidate compound was prepared into a transparent solution (the solvent for the intragastric administration preparation is an aqueous solution of 1% methylcellulose 4000). This project used 4 male CD-1 mice, the dosage was 100 mg / kg, the drug concentration was 10 mg / mL, and plasma samples were collected at 0 h (before administration) and 0.083, 0.25, 0.5, 1, 2, 4, 8, 24 h after administration. At 4°C, centrifuged at 3200 g for 10 minutes, the supernatant was separated to obtain plasma samples. The plasma was transferred to a pre-cooled centrifuge tube, rapidly frozen with dry ice, and then stored in an ultra-low temperature refrigerator at -70±10°C / -60°C or below. The plasma drug concentration was quantitatively analyzed using the LC-MS / MS analysis method, and the pharmacokinetic software of WinNonlin Version 6.3 or above (Pharsight) was used. The non-compartmental model was used to process the plasma drug concentration data of the metabolites of the compound of the present invention. The peak concentration (C max ), half-life (T 1 / 2 ), area under the drug-time curve (AUC 0-inf ) and other pharmacokinetic parameters were calculated, and the experimental results are as shown in Tables 3 and 4.

[0137]

Table 3

[0138]

Table 4

[0139] Conclusion: After the compound of the present invention was administered to mice, it was rapidly metabolized into the corresponding active metabolite in the body, exerted its medicinal effect, the drug concentration of the compound of the present invention lasted for a long time, the maintained concentration was also high, which was beneficial for continuous virus inhibition and exertion of medicinal effect.

Claims

1. A compound represented by formula (IV), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. 【Chemical 1】 (However, R ４ is selected from hydrogen and R １ -C(=O)-, and R １ is selected from C １－６ alkyl, C １－４ alkoxy and phenyl, and the C １－６ alkyl, C １－４ alkoxy and phenyl are each independently optionally substituted by one, two, three Rs, R ２ and R ３ each independently represents hydrogen, C １－６ alkyl-C(=O)-, C １－４ alkoxy-C(=O)- and phenyl-C(=O)-, and the C １－６ alkyl, C １－４ alkoxy and phenyl are each independently optionally substituted by one, two or three Rs, each R is independently selected from hydroxyl, halogen, amino, and cyano.)

2. A compound represented by formula (IV) according to Claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from the structures represented by formulas (VI-1) and (VI-2). 【Chemical 2】 (However, R ２ and R ３ each independently represents hydrogen, C １－６ alkyl-C(=O)-, C １－４ alkoxy-C(=O)-, and phenyl-C(=O)-, and the C １－６ alkyl, C １－４ alkoxy, and phenyl are each independently optionally substituted by one, two, or three Rs R ５ is selected from phenyl, said phenyl being optionally substituted by one, two or three R's, R ６ is selected from C １－６ alkyl and C １－４ alkoxy, and the C １－６ alkyl and C １－４ alkoxy are each independently optionally substituted by one, two, three Rs, each R is independently selected from hydroxyl, halogen, amino, and cyano.)

3. A compound according to any one of Claims 1 or 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein each R is independently selected from hydroxyl.

4. R １ is isopropyl, methoxy, tert-butoxy and [Chemical 3] A compound represented by formula (IV) according to Claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from

5. R ４ is hydrogen, isopropyl-C(=O)-, methoxy-C(=O)-, tert-butoxy-C(=O)-, and [Chemical Formula 4] A compound represented by formula (IV) according to Claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from

6. R ５ is [Chemical Formula 5] A compound according to Claim 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from

7. R ６ is the compound according to claim 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, selected from isopropyl, methoxy and tert-butoxy.

8. R ６ is the compound according to claim 7 selected from isopropyl, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

9. R ２ and R ３ are each independently selected from hydrogen, isopropyl-C(=O)-, and phenyl-C(=O)-, and the isopropyl and phenyl are each independently optionally substituted with one, two, or three Rs, the compound, stereoisomer, or pharmaceutically acceptable salt thereof according to any one of claims 1 or 2.

10. R ２ and R ３ are each independently hydrogen, isopropyl-C(=O)-, and 【Chemical Formula 6】 A compound according to Claim 9, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from

11. The following compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. 【Chemical Formula 7】

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