CN118019530A - Methods of treating viral infections - Google Patents

Methods of treating viral infections Download PDF

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Publication number
CN118019530A
CN118019530A CN202280061163.3A CN202280061163A CN118019530A CN 118019530 A CN118019530 A CN 118019530A CN 202280061163 A CN202280061163 A CN 202280061163A CN 118019530 A CN118019530 A CN 118019530A
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days
protein kinase
kinase inhibitor
rho
compound
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郑翔玲
赵焰平
王红军
周丽莹
仲伟婷
柴栋
何伊欣
候翠柳
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Beijing Tide Pharmaceutical Co Ltd
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Beijing Tide Pharmaceutical Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

Methods for preventing and/or treating viral infections are provided which comprise administering to an individual in need thereof an effective amount of a Rho associated protein kinase inhibitor or a combination of an effective amount of a Rho associated protein kinase inhibitor and one or more other drugs.

Description

Methods of treating viral infections Technical Field
The present invention is in the field of biological medicine and in particular relates to a method for preventing and/or treating viral infections comprising administering to a subject in need thereof an effective amount of a Rho related protein kinase inhibitor or a combination of an effective amount of a Rho related protein kinase inhibitor and one or more other drugs.
Background
[ Correction according to rules 26 23.09.2022] human coronaviruses were first discovered in the middle 60 s of the 20 th century, and they are common viruses that infect most people at some time in their lives, often resulting in mild to moderate upper respiratory and gastrointestinal disease. A new coronavirus called "middle east respiratory syndrome coronavirus" (MERS-CoV or MERS) was first reported in sauter arabia in 2012 and has spread to several other countries.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a disease of COVID-19. Currently, there is no effective therapeutic or vaccine against SARS-CoV-2 infection, and disease control relies mainly on the shut-off of the transmission pathway from strict physical isolation, which directly results in serious economic loss.
Summary of The Invention
In one aspect, the invention provides a method of preventing and/or treating a viral infection comprising administering to a subject in need thereof an effective amount of a Rho associated protein kinase inhibitor.
In another aspect, the invention provides the use of a Rho related protein kinase inhibitor in the manufacture of a medicament for the prevention and/or treatment of a viral infection.
In another aspect, the present invention provides Rho related protein kinase inhibitors for use in the prevention and/or treatment of viral infections.
In one aspect, the invention provides a method of preventing and/or treating a viral infection comprising administering to a subject in need thereof an effective amount of a Rho related protein kinase inhibitor in combination with one or more other drugs.
In another aspect, the invention provides the use of a Rho related protein kinase inhibitor in combination with one or more other drugs in the manufacture of a medicament for the prevention and/or treatment of a viral infection.
In another aspect, the invention provides a combination of a Rho related protein kinase inhibitor and one or more other agents for use in the prevention and/or treatment of viral infections.
In another aspect, the invention provides a pharmaceutical composition comprising a Rho related protein kinase inhibitor in combination with one or more other drugs and a pharmaceutically acceptable carrier.
In another aspect, the invention provides a kit comprising a plurality of containers, each container comprising a Rho-associated protein kinase inhibitor or one other drug, and optionally a pharmaceutically acceptable carrier, and optionally instructions.
Brief description of the drawings
FIG. 1A shows inhibition of SARS-CoV2 virus replication after 24 hours treatment with compound 007.
FIG. 1B shows inhibition of SARS-CoV2 virus replication after 48 hours treatment with compound 007.
FIG. 2 shows the protective effect of compound 007 on SARS-CoV-2 virus infected mice.
FIG. 3 shows the effect of compound 007 on inhibition of lung tissue viral load in a model of infection with SARS-COV2 (FIG. 3A shows the viral load in lung tissue infected with the original strain of SARS-CoV-2 (USA-WA 1/2020; FIG. 3B shows the viral load in lung tissue infected with the variant strain of SARS-CoV-2 delta (B.1.617.2)), and FIG. 3C shows the viral load in lung tissue infected with the variant strain of SARS-CoV-2 omicron (BA.1.1.529).
FIG. 4 shows H & E stained pictures of mouse lung tissue after SARS-CoV-2 infection (note: a-d is the picture at day 7 post-infection, E-H is the picture at day 14 post-infection. C, d, g, H is the 2.5-fold magnified picture of a, b, E, f, respectively).
FIG. 5 shows Masson stained pictures of lung tissue of mice after SARS-CoV-2 infection (note: a-d is the picture at day 7 post-infection, e-h is the picture at day 14 post-infection. C, d, g, h is a 2.5-fold magnification picture of a, b, e, f, respectively).
Detailed Description
Definition of the definition
Unless defined otherwise hereinafter, all technical and scientific terms used herein are intended to be identical to what is commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those that are obvious to those skilled in the art or alternatives to equivalent techniques. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
The terms "comprising," "including," "having," "containing," or "involving," and other variations thereof herein, are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps.
As used herein, the term "alkylene" means a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1,2, 3,4, 5 or 6 carbon atoms, such as methylene, ethylene, propylene or butylene.
As used herein, the term "alkyl" is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, for example 1 to 6 carbon atoms. For example, as used herein, the term "C 1-6 alkyl" refers to a linear or branched group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or n-hexyl) optionally substituted with 1 or more (such as 1 to 3) suitable substituents such as halogen (in which case the group is referred to as "haloalkyl") (e.g., CH 2F、CHF 2、CF 3、CCl 3、C 2F 5、C 2Cl 5、CH 2CF 3、CH 2Cl or-CH 2CH 2CF 3, etc.). The term "C 1-4 alkyl" refers to a linear or branched aliphatic hydrocarbon chain of 1 to 4 carbon atoms (i.e., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl).
As used herein, the term "alkenyl" means a linear or branched monovalent hydrocarbon radical containing one double bond and having 2 to 6 carbon atoms ("C 2-6 alkenyl"). The alkenyl group is, for example, vinyl, 1-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl and 4-methyl-3-pentenyl. When the compounds of the present invention contain alkenylene, the compounds may exist in pure E (ipsilateral (entgegen)) form, pure Z (ipsilateral (zusammen)) form or any mixture thereof.
As used herein, the term "alkynyl" means a monovalent hydrocarbon group containing one or more triple bonds, preferably having 2, 3, 4, 5 or 6 carbon atoms, such as ethynyl or propynyl.
As used herein, the term "cycloalkyl" refers to a saturated monocyclic or multicyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spiro, fused or bridged systems (such as bicyclo [1.1.1] pentyl, bicyclo [2.2.1] heptyl, bicyclo [3.2.1] octyl, or bicyclo [5.2.0] nonyl, decalinyl, etc.)) optionally substituted with 1 or more (such as 1 to 3) suitable substituents. The cycloalkyl group has 3 to 15 carbon atoms. For example, the term "C 3-6 cycloalkyl" refers to a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring of 3 to 6 ring-forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), optionally substituted with 1 or more (such as 1 to 3) suitable substituents, for example methyl substituted cyclopropyl.
As used herein, the terms "cycloalkylene", "cyclic hydrocarbon" and "hydrocarbon ring" refer to a saturated (i.e., "cycloalkylene" and "cycloalkyl") or unsaturated (i.e., having one or more double and/or triple bonds within the ring) mono-or polycyclic hydrocarbon ring having, for example, 3-10 (suitably 3-8, more suitably 3-6) ring carbon atoms, including, but not limited to, cyclopropyl (cyclo), (cyclobutyl (cyclo) ene, (cyclopentyl (cyclo) ene), (cyclohexyl (cyclo) ene), (cycloheptyl (cyclo), (cyclooctyl (cyclo), (cyclonon) (cyclohexenyl (cyclo) ene), and the like.
As used herein, the terms "heterocyclyl", "heterocyclylene" and "heterocycle" refer to a saturated (i.e., heterocycloalkyl) or partially unsaturated (i.e., having one or more double and/or triple bonds within the ring) cyclic group having, for example, 3-10 (suitably 3-8, more suitably 3-6) ring atoms, at least one of which is a heteroatom selected from N, O and S, and the remaining ring atoms being C. For example, a "3-10 membered (sub) heterocyclic (group)" is a saturated or partially unsaturated (sub) heterocyclic (group) having 2-9 (e.g., 2,3, 4,5, 6,7, 8, or 9) ring carbon atoms and one or more (e.g., 1,2, 3, or 4) heteroatoms independently selected from N, O and S. Examples of heterocyclylene and heterocyclic (groups) include, but are not limited to: ethylene oxide, (subunit) aziridinyl, (subunit) azetidinyl (azetidinyl), (subunit) oxetanyl (oxetanyl), (subunit) tetrahydrofuranyl, (subunit) dioxolyl (dioxolinyl), (subunit) pyrrolidinyl, (subunit) pyrrolidinonyl, (subunit) imidazolidinyl, (subunit) pyrazolidinyl, (subunit) pyrrolinyl, (subunit) tetrahydropyranyl, (subunit) piperidinyl, (subunit) morpholinyl, (subunit) dithianyl (dithianyl), (subunit) thiomorpholinyl, (subunit) piperazinyl, or (subunit) trithianyl (trithianyl). The groups also encompass bicyclic systems including spiro, fused or bridged systems (such as 8-azaspiro [4.5] decane, 3, 9-diazaspiro [5.5] undecane, 2-azabicyclo [2.2.2] octane, and the like). The heterocyclylene and heterocyclic (groups) may be optionally substituted with one or more (e.g., 1,2, 3 or 4) suitable substituents.
As used herein, the terms "(arylene) and" aromatic ring "refer to an all-carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated pi-electron system. For example, as used herein, the terms "C 6-10 (arylene) and" C 6-10 aromatic ring "mean an aromatic group containing 6 to 10 carbon atoms, such as a phenyl (phenylene) or a naphthyl (phenylene) group. The aryl (ene) and aromatic rings are optionally substituted with 1 or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO 2、C 1-6 alkyl, etc.).
As used herein, the terms "(arylene) heteroaryl" and "heteroaryl ring" refer to a monocyclic, bicyclic or tricyclic aromatic ring system having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms, particularly 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms, and which contains at least one heteroatom (which may be the same or different, such as oxygen, nitrogen or sulfur), and which may additionally be benzo-fused in each case. In particular, "(arylene) heteroaryl" or "heteroaryl ring" is selected from thienyl (ene) furyl (ene) pyrrolyl (ene) oxazolyl (ene) thiazolyl (ene) imidazolyl (ene) pyrazolyl (ene) isoxazolyl (ene) isothiazolyl (ene) oxadiazolyl (ene) triazolyl (ene) thiadiazolyl, and the like, and benzo derivatives thereof; or (sub) pyridyl, (sub) pyridazinyl, (sub) pyrimidinyl, (sub) pyrazinyl, (sub) triazinyl, etc., and their benzo derivatives.
As used herein, the term "aralkyl" preferably denotes aryl or heteroaryl substituted alkyl, wherein the aryl, heteroaryl and alkyl are as defined herein. Typically, the aryl group may have 6 to 14 carbon atoms, the heteroaryl group may have 5 to 14 ring atoms, and the alkyl group may have 1 to 6 carbon atoms. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, phenylpropyl, phenylbutyl.
As used herein, the term "halo" or "halogen" group is defined to include F, cl, br or I.
As used herein, the term "nitrogen-containing heterocycle" refers to a saturated or unsaturated monocyclic or bicyclic group having 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms and at least one nitrogen atom in the ring, which may optionally further comprise one or more (e.g., one, two, three, or four) ring members selected from N, O, C = O, S, S = O and S (=o) 2, which are attached to the remainder of the molecule through a nitrogen atom in the nitrogen-containing heterocycle and any remaining ring atoms, the nitrogen-containing heterocycle optionally being benzo-fused, and preferably through a nitrogen atom in the nitrogen-containing heterocycle and any carbon atom in the fused benzene ring.
The term "substitution" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom are replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution forms a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
If a substituent is described as "optionally substituted," the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as optionally substituted with one or more of the list of substituents, one or more hydrogens on the carbon (to the extent any hydrogens are present) may be replaced with an independently selected optional substituent, alone and/or together. If the nitrogen of a substituent is described as optionally substituted with one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent any hydrogens are present) may each be replaced with an independently selected optional substituent.
If substituents are described as "independently selected from" a group, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from another (other) substituent.
The term "one or more" as used herein means 1 or more than 1, such as 2,3,4, 5 or 10, under reasonable conditions.
As used herein, unless indicated, the point of attachment of a substituent may be from any suitable position of the substituent.
When the bond of a substituent is shown as a bond through the ring connecting two atoms, then such substituent may be bonded to any ring-forming atom in the substitutable ring.
The invention also includes all pharmaceutically acceptable isotopically-labelled compounds which are identical to those of the present invention except that one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevailing in nature. Examples of isotopes suitable for inclusion in compounds of the invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium (2 H), tritium (3 H)); isotopes of carbon (e.g., 11C、 13 C and 14 C); isotopes of chlorine (e.g., 36 Cl); isotopes of fluorine (e.g., 18 F); isotopes of iodine (e.g., 123 I and 125 I); isotopes of nitrogen (e.g., 13 N and 15 N); isotopes of oxygen (e.g., 15O、 17 O and 18 O); isotopes of phosphorus (e.g., 32 P); and isotopes of sulfur (e.g., 35 S). Certain isotopically-labeled compounds of the present invention (e.g., those into which a radioisotope is incorporated) are useful in pharmaceutical and/or substrate tissue distribution studies (e.g., assays). The radioisotope tritium (i.e., 3 H) and carbon-14 (i.e., 14 C) are particularly useful for this purpose because of their ease of incorporation and ease of detection. Substitution with positron emitting isotopes (such as 11C、 18F、 15 O and 13 N) can be used in Positron Emission Tomography (PET) studies to examine substrate receptor occupancy. Isotopically-labeled compounds of the present invention can be prepared by processes analogous to those described in the accompanying schemes and/or in the examples and preparations by substituting an appropriate isotopically-labeled reagent for the non-labeled reagent previously employed. Pharmaceutically acceptable solvates of the invention include those in which the crystallization solvent may be isotopically substituted, for example, D 2 O, acetone-D 6 or DMSO-D 6.
The term "stereoisomer" refers to an isomer formed as a result of at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, they can produce racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. Specific individual molecules may also exist as geometric isomers (cis/trans). Similarly, the compounds of the application may exist as a mixture of two or more structurally distinct forms (commonly referred to as tautomers) in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. It is to be understood that the scope of the present application encompasses all such isomers in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) or mixtures thereof.
Solid lines (-), solid wedges may be used hereinOr virtual wedge shapeDepicting the chemical bond of the compounds of the present invention. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers at that carbon atom (e.g., particular enantiomers, racemic mixtures, etc.) are included. The use of a solid or virtual wedge to depict a bond to an asymmetric carbon atom is intended to indicate the presence of the stereoisomers shown. When present in a racemic mixture, real and imaginary wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist as stereoisomers (which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).
The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of any ratio of more than one polymorphs.
It will also be appreciated that certain compounds of the invention may exist in free form for use in therapy or, where appropriate, in the form of pharmaceutically acceptable derivatives thereof. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites or prodrugs which, upon administration to a patient in need thereof, are capable of providing the compounds of the invention or metabolites or residues thereof, either directly or indirectly. Thus, when reference is made herein to "a compound of the invention" it is also intended to encompass the various derivative forms of the compounds described above.
Pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.
Suitable acid addition salts are formed from acids that form pharmaceutically acceptable salts. Examples include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclamate, ethanedisulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hyparate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate (naphthylate), 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and xinafoate (xinofoate).
Suitable base addition salts are formed from bases that form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts.
For a review of suitable salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts:properties, selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.
As used herein, the term "ester" means an ester derived from each of the compounds of the general formula in the present application, including physiologically hydrolyzable esters (compounds of the present application that can be hydrolyzed under physiological conditions to release the free acid or alcohol form). The compounds of the application may themselves be esters.
The compounds of the invention may be present in the form of solvates (preferably hydrates) wherein the compounds of the invention comprise a polar solvent as a structural element of the compound lattice, in particular for example water, methanol or ethanol. The polar solvent, in particular water, may be present in stoichiometric or non-stoichiometric amounts.
Those skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides, as nitrogen requires available lone pairs to oxidize to oxides; those skilled in the art will recognize nitrogen-containing heterocycles capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidizing heterocycles and tertiary amines with peroxyacids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as t-butyl hydroperoxide, sodium perborate, and dioxiranes (dioxirane) such as dimethyl dioxirane. These methods for preparing N-oxides have been widely described and reviewed in the literature, see for example: T.L. Gilchrist, comprehensive Organic Synthesis, vol.7, pp 748-750; katritzky and a.j. Boulton, eds., ACADEMIC PRESS; and g.w.h.cheeseman and e.s.g.werstiuk, ADVANCES IN Heterocyclic Chemistry, vol.22, pp 390-392, a.r.katritzky and a.j.boulton, eds., ACADEMIC PRESS.
Also included within the scope of the invention are metabolites of the compounds of the invention, i.e., substances that form in vivo upon administration of the compounds of the invention. Such products may result from, for example, oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc. of the compound being administered. Accordingly, the present invention includes metabolites of the compounds of the present invention, including compounds made by a process of contacting a compound of the present invention with a mammal for a time sufficient to produce the metabolites thereof.
The invention further includes within its scope prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention which may themselves have little or no pharmacological activity, which, when administered into or onto the body, may be converted into the compounds of the invention having the desired activity by, for example, hydrolytic cleavage. Typically such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Additional information regarding the use of prodrugs can be found in "Pro-drugs as Novel DELIVERY SYSTEMS", vol.14, ACS Symposium Series (T.Higuchi and V.stilla). Prodrugs of the invention may be prepared, for example, by replacing the appropriate functional groups present in the compounds of the invention with certain moieties known to those skilled in the art as "pro-moieties" (e.g., "Design of Prodrugs", described in H. Bundgaard (Elsevier, 1985) ".
The invention also encompasses compounds of the invention containing a protecting group. During any process of preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules of interest, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example those described in T.W.Greene & P.G.M.Wuts, protective Groups in Organic Synthesis, john Wiley & Sons,1991, which references are incorporated herein by reference. The protecting group may be removed at a suitable subsequent stage using methods known in the art.
The term "about" means within + -10%, preferably within + -5%, more preferably within + -2% of the stated value.
By "pharmaceutically acceptable carrier" is meant a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting the tissues of humans and/or other animals within the scope of sound medical judgment without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions or kits of the invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, in particular for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The pharmaceutical composition may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, as desired. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).
The components of the pharmaceutical compositions and kits of the present invention may act systematically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of an ophthalmic formulation or by inhalation.
For these routes of administration, the components of the pharmaceutical compositions and kits of the invention may be administered in suitable dosage forms.
Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.
The term "container" as used herein is a container for containing a pharmaceutical composition. Such a container may be used for preparation, storage, transportation and/or independent/batch sales, which is intended to encompass bottles, cans, vials, flasks, syringes, tubes (e.g. for cream products), or any other container for preparing, containing, storing or dispensing a pharmaceutical product.
The term "instructions" as used herein refers to inserts, labels, logos, etc. that enumerate information about the pharmaceutical composition located within the container. The information listed is typically determined by a regulatory agency (e.g., the U.S. food and drug administration) that governs the area in which the article is to be sold. Preferably, the package insert specifically lists the indication for which the pharmaceutical composition is approved. The package insert may be made of any material from which information contained therein or thereon may be read. Preferably the package insert is a printable material (e.g. paper, plastic, cardboard, foil, adhesive paper or plastic, etc.) on which the desired information can be formed (e.g. printed or applied).
The term "effective amount" refers to an amount sufficient to achieve the desired therapeutic effect under the conditions of administration that results in a pathological condition, disease progression, improvement in physiological conditions associated therewith, or induction of resistance to the aforementioned diseases.
As used herein, unless otherwise indicated, the term "treating" means reversing, alleviating, inhibiting the progression of, or preventing such disorder or condition, or one or more symptoms of such disorder or condition to which such term applies.
As used herein, "individual" includes human or non-human animals. Exemplary human individuals include human individuals (referred to as patients) or normal individuals suffering from a disease (e.g., a disease described herein). "non-human animals" in the context of the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).
The combination therapy of Rho related protein kinase inhibitors of this invention with one or more other agents may provide "synergy" and "synergy", i.e., an effect that is greater than the sum of the effects produced by the compounds alone when used together. Synergistic antiviral effects mean antiviral effects which are greater than the predicted net additive effects of the individual active ingredients in the combination.
Therapeutic methods and uses
In some embodiments, the invention provides methods of preventing and/or treating a viral infection comprising administering to a subject in need thereof an effective amount of a Rho associated protein kinase inhibitor.
In other embodiments, the invention provides the use of a Rho related protein kinase inhibitor in the manufacture of a medicament for the prevention and/or treatment of a viral infection.
In other embodiments, the invention provides Rho related protein kinase inhibitors for use in the prevention and/or treatment of viral infections.
In some embodiments, the invention provides methods of preventing and/or treating viral infections comprising administering to an individual in need thereof an effective amount of a Rho related protein kinase inhibitor in combination with one or more other drugs.
In other embodiments, the invention provides the use of a Rho related protein kinase inhibitor in combination with one or more other agents for the manufacture of a medicament for the prevention and/or treatment of a viral infection.
In other embodiments, the invention provides a combination of a Rho related protein kinase inhibitor and one or more other agents for use in preventing and/or treating a viral infection.
In some embodiments, the viral infection is a coronaviridae infection, and the coronaviridae infection is caused by a coronaviridae virus.
In a preferred embodiment, the Rho related protein kinase inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:
Wherein:
Ring A is The above groups are linked to the pyrimidine ring through one of the two positions marked or marked and the other position is linked to the carbonyl group;
R is selected from H and C 1-6 alkyl;
R 1 is
R 2 is selected from H and C 1-6 alkyl;
R 3、R 4、R 7 and R 8 are each independently at each occurrence selected from H, halogen, -NR 5R 6、-OH、C 1-6 alkyl, and-OR 5;
R 9 and R 10 are each independently at each occurrence selected from H, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, C 6-12 aralkyl, -C (=O) R 5, and-C 1-6 alkylene-O (P=O) (OH) 2;
The above alkylene, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and aralkyl groups are each optionally substituted at each occurrence with one OR more substituents independently selected from halogen, C 1-6 alkyl, and-OR 5;
R 5 and R 6 are each independently at each occurrence selected from H, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, and C 6-12 aralkyl;
m is independently at each occurrence an integer of 0, 1, 2 or 3;
n is an integer of 0, 1 or 2 independently at each occurrence.
In a preferred embodiment, ring A isThe above groups are linked to the pyrimidine ring via a position of the label and to the carbonyl group via a position of the label, wherein R 10 is selected from H and C 1-6 alkyl, preferably H or methyl.
In a preferred embodiment, ring A is preferablyThe above groups are linked to the pyrimidine ring via the position of the label and to the carbonyl via the position of the label.
In a preferred embodiment, R is H.
In a preferred embodiment, R 2 is H.
In a preferred embodiment, R 5 and R 6 are each independently selected from H, methyl and ethyl at each occurrence.
In a preferred embodiment, R 3、R 4、R 7 and R 8 are each independently selected at each occurrence from H, F, cl, br, I, -NH 2, -OH, methyl, trifluoromethyl, -CH 2 -Ph, methoxy, ethoxy, and-CH 2OCH 3.
In a preferred embodiment, R 3 is H.
In a preferred embodiment, R 4 is selected from H and halogen (e.g., F, cl, br or I), preferably H or F.
In a preferred embodiment, R 7 is selected from H and halogen (e.g., F, cl, br or I), preferably H or F.
In a preferred embodiment, R 8 is H.
In a preferred embodiment, R 9 and R 10 are each independently at each occurrence selected from H, F, cl, br, methyl, ethyl, n-propyl, isopropyl, vinyl, cyclopropyl, cyclobutyl, cyclopentyl, oxetanyl, monofluoromethyl, difluoromethyl, trifluoromethyl, acetyl 、-CH 2CHF 2、-CH 2OH、-CH 2OCH 3、-CH 2CH 2OCH 3、-CH 2-O(P＝O)(OH) 2、
In a preferred embodiment, R 9 is independently at each occurrence selected from H, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, and C 6-12 aralkyl, preferably H.
In a preferred embodiment, R 10 is independently selected at each occurrence from H and C 1-6 alkyl, preferably H, methyl, ethyl, n-propyl or isopropyl, most preferably H or methyl.
In a preferred embodiment, the present invention provides a method for preventing and/or treating viral infection, wherein the Rho related protein kinase inhibitor is a compound having the structure of formula (II):
wherein each group is as defined above.
In a preferred embodiment, the present invention provides a method for preventing and/or treating viral infection, wherein the Rho related protein kinase inhibitor is a compound having the structure of formula (III):
Wherein R 10 is H or methyl, preferably methyl.
In a preferred embodiment, the Rho related protein kinase inhibitor is a compound having the structure or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:
In some embodiments, the Rho related protein kinase inhibitor is prepared according to the methods disclosed in WO 2019/001572 A1 (which is incorporated herein by reference).
In some embodiments, the invention provides methods of preventing and/or treating viral infections comprising administering to a subject in need thereof an effective amount of a Rho associated protein kinase inhibitor in combination with one or more other drugs;
Wherein the other drug is preferably one or more selected from the group consisting of a small molecule drug, a cytokine drug, a therapeutic antibody, an inflammatory factor storm therapeutic drug, a small interfering RNA and a vaccine for preventing or treating a viral infection.
In some embodiments, the small molecule drug that prevents or treats a viral infection is one or more selected from the group consisting of RNA polymerase inhibitors, endoprotease inhibitors, HIV protease inhibitors, coronavirus primary protease inhibitors, coronavirus 3C-like protease (3C-like proteinase) inhibitors, SARS-Cov-2 3-chymotrypsin (3 CLpro) inhibitors, spike protease (S protease) inhibitors, transmembrane serine protease 2 (TMPRSS 2) inhibitors, angiotensin converting enzyme 2 (ACE 2) blocking inhibitors, viral membrane fusion and endocytosis inhibitors, and capsular blockers.
In some embodiments, the small molecule drug that prevents or treats a viral infection is selected from ribavirin, fpirane Weiba lo Sha Wei, BCX4430 (which is of the structure) One or more of pimodivir (VX-787), nitazoxanide (also known as "nitazoxanide"), darunavir, lopinavir, ritonavir, nelfinavir, arbidol, chloroquine, hydroxychloroquine, radciclovir, vitamin B complex, vitamin C, vitamin D, and zinc supplements.
In some embodiments, the cytokine drug is one or more selected from the group consisting of interferons (e.g., natural/recombinant interferons, spatially altered interferons, interferon conjugates, or interferon fusion proteins), interleukins, colony stimulating factors, tumor necrosis factors, and growth factors.
In some embodiments, the therapeutic antibody is one or more selected from the group consisting of SARS-CoV mab, SARS-CoV-2 mab, and MERS-CoV mab.
In some embodiments, the inflammatory factor storm therapeutic agent is one or more selected from Vesatolimod, ketotifen, glatiramer acetate, polycyclocetin, roflumilast, IL-6 mab, IL-6R mab (e.g., tolizumab), TLR3 mab, mammalian defensins, nickel-striated protein/IL-41 fragment, glucocorticoids (e.g., dexamethasone, prednisone, methylprednisone, and hydrocortisone), and broad spectrum antibiotics (e.g., azithromycin).
In some embodiments, the vaccine is a vaccine against a coronaviridae virus, in particular an inactivated vaccine, a nucleic acid vaccine, a recombinant protein/subunit vaccine, a live viral vector vaccine, an attenuated vaccine or a vaccine developed based on a DNA or RNA strategy or a replicative or non-replicative vector strategy.
In some embodiments, the coronaviridae is selected from the group consisting of SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 and variants thereof.
In some embodiments, variants of SARS-CoV-2 include, but are not limited to, alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and Omicron (B.1.1.529).
In some embodiments, the disease caused by the coronavirus infection is middle east respiratory syndrome, severe acute respiratory syndrome, or COVID-19.
In some embodiments, the coronaviridae virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or 2019-nCoV) that causes a disease of COVID-19.
In some embodiments, each active ingredient of the Rho related protein kinase inhibitor or the combination is administered in an amount of about 0.005 mg/day to about 5000 mg/day, for example, about 0.005、0.05、0.5、5、10、20、30、40、50、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1500、2000、2500、3000、3500、4000、4500 or 5000 mg/day.
In some embodiments, each active ingredient of the Rho related protein kinase inhibitor or the combination is administered in an amount of about 1ng/kg to about 200mg/kg, about 1 μg/kg to about 100mg/kg, or about 1mg/kg to about 50mg/kg body weight per day, for example, about 1 μg/kg, about 10 μg/kg, about 25 μg/kg, about 50 μg/kg, about 75 μg/kg, about 100 μg/kg, about 125 μg/kg, about 150 μg/kg, about 175 μg/kg, about 200 μg/kg, about 225 μg/kg, about 250 μg/kg, about 275 μg/kg, about 300 μg/kg, about 325 μg/kg, about 350 μg/kg, about 375 μg/kg, about 400 μg/kg, about 425 μg/kg, about 450 μg/kg, about 475 μg/kg, about 500 μg/kg, about 525 μg/kg about 550. Mu.g/kg, about 575. Mu.g/kg, about 600. Mu.g/kg, about 625. Mu.g/kg, about 650. Mu.g/kg, about 675. Mu.g/kg, about 700. Mu.g/kg, about 725. Mu.g/kg, about 750. Mu.g/kg, about 775. Mu.g/kg, about 800. Mu.g/kg, about 825. Mu.g/kg, about 850. Mu.g/kg, about 875. Mu.g/kg, about 900. Mu.g/kg, about 925. Mu.g/kg, about 950. Mu.g/kg, about 975. Mu.g/kg, about 1mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 60mg/kg, about 70mg/kg, about 80mg/kg, about 90mg/kg, about 100mg/kg, about 125mg/kg, about 150mg/kg, about 175mg/kg, about 200mg/kg, or about 300mg/kg of body weight.
In some embodiments, the daily dose of each active ingredient in the Rho related protein kinase inhibitor or the combination is administered once or in two, three or four administrations.
In some embodiments, the active ingredients in the combination are administered simultaneously, sequentially or alternately.
In some embodiments, each active ingredient in the Rho related protein kinase inhibitor or the combination is administered continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, or at least 2 years.
In some embodiments, each active ingredient in the Rho-associated protein kinase inhibitor or the combination is administered for one or more (e.g., 1,2,3,4, 5, 6, 7, 8, 9, or 10) courses of treatment, wherein each course of treatment lasts for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days; and every two courses of treatment are spaced 0,1,2, 3,4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks or four weeks apart.
In some embodiments, each of the active ingredients in the Rho-associated protein kinase inhibitor or the combination is administered by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of an ophthalmic formulation or by inhalation, and the route of administration of the active ingredients in the combination may be the same or different.
The invention encompasses any combination of the above embodiments.
Pharmaceutical composition and kit
In another embodiment, the invention provides a pharmaceutical composition comprising a combination of a Rho-associated protein kinase inhibitor and one or more other drugs, and a pharmaceutically acceptable carrier; wherein the Rho related protein kinase inhibitor and the other drugs are each as defined above.
In another embodiment, the invention provides a kit comprising a plurality (e.g., 2, 3, or 4) of containers, each container comprising a Rho-associated protein kinase inhibitor or one other drug, and optionally a pharmaceutically acceptable carrier; wherein the Rho related protein kinase inhibitor and the other drugs are each as defined above.
Examples
In order to make the objects and technical solutions of the present invention more clear, the present invention is further described below with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Moreover, the specific experimental methods not mentioned in the following examples were carried out according to conventional experimental methods.
The compound 128 used in the examples has the following structure, which was prepared according to the method disclosed in WO 2019/001572 A1.
EXAMPLE 1 detection of ROCK2 kinase Activity
Kinase IC 50 assays were performed using the commercial CISBIO kinase assay kit HTRF KINEASE-STK S2 kit (62 ST2 PEC). The kinase ROCK2 (01-119) used for the reaction was purchased from Carna Biosciences.
Before the experiment starts, the following working solutions are prepared according to the requirements and the specifications of the kinase detection kit by using corresponding reagents: 1 Xkinase buffer, 5 XSTK-S2 substrate working solution (1.5. Mu.M) and 5 XSTP working solution (1.5. Mu.M), 5 XSOCK 2 kinase working solution, 4 XSTK-Ab-cave compound 2 detection solution were then subjected to the following procedure.
Preparing 10000nM compound solution by 1 Xkinase buffer with DMSO concentration of 2.5%, and gradually diluting the compound by using the kinase buffer containing DMSO to obtain 9 compound solutions with different concentrations to be tested. In addition to the test compound wells, positive wells (containing all reagents except the compound) and negative wells (containing all reagents except the test compound and kinase) were set. To all wells except the control wells (positive and negative wells), 4. Mu.L of test compound solution was added and to the control wells, 4. Mu.L of 2.5% DMSO solution was added. Then, 2. Mu.M substrate (i.e., 2. Mu.L of 5 XSTK-S2 substrate working solution) was added to all the reaction wells. To all wells except the negative wells, 2. Mu.L of 5 XROCK 2 kinase working solution (containing 1.4ng ROCK2 kinase) was added, and the negative wells were made up to volume with 2. Mu.L of 1 Xkinase buffer. To all wells, 2. Mu.L of 5 XATP working solution was added and incubated for 2 hours at room temperature. After the kinase reaction was completed, 5. Mu.L of 4 Xstreptavidin-XL 665 working solution was added to all the reaction wells, and after mixing, 5. Mu.L of 4 XSTK-Ab-cave compound 2 detection solution was added immediately, and after incubation at room temperature for 1 hour, fluorescence signals were detected by ENVISION (Perkinelmer) instrument (excitation wavelength was 320nm, emission wavelengths were 665nm and 615 nm). The inhibition rate in each well was calculated from the fluorescence intensity values of the wells: ER (Emission Ratio) = (fluorescence intensity at 665 nm/fluorescence intensity at 615 nm); inhibition = (ER positive-ER test compound)/(ER positive-ER negative) 100%, a graph was drawn with software PRISM 5.0, and a median inhibitory concentration (IC 50) of each test compound was obtained by fitting with PRISM 5.0. Compound IC 50 values are shown in the table below.
TABLE 1
Compounds of formula (I) ROCK2 IC 50nM Compounds of formula (I) ROCK2 IC 50nM
Compound 006 34 Compound 011 9
Compound 007 33 Compound 020 44
Compound 008 24 Compound 021 45
Compound 009 12 Compound 022 75
Compound 010 61 Compound 128 27
Example 2: compound 007 in vitro anti-SARS-CoV-2 action
Human airway epithelial cells Calu-3 (ATCC, HTB-55) were purchased from ATCC; SARS-CoV-2 virus (2019-nCoV/USA-WA 1/2020 strain) WAs isolated from Washington 1, USA, no. 2020, month 1, COVID-19, confirmed diagnosis of patient pharyngeal swabs (BEI Resources, NR-52281). Calu-3 was cultured overnight and used for experiments until the degree of fusion reached 80-90%. Appropriate amount of cells were inoculated into 96-well plates, 10. Mu.L of supernatant containing SARS-CoV-2 virus was added for infection, 1,5, 10 and 25. Mu.M compound 007 were added simultaneously, the supernatants were taken after 24 hours and 48 hours of culture, the viral RNA level was detected by QPCR method, and the compound 007 in vitro antiviral effect was evaluated, with 0.1% DMSO as a negative control. The results showed that 1,5, 10 and 25 μm compound 007 significantly inhibited SARS-CoV-2 virus proliferation (p <0.0001 compared to the negative control) with a 1-25 μm compound 007 inhibition of 90% or more (see fig. 1A and 1B).
Example 3: protection of SARS-CoV-2 virus infected mice by Compound 007
2019-NCoV/USA-WA1/2020 strain WAs isolated from Washington 1, 2020, month COVID-19, USA, with confirmed diagnosis of patient pharyngeal swabs (BEI Resources, NR-52281). Humanized ACE2 transgenic mice were obtained from jackson laboratories in the united states.
The 12 animals were randomly divided into 2 groups (6/group): SARS-CoV-2 virus infection group (i.e., a "model group") and compound 007 treatment group (300 mpk). All animals were vaccinated with 10 4 PFU SARS-CoV-2 virus and the animals of the treatment group were given a 300mpk dose of compound 007 orally and gastrally once daily for 14 days; model groups were given equal volumes of 0.5% cmc-Na in parallel. Animal survival was recorded daily.
The results show that: animals in the model group all died on day 10 post-viral infection, and only 1 animal in the compound 007 treated group died on day 11 with an animal survival rate of 83.3% on day 14 (see figure 2). The results show that the compound 007 has remarkable antiviral effect in vivo and can remarkably reduce the death rate of animals.
Example 4: antiviral Effect of Compound 007 on SARS-CoV2 in Vero E6 cells
Vero E6 cells were first seeded in 96-well plates, compound 007 (0.04-20 μm) or adefovir (0.04-20 μm) at various doses was added, cell viability was detected by CCK-8 after 72h incubation, the inhibition of Vero E6 cells by the compound (CC 50) was calculated, and the effect of the compound on cell growth was assessed. The inhibition rate calculation formula is: inhibition (%) =100× (a Control wells -A Detection hole )/A Control wells , CC 50 values for inhibition of cell growth by compounds were calculated using GRAPH PRISM 8.0 software.
Vero E6 cells were inoculated into 48 well plates, 100 μl of either the original strain (accession number NIMDCN0000 HUI) or Delta variant (accession number NMDC 60042793) virus dilution (moi=0.01) was added to each well, incubated with 100 μl of test compound 007 (0.04-20 μm) or radciclovir (0.02-10 μm) at different concentrations, 37 ℃,5% co 2 for 1h, virus-sample mixed medium was pipetted, and after washing twice with 1×pbs, the culture was changed to medium containing only compound 007 or radciclovir and continued. After culturing at 37℃for 48h with 5% CO 2, the cell supernatant was collected and viral RNA was extracted for Real-time PCR virus quantification, and the inhibition ratio (EC 50) of the test sample on viral replication was calculated. The viral replication inhibition was calculated as: viral replication inhibition (%) = (control group virus amount-sample group virus amount)/control group virus amount x 100%, fitting curves were drawn using GRAPH PRISM 8.0.0 software, and EC 50 values of compound antiviral were calculated.
The results show that compound 007 and adefovir both have a CC 50 >20 μm for Vero E6 cells as shown in table 2; compound 007 had EC 50 values of <40nM (SI > 500) for both the original strain and Delta variant and adefovir had EC 50 values of 150nM (SI > 133) and 60nM (SI > 333) for both the original strain and Delta variant, respectively.
TABLE 2 antiviral Effect of Compounds in Vero E6 cells on SARS-CoV-2
Example 5: in vivo antiviral action of Compound 007 on SARS-CoV-2
Humanized K18-hACE2 transgenic mice (purchased from Jackson laboratories, USA). The 1000PFU SARS-COV-2 original strain (USA-WA 1/2020), delta variant (B.1.617.2) or Omacron variant (BA.1.1.529) WAs nasally inoculated, and on the day of virus inoculation, vehicle (0.5% CMC-Na aqueous solution, model group), 100mg/kg, 300mg/kg of compound 007, or 25mg/kg of RedeSivir WAs subcutaneously administered 1 time a day for 21 consecutive days, respectively. Mice lung tissue was assayed at days 7, 14 and 21, respectively, homogenized and assayed for lung tissue viral load in each group using qPCR. Meanwhile, in the 1000PFU SARS-CoV-2 original strain infection model, animal lung tissues of the model group and the compound 007 treatment group are fixed, paraffin embedded and sectioned, and lung tissue sections are respectively subjected to H & E staining and Masson staining, so that the anti-inflammatory and anti-fibrosis effects of the compound 007 are evaluated.
The results show that 100mg/kg and 300mg/kg of compound 007, rituximab treatment for 7 days, 14 days or 21 days significantly reduced the viral load of mice compared to vehicle group in the SARS-COV-2 original strain (USA-WA 1/2020), delta variant (B.1.617.2) or Omicron variant (BA.1.1.529) infection model, as detailed in FIGS. 3A, 3B and 3C.
The results of H & E staining of tissue sections are shown in FIG. 4. The results showed that after 7 days (FIGS. 4 a-c) and 14 days (FIGS. 4 e-g) of infection with 1000PFU SARS-CoV-2 original strain, the pathological changes in lung tissue in lung interstitial inflammatory cell infiltration, alveolar wall thickening, alveolar collapse, necrotic cell debris, etc. were significantly reduced in the group of mice treated with compound 007.
The results of Masson staining of tissue sections are shown in FIG. 5. After the 1000PFU SARS-CoV-2 original strain is infected for 7 days and 14 days, obvious collagen accumulation can be seen in the lung tissue of mice in the model group; whereas in the compound 007 treated group, the collagen accumulation in the lung tissue of mice caused by SARS-CoV-2 infection was significantly reduced.
Various modifications of the application, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this disclosure (including all patents, patent applications, journal articles, books, and any other publications) is hereby incorporated by reference in its entirety.

Claims (21)

  1. A method for preventing and/or treating a viral infection comprising administering to a subject in need thereof an effective amount of a Rho associated protein kinase inhibitor,
    Wherein the Rho related protein kinase inhibitor is preferably a compound of formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:
    Wherein:
    Ring A is The above groups are linked to the pyrimidine ring through one of the two positions marked or marked and the other position is linked to the carbonyl group; preferably, ring A isThe above groups are linked to the pyrimidine ring through a position marked and to the carbonyl through a position marked, wherein R 10 is selected from H and C 1-6 alkyl, preferably H or methyl;
    R is selected from H and C 1-6 alkyl;
    R 1 is
    R 2 is selected from H and C 1-6 alkyl;
    R 3、R 4、R 7 and R 8 are each independently at each occurrence selected from H, halogen (e.g., F, cl, br OR I), -NR 5R 6、-OH、C 1-6 alkyl, and-OR 5;
    R 9 and R 10 are each independently at each occurrence selected from H, halogen, C 1-6 alkyl (e.g., methyl), C 2-6 alkenyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, C 6-12 aralkyl, -C (=o) R 5, and-C 1-6 alkylene-O (p=o) (OH) 2;
    The above alkylene, alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and aralkyl groups are each optionally substituted at each occurrence with one OR more substituents independently selected from halogen, C 1-6 alkyl, and-OR 5;
    R 5 and R 6 are each independently at each occurrence selected from H, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-14 membered heteroaryl, and C 6-12 aralkyl;
    m is independently at each occurrence an integer of 0, 1, 2 or 3;
    n is an integer of 0, 1 or 2 independently at each occurrence; and
    The viral infection is preferably a coronaviridae infection, and the coronaviridae infection is caused by a coronaviridae virus.
  2. The method of claim 1, wherein the Rho related protein kinase inhibitor is a compound having the structure of formula (II) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:
    wherein each group is as defined in claim 1;
    Preferably, the Rho related protein kinase inhibitor is a compound having the structure of formula (III) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:
    Wherein R 10 is H or methyl, preferably methyl.
  3. The method of claim 1 or 2, wherein the Rho related protein kinase inhibitor is a compound having the structure or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof:
  4. the method of any one of claims 1-3, comprising administering to a subject in need thereof an effective amount of a Rho associated protein kinase inhibitor in combination with one or more other drugs;
    Wherein the other drug is preferably one or more selected from the group consisting of a small molecule drug, a cytokine drug, a therapeutic antibody, an inflammatory factor storm therapeutic drug, a small interfering RNA and a vaccine for preventing or treating a viral infection.
  5. The method of claim 4, wherein the small molecule drug that prevents or treats a viral infection is one or more selected from the group consisting of RNA polymerase inhibitors, endo-enzyme inhibitors, HIV protease inhibitors, coronavirus primary protease inhibitors, coronavirus 3C-like protease inhibitors, SARS-Cov-2 3-chymotrypsin (3 CLpro) inhibitors, spike protease (S protease) inhibitors, transmembrane serine protease 2 (TMPRSS 2) inhibitors, angiotensin converting enzyme 2 (ACE 2) blocking inhibitors, viral membrane fusion and endocytosis inhibitors, and capsular blockers.
  6. The method of claim 4 or 5, wherein the small molecule drug that prevents or treats a viral infection is one or more selected from ribavirin, fampica Weiba lo Sha Wei, BCX4430, pimodivir (VX-787), nitazoxanide, darunavir, lopinavir, ritonavir, nelfinavir, arbidol, chloroquine, hydroxychloroquine, radciclovir, vitamin B complex, vitamin C, vitamin D, and zinc supplements.
  7. The method of claim 4, wherein the cytokine drug is one or more selected from the group consisting of interferons (e.g., natural/recombinant interferons, spatially altered interferons, interferon conjugates, or interferon fusion proteins), interleukins, colony stimulating factors, tumor necrosis factors, and growth factors.
  8. The method of claim 4, wherein the therapeutic antibody is one or more selected from the group consisting of SARS-CoV mab, SARS-CoV-2 mab, and MERS-CoV mab.
  9. The method of claim 4, wherein the inflammatory factor storm therapeutic agent is one or more selected from Vesatolimod, ketotifen, glatiramer acetate, polycyclocetin, roflumilast, IL-6 mab, IL-6R mab (e.g., tolizumab), TLR3 mab, mammalian defensins, nickel-striated protein/IL-41 fragment, glucocorticoids (e.g., dexamethasone, prednisone, methylprednisone, and hydrocortisone), and broad spectrum antibiotics (e.g., azithromycin).
  10. The method of claim 4, wherein the vaccine is a vaccine against a virus of the coronaviridae family, in particular an inactivated vaccine, a nucleic acid vaccine, a recombinant protein/subunit vaccine, a live viral vector vaccine, an attenuated vaccine or a vaccine developed based on a DNA or RNA strategy or a replicative or non-replicative vector strategy.
  11. The method of any one of claims 1-10, wherein the coronaviridae virus is selected from the group consisting of SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, and variants thereof;
    Preferably, the variant of SARS-CoV-2 is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) or Omicron (B.1.1.529).
  12. The method of any one of claims 1-11, wherein the disease caused by the coronavirus infection is middle east respiratory syndrome, severe acute respiratory syndrome, or COVID-19.
  13. The method of any one of claims 1-12, wherein the Rho related protein kinase inhibitor or each active ingredient in the combination is administered in an amount of about 0.005 mg/day to about 5000 mg/day, such as about 0.005、0.05、0.5、5、10、20、30、40、50、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1500、2000、2500、3000、3500、4000、4500 or 5000 mg/day.
  14. The method of any one of claims 1-13, wherein each active ingredient of said Rho related protein kinase inhibitor or said combination is administered in an amount of about 1ng/kg to about 200mg/kg, about 1 μg/kg to about 100mg/kg, or about 1mg/kg to about 50mg/kg body weight per day, for example, at about 1 μg/kg, about 10 μg/kg, about 25 μg/kg, about 50 μg/kg, about 75 μg/kg, about 100 μg/kg, about 125 μg/kg, about 150 μg/kg, about 175 μg/kg, about 200 μg/kg, about 225 μg/kg, about 250 μg/kg, about 275 μg/kg, about 300 μg/kg, about 325 μg/kg, about 350 μg/kg, about 375 μg/kg, about 400 μg/kg, about 425 μg/kg, about 450 μg/kg, about 75 μg/kg, about 500 μg/kg, about 525 μg/kg, about 550 μg/kg, about 575 μg/kg, about 600 μg/kg, about 625 μg/kg, about 650 μg/kg, about 675 g/kg, about 725 μg/kg, about 750 μg/kg, about 775 μg/kg, about 75mg, about 5mg, about 25mg, about 97 mg, about 25mg, about 75mg, about 5mg, about 25mg, about 75mg About 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 60mg/kg, about 70mg/kg, about 80mg/kg, about 90mg/kg, about 100mg/kg, about 125mg/kg, about 150mg/kg, about 175mg/kg, about 200mg/kg, or about 300mg/kg of body weight.
  15. The method of any one of claims 1-14, wherein the daily dose of each active ingredient in the Rho related protein kinase inhibitor or the combination is administered once or in two, three or four administrations.
  16. The method of any one of claims 4-15, wherein the active ingredients in the combination are administered simultaneously, sequentially or alternately.
  17. The method of any one of claims 1-16, wherein each active ingredient of the Rho related protein kinase inhibitor or the combination is administered continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, or at least 2 years.
  18. The method of any one of claims 1-17, wherein the Rho-associated protein kinase inhibitor or each active ingredient in the combination is administered one or more (e.g., 1,2,3,4, 5, 6, 7, 8, 9, or 10) courses, wherein each course lasts for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, or at least 50 days; and every two courses of treatment are spaced 0,1,2, 3,4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks or four weeks apart.
  19. The method of any one of claims 1-18, wherein the Rho-associated protein kinase inhibitor or each active ingredient in the combination is administered by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of an ophthalmic formulation or by inhalation, and the route of administration of the active ingredients in the combination may be the same or different.
  20. A pharmaceutical composition comprising a Rho related protein kinase inhibitor in combination with one or more other drugs and a pharmaceutically acceptable carrier;
    Wherein:
    The Rho related protein kinase inhibitor is as defined in any one of claims 1-3; and
    The other medicament is as defined in any one of claims 4 to 10.
  21. A kit comprising a plurality (e.g., 2,3 or 4) of containers, each container comprising a Rho-associated protein kinase inhibitor or one other drug, and optionally a pharmaceutically acceptable carrier, and optionally instructions;
    Wherein:
    The Rho related protein kinase inhibitor is as defined in any one of claims 1-3; and
    The other medicament is as defined in any one of claims 4 to 10.
CN202280061163.3A 2021-09-17 2022-09-16 Methods of treating viral infections Pending CN118019530A (en)

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