WO2022184102A1 - 〈杨梅素和二氢杨梅素磷酸酯类化合物在防治新冠肺炎药物中的应用〉 - Google Patents

〈杨梅素和二氢杨梅素磷酸酯类化合物在防治新冠肺炎药物中的应用〉 Download PDF

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WO2022184102A1
WO2022184102A1 PCT/CN2022/078851 CN2022078851W WO2022184102A1 WO 2022184102 A1 WO2022184102 A1 WO 2022184102A1 CN 2022078851 W CN2022078851 W CN 2022078851W WO 2022184102 A1 WO2022184102 A1 WO 2022184102A1
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active ingredient
myricetin
coronavirus
acid
phosphate
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PCT/CN2022/078851
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English (en)
French (fr)
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叶阳
许叶春
姚胜
苏海霞
张磊砢
肖庚富
柯昌强
赵文峰
冯璐
谢航
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中国科学院上海药物研究所
中国科学院武汉病毒研究所
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Publication of WO2022184102A1 publication Critical patent/WO2022184102A1/zh

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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the invention relates to the field of medicine, in particular to the preparation of a class of myricetin and dihydromyricetin phosphate prodrug compounds and their application in medicines for preventing and treating new coronary pneumonia
  • Coronaviruses are single-stranded positive-stranded RNA viruses that belong to the subfamily Orthocoronavirinae of the Coronaviridae family of the order Nidovirales and can infect humans, bats, pigs, mice, cattle, horses, and goats , monkeys and other species.
  • Six coronaviruses (HCoV) are known to infect humans, including Middle East respiratory syndrome-related coronavirus (MERSr-CoV) and severe acute respiratory syndrome-related coronavirus (SARSr-CoV).
  • MERSr-CoV Middle East respiratory syndrome-related coronavirus
  • SARSr-CoV severe acute respiratory syndrome-related coronavirus
  • Novel coronavirus 2019-nCoV (SARS-CoV-2) is the seventh coronavirus to infect humans and can cause severe pneumonia.
  • the transmission route of the 2019-nCoV virus is not fully understood, it is known to be transmitted through droplets and contact, and there is human-to-human transmission, medical staff infection, a certain risk of community transmission, and the virus may mutate. At present, there is no specific prevention and treatment method for the disease caused by the new coronavirus.
  • the 2019-nCoV coronavirus belongs to the genus Coronaviridae and is a single-stranded positive-sense RNA virus with an envelope. Similar to other known coronaviruses, the 2019-nCoV coronavirus also completes the proliferation of progeny viruses through several processes such as adsorption, penetration, shelling, biosynthesis, and assembly and release of progeny viruses.
  • 2019-nCoV coronavirus infection of host cells begins with the binding of the spike glycoprotein on the surface of the viral envelope to receptors on the surface of the host cell, followed by membrane fusion, the virus enters the host cell, and is released under the action of cell lysosomes and other organelles
  • the single-stranded positive-sense RNA of the genetic material of the virus is produced, and under the action of the host cell's mitochondria, ribosomes and other protein synthesis elements and necessary raw materials, it is translated to produce polyproteins.
  • Acid proteases papain-like protease (PL pro ) and 3C-like protease (3C-like protease, 3CL pro ) cleave and process polyprotein precursors at specific sites, producing multiple proteins that are very important for the viral life cycle. nonstructural proteins. Under the action of these non-structural proteins, viral RNA replicates progeny viral nucleic acid material, and a large number of required structural proteins are translated to complete the assembly and release of progeny viruses.
  • PL pro papain-like protease
  • 3C-like protease 3C-like protease, 3CL pro
  • Any link or key enzyme in the life cycle of 2019-nCoV coronavirus-infected cells can be used as research targets for antiviral drugs, such as cysteine proteases PL pro and 3CL pro that hydrolyze and cleave polyprotein precursors, which are responsible for completing the RNA polymerase that replicates viral genetic material, etc.
  • antiviral drugs such as cysteine proteases PL pro and 3CL pro that hydrolyze and cleave polyprotein precursors, which are responsible for completing the RNA polymerase that replicates viral genetic material, etc.
  • the purpose of the present invention is to provide the new application of myricetin phosphate compound in anti-novel coronavirus.
  • the present invention provides myricetin 3'-O-diisopropyl phosphate, myricetin 7-O-2,2-dimethyl-1,3 propanediol phosphate, myricetin 7-O-
  • myricetin phosphate compounds represented by diphenyl phosphate and myricetin 7-O-phosphate bis(p-chlorophenyl) ester are used in the preparation of novel coronavirus 3CL protease inhibitors, as well as the treatment, prevention and relief of novel coronaviruses.
  • the use of an active ingredient or a preparation containing the active ingredient is a compound represented by formula I or a solvate or a prodrug or a composition thereof;
  • R 1 and R 2 are each independently selected from: OH, -OC 1 -C 6 alkyl, -OC 3 -C 6 cycloalkyl, alkyl, cycloalkyl, and at least one of R 1 and R 2 is
  • R 8 and R 9 are each independently selected from: H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 6 cycloalkyl, substituted or unsubstituted phenyl; wherein, Said substitution means being substituted by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl;
  • R 8 , R 9 and the connected -OPO- together form a substituted or unsubstituted 6-7 membered heterocycle, and the heterocycle contains 2 O heteroatoms, 1 P heteroatom and 0-1 N heteroatoms atom, said substitution means substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, C1-C3 haloalkyl, C3-C6 cycloalkyl, benzyl, halobenzyl, Alk(oxy)-substituted benzyl;
  • the preparation of the active ingredient or the pharmaceutical composition containing the active ingredient is used to prepare (a) an inhibitor that inhibits the activity of the novel coronavirus 3CL protease; and/or (b) treatment and/or prevention, alleviation Drugs for related diseases caused by novel coronavirus infection.
  • R 8 and R 9 are each independently selected from the group consisting of Cl-substituted phenyl, Br-substituted phenyl, and I-substituted phenyl.
  • the related disease caused by novel coronavirus infection is selected from the group consisting of respiratory tract infection, pneumonia and complications thereof, or a combination thereof.
  • R 1 and R 2 are as defined above.
  • the formulation containing the active ingredient may also contain other antiviral drugs.
  • the active ingredient-containing preparation further includes additional components selected from the group consisting of anti-lung injury drugs, anti-inflammatory drugs or drugs with immunomodulatory effects.
  • the active ingredient-containing formulation further includes additional components selected from the group consisting of zinc (Zinc), Fingolimod, vitamin C (Vitamin C), olmesartan medoxomil (Olmesartan Medoxomil), Valsartan (valsartan), Losartan (Losartan), Thalidomide (Thalidomide), glycyrrhizic acid (glycyrrhizic acid), Artemisinin (Artemisinin), dihydroartemisinin (dihydroartemisinin), Artesunate, Artemisone, Azithromycin, Escin, Naproxen, or a combination thereof.
  • the active ingredient is selected from the following group:
  • the preparations include: oral preparations and non-oral preparations.
  • the preparations include: powders, granules, capsules, injections, tinctures, oral liquids, tablets, lozenges, or drop pills.
  • a pharmaceutical composition in a second aspect of the present invention, contains:
  • the first active ingredient, the first active ingredient is the compound represented by formula I or its solvate or its prodrug or its composition
  • An optional second active ingredient, the second active ingredient is an antiviral drug selected from the group consisting of interferons, RNA-dependent RNA polymerase inhibitors (such as Remdesivir (Remdesivir or GS) -5734), favipiravir, Galidesivir, GS-441524); 3CL protease inhibitors (such as GC-376), Lopinavir, Ritonavir, nelfinavir (Nelfinavir); chloroquine (Chloroquine, Sigma-C6628), hydroxychloroquine (hydroxychloroquine), cyclosporine (cyclosporine), carrimycin (Carrimycin), baicalin (baicalin), baicalein (baicalein), naphthoquine (Naphthoquine), Ciclesonide, Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat ), nitazoxanide, Darunavir, Ar
  • the second active ingredient is selected from the group consisting of: Zinc (Zinc), Fingolimod (Fingolimod), Vitamin C (Vitamin C), Olmesartan Medoxomil (Olmesartan Medoxomil), Valsartan (valsartan) , Losartan, Thalidomide, Glycyrrhizic acid, Artemisinin, Dihydroartemisinin, Artesunate, Artemisinone (Artemisone), Azithromycin (Azithromycin), Escin (Escin), Naproxen (Naproxen), or a combination thereof;
  • the first active ingredient is a compound represented by formula II or a solvate or a prodrug or a composition thereof.
  • the first active ingredient is selected from:
  • the pharmaceutical composition is used to inhibit the activity of the novel coronavirus 3CL protease.
  • a use of the pharmaceutical composition described in the second aspect for preparing (a) an inhibitor of novel coronavirus 3CL protease; and/or (b) treatment and/or prevention , Drugs for relieving related diseases caused by novel coronavirus infection.
  • an active ingredient is the compound represented by formula II or its solvate or its prodrug or its composition, wherein formula II is as defined above.
  • the active ingredient is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-oxide, N-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • a method for inhibiting novel coronavirus 3CL protease in vitro comprising the steps of:
  • coronavirus SARS-CoV-2
  • the said first active ingredient is the compound represented by formula I or its solvate or its composition.
  • the one active ingredient is a compound represented by formula II or a solvate or a composition thereof, wherein formula II is as defined above.
  • the first active ingredient is selected from the following group:
  • the method is an in vitro method.
  • the method is non-therapeutic and non-diagnostic.
  • Figure 1 Curve of inhibitory activity of compound 1 against 2019-nCoV-3CLpro.
  • Figure 2 Curve of inhibitory activity of compound 2 on 2019-nCoV-3CLpro.
  • Figure 3 Inhibitory activity curve of compound 3 on 2019-nCoV-3CLpro.
  • Figure 4 Inhibitory activity curve of compound 4 against 2019-nCoV-3CLpro.
  • Figure 5 Curve of myricetin's inhibitory activity against 2019 novel coronavirus replication.
  • Figure 6 Curve of compound 3's inhibitory activity against 2019 novel coronavirus replication.
  • Figure 7 Graph of the inhibitory activity of dihydromyricetin on 2019 novel coronavirus replication.
  • Figure 8 Curve of compound 5's inhibitory activity against 2019 novel coronavirus replication.
  • the present invention is prepared with myricetin 3'-O-diisopropyl phosphate, myricetin 7-O-2,2-dimethyl-1,3 propanediol phosphate, myricetin 7-O- Diphenyl phosphate, myricetin 7-O-phosphate bis(p-chlorophenyl) ester, dihydromyricetin 7-O-diphenyl phosphate, dihydromyricetin 7-O-phosphate bis(p-chlorobenzene)
  • the myricetin and dihydromyricetin phosphate compounds represented by the myricetin and dihydromyricetin phosphate compounds were found to effectively inhibit the activity of the 3CL protease of the new coronavirus, and can inhibit the replication of the new coronavirus.
  • the cell permeability of these compounds is also very beneficial and has good medicinal prospects.
  • pharmaceutical active ingredient of the present invention refers to myricetin phosphates compound.
  • formulation of the present invention refers to a formulation containing an active ingredient of the present invention.
  • novel coronavirus As used herein, the terms "novel coronavirus”, “2019-nCov” or “SARS-CoV-2” are used interchangeably, which is the seventh coronavirus known to infect humans and cause COVID-19 (COVID-19), is one of the serious infectious diseases threatening human health worldwide.
  • Coronavirus belongs to the family Coronaviridae of the order Nidovirales. It is an enveloped positive-strand RNA virus.
  • HCoV-229E and HCoV-NL63 belong to alpha coronaviruses, and HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all beta coronaviruses Virus.
  • SARS-CoV-2 is also known as 2019-nCov.
  • the genome of this virus is a single-stranded positive-stranded RNA, which is one of the largest RNA viruses in the genome, encoding replicase, spike protein, envelope protein, envelope protein and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to generate non-structural proteins (such as RNA polymerase and unwinding). enzymes) and structural proteins (such as spike proteins) and accessory proteins.
  • an active ingredient that can effectively inhibit the activity of coronavirus 3CL protease is provided.
  • the active ingredient is myricetin phosphate compound or its solvate or its composition.
  • the active ingredient of the present invention is selected from:
  • pharmaceutical active ingredient of the present invention As used herein, “pharmaceutical active ingredient of the present invention”, “active ingredient of the present invention”, “active compound of the present invention”, and “active compound of the present invention that inhibits the replication of coronavirus” are used interchangeably and refer to those with excellent inhibition of coronavirus Viral replication active compounds.
  • active ingredients of the present invention include myricetin phosphate compounds or solvates or compositions thereof.
  • the active ingredients of the present invention also include crystalline forms, amorphous compounds, and deuterated compounds of the active compounds of the present invention.
  • the "pharmaceutically acceptable salts” are conventional non-toxic salts formed by the reaction of the active compounds of the present invention with inorganic or organic acids.
  • conventional non-toxic salts can be prepared by reacting the active compounds of the present invention with inorganic or organic acids, including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid, and the like.
  • Acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid , Fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the active compound of the present invention is mixed with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid
  • the active ingredient of the present invention is also particularly suitable for use in combination with other anti-coronavirus drugs.
  • Representative other anti-coronavirus drugs include (but are not limited to): interferon, RNA-dependent RNA polymerase inhibitors (such as Remdesivir (Remdesivir or GS-5734), favipiravir, Galidesivir, GS-441524); 3CL protease inhibitors (such as GC-376), Lopinavir, Ritonavir, Nelfinavir; Chloroquine (Chloroquine, Sigma-C6628) , Hydroxychloroquine, Cyclosporine, Carrimycin, Baicalin, Baicalein, Naphthoquine, Ciclesonide, Ribavir Ribavirin, Penciclovir, Leflunomide, Teriflunomide, Nafamostat, Nitazoxanide, Darunavir ( Darunavir, Arbidol, Camostat, Niclosamide, Barici
  • the active ingredients of the present invention are also particularly suitable for use in combination with drugs that have ameliorating acute lung injury, anti-inflammatory effects or immune-modulating effects.
  • Representative drugs include, but are not limited to, Zinc, Fingolimod, Vitamin C, Olmesartan Medoxomil, valsartan, Losartan , Thalidomide, Glycyrrhizic acid, Artemisinin, Dihydroartemisinin, Artesunate, Artemisone, Azithromycin ), aescin (Escin), naproxen (Naproxen).
  • the present invention also provides a mixture of the active compound for inhibiting the replication of coronavirus of the present invention, or a pharmaceutically acceptable salt thereof, or a mixture of one or more of its prodrugs as an active ingredient, in the preparation of treatment and/or prevention, Use in medicines for relieving respiratory infections, pneumonia and other related diseases caused by 2019 novel coronavirus and other coronavirus infections.
  • the pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, and the preferred ratio is that the active compound of the present invention, as an active ingredient, accounts for 0.1wt%-90wt% or 1wt%-50wt% of the total weight, The remainder is a pharmaceutically acceptable carrier, diluent or solution or saline solution.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and the like conventional in the pharmaceutical field.
  • the compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in suitable solid or liquid carriers or diluents Neutralize in suitable sterile equipment for injection or instillation.
  • Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field.
  • the unit measurement of the formulation usually contains 0.05-400 mg of the active compound of the present invention, preferably, the unit measurement of the formulation contains 1 mg-500 mg of the active compound of the present invention.
  • the compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, by oral, nasal, dermal, pulmonary, or gastrointestinal routes of administration. Most preferred is oral administration.
  • the most preferred daily dose is 0.01-400 mg/kg body weight in a single dose, or 0.01-200 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dose for an individual will depend on the specific treatment. It is common to start with a small dose and gradually increase the dose until the most suitable dose is found.
  • the drug or inhibitor of the present invention can be administered in various ways, for example, it can be introduced into the body such as muscle, intradermally, subcutaneously, intravenously by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated methods , mucosal tissue; or mixed or packaged by other substances into the body.
  • the active ingredient of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, Eyes, lungs and respiratory tract, skin, vagina, rectum, etc.
  • the dosage form for administration can be a liquid dosage form, a solid dosage form or a semi-solid dosage form.
  • Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w, w/o and double emulsion), suspensions, injections (including water injection, powder injection and infusion), eye drops solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules ( Including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, drop pills, suppositories, films, patches, gas (powder) aerosols, sprays, etc.; semi-solid dosage forms can be ointments, Gels, pastes, etc.
  • the active ingredients of the present invention can be prepared into ordinary preparations, as well as into sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
  • diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the wetting agent can be water, ethanol, Isopropyl alcohol, etc.
  • the binder can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin pulp, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry
  • the tablets can also be further prepared as coated tablets, such as sugar-coated, film-coated, enteric-coated, or bilayer and multi-layer tablets.
  • the active ingredients of the present invention can be mixed with diluents and glidants, and the mixture can be directly placed in hard capsules or soft capsules.
  • the active ingredients can also be made into granules or pellets with diluents, binders and disintegrating agents, and then placed in hard capsules or soft capsules.
  • Various diluents, binders, wetting agents, disintegrants, and glidants used for preparing the tablets of the present invention can also be used for preparing the capsules of the present invention.
  • solubilizers, cosolvents, pH adjusters and osmotic pressure adjusters commonly used in the art can be added.
  • Solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl- ⁇ -cyclodextrin, etc.
  • PH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.
  • osmotic pressure adjuster can be It is sodium chloride, mannitol, glucose, phosphate, acetate, etc.
  • mannitol, glucose, etc. can also be added as proppant.
  • colorants preservatives, fragrances, flavors, or other additives can also be added to the pharmaceutical preparations, if desired.
  • the active ingredient or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs.
  • the active ingredient of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
  • the active ingredient of the present invention can effectively inhibit the activity of the 3CL protease of the novel coronavirus.
  • the IC 50 values of -2 3CLpro were 27.58 ⁇ M, 6.14 ⁇ M, 2.70 ⁇ M, 2.11 ⁇ M, 1.88 ⁇ M and 8.59 ⁇ M, respectively.
  • the active ingredient of the present invention can effectively inhibit the replication of the novel coronavirus.
  • the representative compound myricetin 7-O-diphenyl phosphate inhibits the new coronavirus with an EC 50 value of about 2.98 ⁇ M.
  • the cell permeability of the active ingredient of the present invention is good.
  • the average absorption bioavailability of the representative compound myricetin 7-O-diphenyl phosphate is 82%, and the permeability is good.
  • the oral exposure of the active ingredient of the present invention is higher.
  • the representative compound dihydromyricetin 7-O-diphenyl phosphate (5) under the oral dose of 30mg/kg, the maximum blood concentration of dihydromyricetin released (C max ) and the area under the drug-time curve ( AUC last ) was comparable to the maximum plasma concentration (C max ) and the area under the curve (AUC last ) of dihydromyricetin when administered orally at 100 mg/kg.
  • Embodiment 1 the synthesis of myricetin phosphate compound
  • the inhibitory activity of myricetin and its phosphate compounds on SARS-CoV-2 3CLpro enzyme activity was evaluated by fluorescence resonance energy transfer method.
  • the volume of the entire enzymatic reaction system was 120 ⁇ L, the final concentration of protease was 30 nM, and the final concentration of substrate was 20 ⁇ M.
  • Buffers of the reaction system included 50 mM Tris pH 7.3, 1 mM EDTA.
  • SARS-CoV-2 3CLpro protease and different concentrations of compounds were added to a 96-well plate, incubated at 30°C for 10 min, added substrate and quickly put into a microplate reader for reading. The excitation and emission light were 320 nM and 405 nM, respectively.
  • Figures 1-4 are the inhibition curves of myricetin phosphate compounds 1-4 against SARS-CoV-2 3CLpro, respectively.
  • Embodiment 3 Myricetin and myricetin-7-O-bisphenyl phosphate (3) Caco-2 cell permeability test
  • Caco-2 cells were cultured in high-glucose DMEM medium in an incubator at 37°C, 5% CO2, and 90% relative air humidity.
  • the medium was supplemented with 10% fetal bovine serum, 10 mmol/L HEPES, and 1 mmol/L acetone.
  • One generation was passed every 7 days, and the passage ratio was 1:10.
  • the experiments used cells between passages 40 and 60. After 21 days of culture, the compactness of the cell monolayer (must be greater than 400 ⁇ cm 2 ) was detected by the TEER value.
  • the drug transport from the apical layer (A side) to the basal layer (B side) and from the B side to the A side was also investigated.
  • the test method was as follows: after washing the cells three times with HBSS, 20 ⁇ M of the compound and the compound plus inhibitor (GF120918) were added to the corresponding cell wells (A side pH 6.8, B side pH 7.4). Incubate in a 37°C incubator for 95 minutes, and sample the dosing side at 5 and 95 minutes, and the receiving side at 35 and 95 minutes, respectively. The concentration of the samples was checked by LC-MS/MS. The Papp and Efflux values of the test compounds myricetin and myricetin-7-O-bisphenyl phosphate (3) were determined. Table 2 shows the test results of myricetin and myricetin phosphate compound (3) Caco-2 cell permeability.
  • Permeability criterion Fab%>70%, good permeability; 30% ⁇ Fab% ⁇ 70%, medium permeability; Fab% ⁇ 30%, poor permeability.
  • Embodiment 4 Comparison of pharmacokinetic parameters of dihydromyricetin and its phosphate prodrugs
  • ICR mice male, weighing 18-22 g, were randomly divided into 2 groups with 3 mice in each group. Test compounds were administered by gavage according to the following protocol.
  • the rats were fasted for 12 h before the test and had free access to water. 2h after the administration of unified food.
  • Gavage solutions were formulated to final concentrations in DMSO/0.5% HPMC (5/95, v/v). The dosing solution was reserved (before and after administration, 50 ⁇ L of the medicinal solution was mixed with 50 ⁇ L of DMSO) for testing.
  • the drug was administered at the above-mentioned dosage, the administration time was recorded, and 40 ⁇ L of blood was collected from the mouse femoral plexus at the time point set above and placed in a heparinized test tube. Immediately centrifuge at 4 degrees Celsius for 5 min (rotation speed 11000 rpm), then immediately aspirate 20 ⁇ L of plasma and add 2 ⁇ L of 20% vitamin C aqueous solution, then add 200 ⁇ L of acetonitrile (containing 1% formic acid) solution to precipitate and mix, and freeze at -60 °C To be tested.
  • Embodiment 5 Myricetin and myricetin-7-O-bisphenyl phosphate (3), dihydromyricetin and dihydromyricetin-7-O-bisphenyl phosphate (5) against 2019 novel coronavirus Replication inhibitory activity evaluation
  • Table 3 Test results of myricetin and myricetin-7-O-bisphenyl phosphate (3) on the replication inhibitory activity of 2019 novel coronavirus
  • the present invention by introducing a phosphate group into the structure of myricetin, it is unexpectedly found that the permeability of the obtained myricetin phosphate, especially the myricetin 7-O-diphenyl phosphate, has been significantly improved, and the compound has a significant effect on the cell membrane. The permeability is improved, thereby enhancing the compound's replication-inhibitory activity against the novel coronavirus.
  • the present invention unexpectedly finds the obtained dihydromyricetin phosphate, especially dihydromyricetin 7-O-diphenyl phosphate, by introducing a phosphate group into the structure of dihydromyricetin. Both oral exposure and inhibitory activity against 2019-nCoV were significantly improved.

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Abstract

本发明涉及一类杨梅素、二氢杨梅素磷酸酯类前药化合物的制备及其在防治新冠肺炎药物中的应用。具体而言，本发明涉及杨梅素磷、二氢杨梅素酸酯类前药化合物及其药物组合物在(a)制备2019新型冠状病毒(SARS-CoV-2)3CL蛋白酶抑制剂；(b)制备治疗和/或预防和/或缓解由2019新型冠状病毒感染引起的呼吸道感染、肺炎等相关疾病的药物中的用途。

Description

〈杨梅素和二氢杨梅素磷酸酯类化合物在防治新冠肺炎药物中的应用〉 技术领域
本发明涉及医药领域，具体地涉及一类杨梅素和二氢杨梅素磷酸酯类前药化合物的制备及其在防治新冠肺炎药物中的应用
背景技术
在急性传染病中，绝大部分都是病毒性传染病，病毒性传染病的发病率高，死亡率也很高。由于检测和诊断手段有限，导致新病毒引发的新疫情爆发往往具有突发性、随机性和不可预测性等特点，一旦爆发，如无有效的防治手段，极易造成大规模流行，严重威胁人民健康生命安全。
冠状病毒(Coronaviruses)是单股正链RNA病毒，属于巢病毒目(Nidovirales)冠状病毒科(Coronaviridae)正冠状病毒亚科(Orthocoronavirinae)，可以感染人、蝙蝠、猪、老鼠、牛、马、山羊、猴子等多种物种。已知感染人的冠状病毒(HCoV)有6种，包括中东呼吸综合征相关冠状病毒(MERSr-CoV)和严重急性呼吸综合征相关冠状病毒(SARSr-CoV)。
新型冠状病毒2019-nCoV(SARS-CoV-2)是第7种感染人的冠状病毒，可以引起严重肺炎。2019-nCoV病毒传播途径未完全掌握，已知能通过飞沫和接触传播，且存在人传人、医务人员感染，一定社区传播风险，且病毒存在变异的可能。目前对于新型冠状病毒所致疾病没有特异的预防和治疗方法。
2019-nCoV冠状病毒属于冠状病毒科冠状病毒属，为具有包膜的单链正义RNA病毒。和其他已知冠状病毒类似，2019-nCoV冠状病毒也经过吸附、穿入、脱壳、生物合成、子代病毒的组装与释放等几个过程完成子代病毒的增殖。2019-nCoV冠状病毒感染宿主细胞起始于病毒包膜表面的刺突糖蛋白与宿主细胞表面的受体结合，随后发生膜融合，病毒进入宿主细胞，在细胞溶酶体等细胞器作用下，释放出病毒的遗传物质单链正义RNA，在宿主细胞的线粒体、核糖体等蛋白质合成元件以及必须的原料等作用下，翻译产生多聚蛋白，之后，2019-nCoV冠状病毒的两大必需半胱氨酸蛋白酶：木瓜样蛋白酶(papain-like protease,PL  pro)和3C样蛋白酶(3C-like protease,3CL pro)在特定位点切割加工多聚蛋白前体，产生多个对病毒生命周期非常重要的非结构蛋白。在这些非结构蛋白的作用下，病毒RNA复制出子代病毒核酸物质，并大量翻译出所需的结构蛋白，完成子代病毒的组装和释放。2019-nCoV冠状病毒感染细胞的生命周期的任何环节或关键酶均可以作为抗病毒药物的研究靶点，如水解切割多聚蛋白前体的半胱氨酸蛋白酶PL pro和3CL pro，负责完成子代病毒遗传物质复制的RNA聚合酶等。
目前，针对SARS-CoV-2冠状病毒导致的严重肺炎疾病尚无特效的疫苗和抗病毒药物。这些感染性疾病严重影响了人们的生命健康，研发效果好的抗病毒药物迫在眉睫。针对SARS-CoV-2冠状病毒开发出低毒高效的抗病毒药物，以满足国内外SARS-CoV-2 冠状病毒感染患者的临床需求，具有重大的社会意义。
综上所述，本领域迫切需要开发针对SARS-CoV-2冠状病毒的抑制剂以用于治疗新型冠状病毒感染引起的肺炎。
发明内容
本发明的目的是提供杨梅素磷酸酯类化合物在抗新型冠状病毒中的新用途。
具体地，本发明提供了以杨梅素3’-O-磷酸二异丙基酯、杨梅素7-O-2,2-二甲基-1,3丙二醇基磷酸酯、杨梅素7-O-磷酸二苯基酯、杨梅素7-O-磷酸双(对氯苯基)酯为代表的一类杨梅素磷酸酯类化合物在制备新型冠状病毒3CL蛋白酶抑制剂，以及治疗、预防、缓解由新型冠状病毒引起的相关疾病的药物中的用途。
在本发明的第一方面，一种活性成分或含所述活性成分的制剂的用途，所述的活性成分为式I所示化合物或其溶剂化物或其前药或其组合物；
Figure PCTCN2022078851-appb-000001
式中，
Figure PCTCN2022078851-appb-000002
为单键或双键；
R 1和R 2各自独立地选自：OH、-O-C 1-C 6烷基、-O-C 3-C 6环烷基、
Figure PCTCN2022078851-appb-000003
烷基、
Figure PCTCN2022078851-appb-000004
环烷基、
Figure PCTCN2022078851-appb-000005
且R 1、R 2中至少一个为
Figure PCTCN2022078851-appb-000006
R 8、R 9各自独立地选自：H、取代或未取代的C 1-C 6烷基、取代或未取代的C 3-C 6环烷基、取代或未取代的苯基；其中，所述的取代表示被一个或多个选自下组的取代基取代：卤素、C1-C3烷基、C1-C3卤代烷基、C3－C6环烷基；
或者R 8、R 9与相连的－O-P-O－共同构成取代或未取代的6-7元杂环，所述的杂环含有2个O杂原子、1个P杂原子和0-1个N杂原子，所述的取代表示被一个或多个选自下组的取代基取代：卤素、C1-C3烷基、C1-C3卤代烷基、C3－C6环烷基、苄基、卤代苄基、烷(氧)基取代的苄基；
并且，所述的活性成分或含所述活性成分的药物组合物的制剂被用于制备(a)抑制新型冠状病毒3CL蛋白酶活性的抑制剂；和/或(b)治疗和/或预防、缓解由新型冠状病毒感染引起的相关疾病的药物。
在另一优选例中，R 8和R 9各自独立地选自下组：Cl取代的苯基、Br取代的苯基、I取代的苯基。
在另一优选例中，所述由新型冠状病毒感染引起的相关疾病选自下组：呼吸道感染、肺炎及其并发症、或其组合。
在另一优选例中，当式I中
Figure PCTCN2022078851-appb-000007
为单键，所述活性成分为如式II所示化合物或其溶剂化物或其前药或其组合物：
Figure PCTCN2022078851-appb-000008
式中，R 1、R 2如上定义。
在另一优选例中，含所述含活性成分的制剂还可以含有其他抗病毒药。
在另一优选例中，所述的含活性成分的制剂还包括选自下组的额外组分：抗肺损伤的药物、抗炎药物或具有免疫调节作用的药物。
在另一优选例中，所述的含活性成分的制剂还包括选自下组的额外组分：锌(Zinc)、芬戈莫德(Fingolimod)、维生素C(Vitamin C)、奥美沙坦酯(Olmesartan Medoxomil)、缬沙坦(valsartan)、氯沙坦(Losartan)、沙利度胺(Thalidomide)、甘草酸(glycyrrhizic acid)、青蒿素(Artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(Artesunate)、青蒿酮(Artemisone)、阿奇霉素(Azithromycin)、七叶皂苷(Escin)、萘普生(Naproxen)、或其组合。
在另一优选例中，所述的活性成分选自下组：
Figure PCTCN2022078851-appb-000009
Figure PCTCN2022078851-appb-000010
A7 A1-A6的组合。
在另一优选例中，所述的制剂包括：口服制剂和非口服制剂。
在另一优选例中，所述的制剂包括：粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂、含片、或滴丸。
在本发明的第二方面，提供了一种药物组合物，所述的药物组合物含有：
(a1)第一活性成分，所述第一活性成分为式I所示化合物或其溶剂化物或其前药或其组合物；
(a2)任选的第二活性成分，所述的第二活性成分为抗病毒药物，其选自下组：干扰素、RNA依赖的RNA聚合酶抑制剂(如Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524)；3CL蛋白酶抑制剂(如GC-376)、洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、奈非那韦(Nelfinavir)；氯喹(Chloroquine,Sigma-C6628)、羟氯喹(hydroxychloroquine)、环孢菌素(cyclosporine)、可利霉素(Carrimycin)、黄芩苷(baicalin)、黄芩素(baicalein)、萘酚喹(Naphthoquine)、环索奈德(Ciclesonide)、利巴韦林(Ribavirin)、喷昔洛韦(Penciclovir)、来氟米特(Leflunomide)、特立氟胺(Teriflunomide)、萘莫司他(nafamostat)、硝唑尼特(nitazoxanide)、达芦那韦(Darunavir)、阿比多尔(Arbidol)、卡莫司他(Camostat)、氯硝柳胺(Niclosamide)、巴瑞替尼(baricitinib)、芦可替尼(Ruxolitinib)、达沙替尼(Dasatinib)、沙奎那韦(Saquinavir)、Beclabuvir、司美匹韦(Simeprevir)、或其药学上可接受的盐、或其组合；
和/或所述的第二活性成分选自下组：锌(Zinc)、芬戈莫德(Fingolimod)、维生素C(Vitamin C)、奥美沙坦酯(Olmesartan Medoxomil)、缬沙坦(valsartan)、氯沙坦(Losartan)、沙利度胺(Thalidomide)、甘草酸(glycyrrhizic acid)、青蒿素(Artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(Artesunate)、青蒿酮(Artemisone)、阿奇霉素(Azithromycin)、七叶皂苷(Escin)、萘普生(Naproxen)、或其组合；
及(b)药学上可接受的载体。
在另一优选例中，所述第一活性成分为式II所示化合物或其溶剂化物或其前药或其组合物。
在另一优选例中，所述第一活性成分选自：
Figure PCTCN2022078851-appb-000011
A7 A1-A6的组合。
在另一优选例中，所述的药物组合物用于抑制新型冠状病毒3CL蛋白酶活性。
在本发明的第三方面，提供了一种第二方面所述的药物组合物的用途，用于制备(a)抑制新型冠状病毒3CL蛋白酶抑制剂；和/或(b)治疗和/或预防、缓解由新型冠状病毒感染引起的相关疾病的药物。
在本发明的第四方面，提供了一种活性成分，所述活性成分为式II所示的化合物或其或其溶剂化物或其前药或其组合物，其中式II如上定义。
在另一优选例中，所述活性成分为
Figure PCTCN2022078851-appb-000012
在本发明的第五方面，提供了一种体外的抑制新型冠状病毒3CL蛋白酶的方法，包括步骤：
将第一活性成分或含所述第一活性成分的制剂与冠状病毒(SARS-CoV-2)接触，抑制所述冠状病毒3CL蛋白酶的活性，从而抑制冠状病毒的复制；
其中，所述的第一活性成分为式I所示化合物或其溶剂化物或其组合物。
在另一优选例中，所述一活性成分为式II所示化合物或其溶剂化物或其组合物，其中式II如上定义。
在另一优选例中，所述的第一活性成分选自下组：
Figure PCTCN2022078851-appb-000013
Figure PCTCN2022078851-appb-000014
A7 A1-A4的组合。
在另一优选例中，所述方法是体外方法。
在另一优选例中，所述方法是非治疗性和非诊断性的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1：化合物1对2019-nCoV-3CLpro的抑制活性曲线图。
图2：化合物2对2019-nCoV-3CLpro的抑制活性曲线图。
图3：化合物3对2019-nCoV-3CLpro的抑制活性曲线图。
图4：化合物4对2019-nCoV-3CLpro的抑制活性曲线图。
图5：杨梅素对2019新型冠状病毒复制抑制活性曲线图。
图6：化合物3对2019新型冠状病毒复制抑制活性曲线图。
图7：二氢杨梅素对2019新型冠状病毒复制抑制活性曲线图。
图8：化合物5对2019新型冠状病毒复制抑制活性曲线图。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次意外地发现杨梅素磷酸酯类化合物在抑制新型冠状病毒中的作用。在此基础上完成了本发明。
具体地，本发明通过制备以杨梅素3’-O-磷酸二异丙基酯、杨梅素7-O-2,2-二甲基-1,3丙二醇基磷酸酯、杨梅素7-O-磷酸二苯基酯、杨梅素7-O-磷酸双(对氯苯基)酯、二氢杨梅素7-O-磷酸二苯基酯、二氢杨梅素7-O-磷酸双(对氯苯基)酯为代表的杨梅素和二氢杨梅素磷酸酯类化合物，发现这类化合物能有效抑制新型冠状病毒的3CL蛋白酶的活性，并且能够抑制新型冠状病毒的复制。此外该类化合物的细胞渗透性也非常有益，具有良好的药用前景。
术语
如本文所用，“本发明的药物活性成分”、“本发明的活性成分”、“本发明的抑制新型冠状病毒3CL蛋白霉的活性成分”、可互换使用均指的是杨梅素磷酸酯类化合物。
如本文所用，“本发明的制剂”指含有本发明活性成分的制剂。
如本文所用，术语“包括”或其变换形式如“包含”或“包括有”等等，被理解为包括所述的元件或组成部分，而并未排除其它元件或其它组成部分。
如本文所用，术语“新型冠状病毒”、“2019-nCov”或“SARS-CoV-2”可互换使用，该2019新型冠状病毒是已知感染人的第7种冠状病毒，并且造成新冠肺炎(COVID-19)，是威胁全球人类健康的严重传染性疾病之一。
冠状病毒
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。SARS-CoV-2也被称为2019-nCov。
2003年和2012年分别爆发的高致病性冠状病毒“非典”SARS-CoV和“中东呼吸综合征”MERS-CoV均属于β属冠状病毒。2019年年底爆发的新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白(如刺突蛋白)及辅助蛋白。
本发明的活性化合物和活性成分
在本发明中，提供了可有效抑制冠状病毒3CL蛋白酶活性的活性成分。该活性成分为杨梅素磷酸酯类化合物或其溶剂化物或其组合物。
优选地，本发明的活性成分选自：
Figure PCTCN2022078851-appb-000015
Figure PCTCN2022078851-appb-000016
A7 A1-A4的组合。
实验表明，本发明的活性成分可有效地抑制新型冠状病毒的3CL蛋白酶活性，从而预防、治疗和/或缓解新型冠状病毒的相关疾病。
如本文所用，“本发明的药物活性成分”“本发明的活性成分”“本发明的活性化合物”、“本发明的抑制冠状病毒复制的活性化合物”可互换使用，指具有优异的抑制冠状病毒复制的活性的化合物。
应理解，本发明的活性成分包括杨梅素磷酸酯类化合物或其溶剂化物或其组合物。
应理解，本发明的活性成分还包括本发明的活性化合物的晶型、无定形化合物、以及氘代化合物等形式。
所述“药学上可接受的盐”为本发明的活性化合物与无机酸或有机酸反应形成常规的无毒盐。例如，常规的无毒盐可通过本发明的活性化合物与无机酸或有机酸反应制得，所述无机酸包括盐酸、氢溴酸、硫酸、硝酸、胺基磺酸和磷酸等，所述有机酸包括柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者本发明的活性化合物与丙酸、 草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、天冬氨酸或谷氨酸形成酯后再与无机碱形成的钠盐、锌盐、钾盐、钙盐、铝盐或铵盐；或者本发明的活性化合物与赖氨酸、精氨酸、鸟氨酸形成酯后再与盐酸、氢溴酸、氢氟酸、硫酸、硝酸或磷酸形成的对应的无机酸盐或与甲酸、乙酸、苦味酸、甲磺酸或乙磺酸形成的对应的有机酸盐；或者本发明的活性化合物分子中的羧基/酚羟基与无机碱形成的钠盐、锌盐、钾盐、钙盐、铝盐或铵盐。
此外，本发明的活性成分还特别适合与其他抗冠状病毒的药物联用。代表性的其他的抗冠状病毒药物包括(但并不限于)：干扰素、RNA依赖的RNA聚合酶抑制剂(如Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524)；3CL蛋白酶抑制剂(如GC-376)、洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、奈非那韦(Nelfinavir)；氯喹(Chloroquine,Sigma-C6628)、羟氯喹、环孢菌素(cyclosporine)、可利霉素(Carrimycin)、黄芩苷(baicalin)、黄芩素(baicalein)、萘酚喹(Naphthoquine)、环索奈德(Ciclesonide)、利巴韦林(Ribavirin)、喷昔洛韦(Penciclovir)、来氟米特(Leflunomide)、特立氟胺(Teriflunomide)、萘莫司他(nafamostat)、硝唑尼特(nitazoxanide)、达芦那韦(Darunavir)、阿比多尔(Arbidol)、卡莫司他(Camostat)、氯硝柳胺(Niclosamide)、巴瑞替尼(baricitinib)、芦可替尼(Ruxolitinib)、达沙替尼(Dasatinib)、沙奎那韦(Saquinavir)、Beclabuvir、司美匹韦(Simeprevir)、或其药学上可接受的盐、或其组合。所述干扰素包括干扰素α-2a、干扰素α-2b、干扰素α-n1、干扰素α-n3、干扰素β-1a、干扰素β-1b中的一种或多种。
此外，由于SARS-CoV-2感染可引起急性肺损伤、炎症反应甚至细胞因子风暴，本发明的活性成分还特别适合与具有改善急性肺损伤、抗炎作用或调节免疫作用的药物联用。代表性的药物包括但不限于锌(Zinc)、芬戈莫德(Fingolimod)、维生素C(Vitamin C)、奥美沙坦酯(Olmesartan Medoxomil)、缬沙坦(valsartan)、氯沙坦(Losartan)、沙利度胺(Thalidomide)、甘草酸(glycyrrhizic acid)、青蒿素(Artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(Artesunate)、青蒿酮(Artemisone)、阿奇霉素(Azithromycin)、七叶皂苷(Escin)、萘普生(Naproxen)。
药物组合物和应用
本发明还提供了以本发明的抑制冠状病毒复制的活性化合物、或其药学上可接受的盐、或其前药的一种或多种的混合物为有效成分，在制备治疗和/或预防、缓解由2019新型冠状病毒等冠状病毒感染引起的呼吸道感染、肺炎等相关疾病的药物中的用途。
本发明所提供的药物组合物优选含有重量比为0.001-99wt％的活性成份，优选的比例是本发明的活性化合物作为活性成分占总重量的0.1wt％～90wt％或1wt％～50wt％，其余部分为药学可接受的载体、稀释液或溶液或盐溶液。
需要的时候，在本发明药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
本发明所提供的化合物和药物组合物可以是多种形式，如片剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液中和适宜的用于注射或滴注的消毒器具中。
本发明的药物组合物的各种剂型可按照药学领域的常规制备方法制备。其制剂配方的单位计量中通常包含0.05-400mg本发明的活性化合物，优选地，制剂配方的单位计量中包含1mg-500mg本发明的活性化合物。
本发明的化合物和药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服。最优选日剂量为0.01-400mg/kg体重，一次性服用，或0.01-200mg/kg体重分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。
本发明的药物或抑制剂可通过各种不同方式施用，例如可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹导入机体。
典型地，本发明活性成分或含有它的药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明活性成分可以被制成普通制剂、也可以制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明活性成分被制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；黏合剂可以是淀粉浆、糊精、糖浆、蜂 蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明活性成分与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明的胶囊剂。
为将本发明活性成分制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、PH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；PH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其他添加剂。
本发明的活性成分或组合物可单独服用，或与其他治疗药物或对症药物合并使用。
当本发明的活性成分与其他治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明的主要优点包括：
(a)本发明活性成分能有效抑制新型冠状病毒的3CL蛋白酶的活性。杨梅素3’-O-磷酸二异丙基酯、杨梅素7-O-磷酸2,2-二甲基-1,3-丙二醇基酯、杨梅素7-O-磷酸二苯基酯、杨梅素7-O-磷酸双(对氯苯基)酯、二氢杨梅素7-O-磷酸二苯基酯、二氢杨梅素7-O-磷酸双(对氯苯基)酯抑制SARS-CoV-2 3CLpro的IC 50值分别为27.58μM、6.14μM、2.70μM、2.11μM、1.88μM、8.59μM。
(b)本发明活性成分能有效抑制新型冠状病毒的复制。代表化合物杨梅素7-O-磷酸二苯基酯抑制新型冠状病毒的EC 50值约为2.98μM。
(c)本发明活性成分的细胞渗透性好。代表化合物杨梅素7-O-磷酸二苯基酯的平均吸收生物利用度为82％，渗透性较好。
(d)本发明活性成分的口服暴露量更高。代表化合物二氢杨梅素7-O-磷酸二苯基酯(5)在30mg/kg的口服剂量下，其释放出来的二氢杨梅素最大血药浓度(C max)和药时曲线下面积(AUC last)与二氢杨梅素以100mg/kg口服形式给药所达到的最大血药浓度(C max)和药时曲线下面积(AUC last)相当。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例一、杨梅素磷酸酯类化合物的合成
1.1合成杨梅素3’-O-磷酸二异丙基酯(1)
Figure PCTCN2022078851-appb-000017
于25ml的圆底烧瓶中加入318mg的杨梅素(1.0mmol)和10ml的无水THF，待杨梅素完全溶解后加入146μL的无水三乙胺(1.05mmol,1.05当量)，反应瓶置于0度冰水浴中10min后加入氯磷酸二异丙酯(192μL,1.05mmol,1.05当量)，反应液于室温下搅拌3h，然后过滤除去三乙胺的盐酸盐，滤液减压蒸馏除去THF，所得剩余物通过制备型高效液相色谱【乙腈-水(含0.1％甲酸)，30-45％，0-35min】制备纯化，杨梅素3’-O-磷酸二异丙基酯120mg(得率25％)。化合物的质谱和波谱数据如下：ESI-MS m/z 483.02[M+H] +,481.00[M-H] -； 1H NMR(Acetone-d 6,500MHz)δ12.07(1H,s,5-OH),7.77 and 7.68(each 1H,br.s,H-2’,6’),6.50 and 6.25(each 1H,d,J＝2.0Hz,H-5,7),4.85(2H,m,OCH-×2),1.40 and 1.36(each 3H,d,J＝6.3Hz,CH 3×2)； 13C NMR(Acetone-d 6,125MHz)δ175.7,164.3,161.4,156.8,147.2,144.8,139.2(139.1),138.6(138.5),136.4,122.3,112.5(112.5),111.7,103.3,98.4,93.6,74.2,74.2,23.0,23.0,22.9,22.9； 31P NMR(Acetone-d 6,125MHz)δ-5.9.
1.2合成杨梅素7-O-2,2-二甲基-1,3丙二醇基磷酸酯(2)
Figure PCTCN2022078851-appb-000018
于25ml的圆底烧瓶中加入318mg的杨梅素(1.0mmol)和10ml的无水THF，待杨梅素完全溶解后加入146μL的无水三乙胺(1.05mmol,1.05当量)，反应瓶置于0度冰水浴中10min后加入2-氯-5,5-二甲基-1,3,2-二氧杂磷化氢烷2-氧化物(136.8μL,1.05mmol,1.05当量)，反应液于室温下搅拌3h，然后过滤除去三乙胺的盐酸盐，滤液减压蒸馏除去THF，所得剩余物通过制备型高效液相色谱【乙腈-水(含0.1％甲酸)，25-45％，0-60min】制备，得化合物杨梅素7-O-5,5-二甲基-1,3丙二醇基磷酸酯50mg(得率11％)，化合物的质谱和波谱数据如下：ESI-MS m/z 467.00[M+H] +,464.94[M-H] -； 1H NMR(Acetone-d 6,500MHz)δ12.07(1H,s,5-OH),7.30(2H,s,H-2’,6’),7.05 and 6.25(each 1H,d,J＝3.1Hz,H-5,7),4.35(2H,d,J＝10.6Hz,OCH 2-),4.07(2H,dd,J＝10.8 and 10.6Hz,OCH 2-),1.20 and 0.87(each 3H,s,CH 3×2)； 13C NMR(Acetone-d 6,125MHz)δ176.5,161.1,155.4,155.0(154.9),148.8,146.3,146.3,137.2,136.9,120.8,107.9,107.9,107.3,102.3(102.2),98.9(98.8),79.0,78.9,32.4,21.4,19.5； 31P NMR(Acetone-d 6,125MHz)δ-14.6
1.3合成杨梅素7-O-磷酸二苯基酯(3)
Figure PCTCN2022078851-appb-000019
于25ml的圆底烧瓶中加入318mg的杨梅素(1.0mmol)和10ml的无水THF，待杨梅素完全溶解后加入146μL的无水三乙胺(1.05mmol,1.05当量)，反应瓶置于0度冰水浴中10min后加入氯磷酸二苯酯(218μL,1.05mmol,1.05当量)，反应液于室温下搅拌3h，然后过滤除去三乙胺的盐酸盐，滤液减压蒸馏除去THF，所得剩余物通过制备型高效液相色谱【乙腈-水(含0.1％甲酸)，45-65％，0-35min】制备，得化合物杨梅素7-O-磷酸二苯基酯42mg(得率8％)，化合物的质谱和波谱数据如下：ESI-MS m/z 551.20[M+H] +,549.17[M-H] -； 1H NMR (Acetone-d 6,500MHz)δ12.37(1H,s,5-OH),7.47(4H,d,J＝7.5Hz,H-1a,6a×2),7.45(2H,s,H-2’,6’),7.35(4H,dd,J＝7.5 and 1.1Hz,H-2a,5a×2),7.30(2H,dt,J＝7.5 and 1.1Hz,H-3a×2),7.10 and 6.67(each 1H,d,J＝1.3Hz,H-5,7)； 13C NMR(Acetone-d 6,125MHz)δ176.0,161.3,155.7,155.2(155.1),150.0,150.0,147.9,145.7,145.7,136.8,136.1,120.8,130.1,130.1,129.1,129.1,126.0,123.3,120.3,120.2,120.1,120.1,107.9,107.9,107.1,102.0(102.0),99.1(99.0)； 31P NMR(Acetone-d 6,125MHz)δ-18.5
1.4合成杨梅素7-O-磷酸双(对氯苯基)酯(4)
Figure PCTCN2022078851-appb-000020
于25ml的圆底烧瓶中加入318mg的杨梅素(1.0mmol)和10ml的无水THF，待杨梅素完全溶解后加入146μL的无水三乙胺(1.05mmol,1.05当量)，反应瓶置于0度冰水浴中10min后加入氯磷酸二苯酯(242μL,1.05mmol,1.05当量)，反应液于室温下搅拌3h，然后过滤除去三乙胺的盐酸盐，滤液减压蒸馏除去THF，所得剩余物通过制备型高效液相色谱【乙腈-水(含0.1％甲酸)，52-72％，0-35min】制备，得化合物杨梅素7-O-磷酸双(对氯苯基)酯32mg(得率5％)，化合物的质谱和波谱数据如下：ESI-MS m/z 619.11[M+H] +,617.09[M-H] -； 1H NMR(Acetone-d 6,500MHz)δ12.38(1H,s,5-OH),7.62(2H,dt,J＝1.4,8.3Hz,H-2a,6a),6.59(each 2H,dt,J＝1.6,8.1Hz,H-2b,6b),7.47(2H,s,H-2’,6’),7.5(2H,dt,J＝1.6,8.1Hz,H-3b,5b),7.34(2H,dt,J＝1.4,8.3Hz,H-3a,5a),7.17 and 6.74(each 1H,d,J＝1.4Hz,H-5,7)； 13C NMR(Acetone-d 6,125MHz)δ176.9(C-3),162.2(C-5),156.5,155.7(155.7),148.7,147.2,147.1,146.4,146.4,137.8,136.9,131.8,131.8,129.5,129.5,128.2,128.2,126.1,126.0,122.7,122.7,122.2,108.6(C-2’),108.6(C-5’),108.1,103.1(103.0),100.1(100.0)； 31P NMR(Acetone-d 6,125MHz)δ-5.9.
1.5合成杨梅素7-O-磷酸双苯酯(5)
Figure PCTCN2022078851-appb-000021
二氢杨梅素7-O-磷酸双苯酯(5)的合成参照杨梅素7-O-磷酸双苯酯(3)即得。化合物的质谱和波谱数据如下：ESI-MS m/z 553.3[M+H] +, 1H NMR(Acetone-d 6,500MHz)δ11.69(1H,s,5-OH),7.46(4H,dd,J＝7.5Hz,H-2a,6a；2b,6b),7.34(4H,dd,J＝7.5 and 1.1Hz,H-3a,5a；3b,5b),7.30(2H,dt,J＝8.5 and 1.1Hz,H-4a；4b),6.64(2H,s,H-2’,6’),6.46 and 6.44(each 1H,d,J＝2.1Hz,H-5,7),5.10 and 4.73(each 1H,d,J＝11.6Hz,H-2,H-3)； 13C NMR(Acetone-d 6,125MHz)δ199.9,164.2,163.8,158.6(158.5),151.3(151.2),146.4,146.5,134.4,131.0,131.0,131.0,131.0,128.5,126.9,126.9,126.9,121.1,121.1,120.9,120.9,108.1,108.1,105.4,101.7(101.6),100.6(100.5),85.0,73.4； 31P NMR(Acetone-d 6,125MHz)δ-18.7.
1.6合成二氢杨梅素7-O-磷酸双(对氯苯基)酯(6)
Figure PCTCN2022078851-appb-000022
二氢杨梅素7-O-磷酸双(对氯苯基)酯(6)的合成参照杨梅素7-O-磷酸双(对氯苯基)酯(4)即得，化合物的质谱和波谱数据如下：ESI-MS m/z 621.3[M+H] +, 1H NMR(Acetone-d 6,600MHz)δ11.70(1H,s,5-OH),7.59(4H,dd,J＝7.8Hz,H-2a,6a；2b,6b),7.45(2H,dt,J＝1.6,7.8Hz,H-3a,5a),7.34(2H,brt,J＝7.8Hz,H-3b,5b),6.64(2H,s,H-2’,6’),6.52 and 6.49(each 1H,d,J＝2.3Hz,H-5,7)； 13C NMR(Acetone-d 6,125MHz)δ200.0,164.2,163.8,158.2(158.2),147.1(147.1),146.4,146.4,146.4,134.5,131.9,131.9,129.6,129.6,128.5,128.3,128.3,126.1,126.0,122.7,122.7,108.2, 108.2,105.6,101.9(101.8),100.8(100.7),85.0,73.5； 31P NMR(Acetone-d 6,125MHz)δ-19.2.
实施例二、杨梅素磷酸酯类化合物对SARS-CoV-2 3CLpro抑制活性的测试
利用荧光共振能量转移方法评价测定杨梅素及其磷酸酯类化合物对SARS-CoV-2 3CLpro酶活的抑制活性。整个酶促反应体系的体积为120μL，蛋白酶的终浓度为30nM，底物终浓度为20μM。反应体系的缓冲液包括50mM Tris pH7.3、1mM EDTA。在96孔板中加入SARS-CoV-2 3CLpro蛋白酶和不同浓度的化合物，30℃孵育10min，加入底物并迅速放入酶标仪中读数。激发光和发射光分别为320nM和405nM。测试时间为3.5min，每隔35s读一次荧光值。最终结果取前2min的读值拟合出反应速率，并与对照组(DMSO)比较，计算抑制率。利用软件GraphPad Prism 8拟合得到IC 50值以及抑制率曲线。图1-4分别是杨梅素磷酸酯类化合物1-4对SARS-CoV-2 3CLpro的抑制曲线图。
将杨梅素磷酸酯类化合物对SARS-CoV-2 3CLpro抑制的IC 50值列在表中，实验结果如表1所示。
表1：杨梅素磷酸酯类化合物对SARS-CoV-2 3CLpro的抑制作用
Figure PCTCN2022078851-appb-000023
Figure PCTCN2022078851-appb-000024
*杨梅素对SARS-CoV-2 3CLpro抑制IC 50值引自专利CN202010153446.3
实施例三、杨梅素和杨梅素-7-O-磷酸双苯基酯(3)Caco-2细胞渗透性测试
Caco-2细胞用高糖DMEM培养基，置于37℃、5％CO2、空气相对湿度90％的培养箱中培养，培养基中添加10％胎牛血清、10mmol/L HEPES、1mmol/L丙酮酸钠、1％谷氨酰胺、1％非必需氨基酸、100U/mL青霉素和100μg/mL链霉素。每隔7天传一代，传代比率1:10。实验使用40至60代之间的细胞。培养21天以后，用TEER值检测细胞单层的紧密程度(须大于400Ω·cm 2)。同时考察从细胞顶层(A侧)到基底层(B侧)和B侧到A侧的药物转运。试验方法如下：用HBSS洗细胞三次以后，20μM的化合物以及化合物加抑制剂(GF120918)分别加到对应的细胞孔中(A侧pH6.8,B侧pH7.4)。在37℃培养箱中培养95分钟，分别在5分钟和95分钟时给药侧取样，在35分钟和95分钟时接收侧取样。用LC-MS/MS检测样品的浓度。测定被试化合物杨梅素和杨梅素-7-O-磷酸双苯基酯(3)的Papp和Efflux值。杨梅素和杨梅素磷酸酯类化合物(3)Caco-2细胞渗透性的测试结果如表2所示。
表2：杨梅素和杨梅素-7-O-磷酸双苯基酯(3)细胞渗透性测试结果
Figure PCTCN2022078851-appb-000025
渗透性判定标准：Fab％＞70％，渗透性较好；30％<Fab％<70％，渗透性中等；Fab％<30％，渗透性较差。
实验结果显示在杨梅素结构中引入前药基团磷酸酯基明显提高了化合物的渗透性。
实施例四、二氢杨梅素及其磷酸酯前药的药代动力学参数比较
1、色谱条件
色谱柱：ACQUITY UPLC BEH Phenyl(2.1*50mm，1.7μm)
流速：0.5mL/min
A：0.1％FA in H2O
B：ACN
2、给药方案
ICR小鼠6只，雄性，体重18-22g，分别随机分成2组，每组各3只。按照如下方案灌胃给予受试化合物。
试验前禁食12h，自由饮水。给药后2h统一进食。
3、实验分组、采血时间点及样品处理
每个时间点3只动物，分组及采血时间点见下表：
Figure PCTCN2022078851-appb-000026
Figure PCTCN2022078851-appb-000027
灌胃给药溶液以DMSO/0.5％HPMC(5/95,v/v)配制到最终浓度。给药溶液留样(分别在给药前及给药后，取50μL药液与50μL DMSO混匀)待测。
按上述给药剂量给予药物，记录给药时间，并在以上设定的时间点经小鼠股脉丛取血40μL，置肝素化试管中。立即于4摄氏度下离心5min(转速11000rpm)，之后立即准确吸取20μL血浆并加入2μL的20％维生素C水溶液，再加入200μL的乙腈(含1％甲酸)溶液沉淀混匀，冻存于-60℃待测。
4、标准溶液的配制
分别精密称取二氢杨梅素对照品适量，用DMSO溶解稀释配制成二氢杨梅素浓度为3、10、30、100、300、1000、3000和10000ng/mL的标准系列溶液，4℃冰箱保存备用。
5、血浆样品标准曲线的绘制
取小鼠空白血浆18μL，加入二氢杨梅素标准系列溶液2μL和2μL的20％维生素C水溶液，加入加入200μL的乙腈(含1％甲酸)溶液沉淀混匀.于4℃下离心5min(转速11000rpm)，冻存于-60℃待测。
6、血药浓度数据处理
运用WinNonlin 6.4软件对血药浓度数据进行分析处理，采用非房室模型计算药动学参数，实验结果如表3所示。
表3：二氢杨梅素与二氢杨梅素7-O-磷酸二苯基酯(5)口服PK参数比较
Figure PCTCN2022078851-appb-000028
Figure PCTCN2022078851-appb-000029
实验结果表明，二氢杨梅素的磷酸酯前药二氢杨梅素7-O-磷酸二苯基酯(5)在30mg/kg的口服剂量下，其释放出来的二氢杨梅素最大血药浓度(C max)和药时曲线下面积(AUC last)与二氢杨梅素以100mg/kg口服形式给药所达到的最大血药浓度(C max)和药时曲线下面积(AUC last)相当。
因此，在二氢杨梅素结构中引入前药基团磷酸酯基明显提高了化合物的暴露量。
实施例五、杨梅素和杨梅素-7-O-磷酸双苯基酯(3)、二氢杨梅素和二氢杨梅素-7-O-磷酸双苯基酯(5)对2019新型冠状病毒复制抑制活性评价
将Vero E6细胞提前一天铺于48孔板中(50000/孔)，随后每孔加入不同浓度的小分子化合物，1小时后以MOI＝0.01的感染复数加入SARS-CoV-2，孵育一小时后吸走上清，并加入含不同浓度小分子化合物的培养基。感染24小时后，取上清，提取上清中病毒RNA并进行反转，随后利用实时荧光定量PCR方法对上清中病毒RNA拷贝数进行定量，计算不同浓度化合物对病毒RNA的抑制率，利用软件GraphPad Prism 8计算IC 50值并拟合抑制曲线图。杨梅素和杨梅素磷酸酯类化合物(3)、二氢杨梅素和二氢杨梅素磷酸酯(5)对2019新型冠状病毒复制抑制活性测试结果如表3所示，杨梅素及其磷酸酯化合物3、二氢杨梅素及其磷酸酯化合物5对2019新型冠状病毒复制抑制的曲线图分别如图5-8所示。
表3：杨梅素和杨梅素-7-O-磷酸双苯基酯(3)对2019新型冠状病毒复制抑制活性测试结果
Figure PCTCN2022078851-appb-000030
Figure PCTCN2022078851-appb-000031
实验结果显示在杨梅素、二氢杨梅素结构中引入前药基团磷酸酯基有助于提高化合物对新型冠状病毒复制抑制活性。
讨论
在化合物中引入磷酸酯或磷酸及其钠盐作为前药在药物研究与开发中是一个极其重要的策略。在前述发明中，我们发现杨梅素和二氢杨梅素对新冠病毒主蛋白酶SARS-CoV-2 3CLpro有明显的抑制作用，IC 50值分别为0.64和1.14μM。但由于杨梅素结构上有6个羟基，造成杨梅素渗透性仅为中等。本发明通过在杨梅素的结构中引入磷酸酯基团，意外的发现所得杨梅素磷酸酯，尤其是杨梅素7-O-磷酸二苯基酯的渗透性有了明显的改善，化合物对细胞膜的通透性得到了提高，从而提高了化合物对新型冠状病毒复制抑制活性。对于二氢杨梅素，本发明通过在二氢杨梅素的结构中引入磷酸酯基团，意外的发现所得二氢杨梅素磷酸酯，尤其是二氢杨梅素7-O-磷酸二苯基酯的口服暴露量和对新型冠状病毒复制抑制活性均有了明显的改善。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种活性成分或含所述活性成分的制剂的用途，其特征在于，所述的活性成分为式I所示化合物、或其药学上可接受的盐、或其溶剂化物或其前药；
    Figure PCTCN2022078851-appb-100001
    式中，
    Figure PCTCN2022078851-appb-100002
    为单键或双键；
    R 1和R 2各自独立地选自：OH、-O-C 1-C 6烷基、-O-C 3-C 6环烷基、
    Figure PCTCN2022078851-appb-100003
    Figure PCTCN2022078851-appb-100004
    且R 1、R 2中至少一个为
    Figure PCTCN2022078851-appb-100005
    R 8、R 9各自独立地选自：H、取代或未取代的C 1-C 6烷基、取代或未取代的C 3-C 6环烷基、取代或未取代的苯基；其中，所述的取代表示被一个或多个选自下组的取代基取代：卤素、C1-C3烷基、C1-C3卤代烷基、C3－C6环烷基；
    或者R 8、R 9与相连的－O-P-O－共同构成取代或未取代的6-7元杂环，所述的杂环含有2个O杂原子、1个P杂原子和0-1个N杂原子，所述的取代表示被一个或多个选自下组的取代基取代：卤素、C1-C3烷基、C1-C3卤代烷基、C3－C6环烷基、苄基、卤代苄基、烷(氧)基取代的苄基；
    并且，所述的活性成分或含所述活性成分的药物组合物的制剂被用于制备(a)抑制新型冠状病毒3CL蛋白酶活性的抑制剂；和/或(b)治疗和/或预防、缓解由新型冠状病毒感染引起的相关疾病的药物。
  2. 如权利要求1所述的用途，所述由新型冠状病毒感染引起的相关疾病选自下组：呼吸道感染、肺炎及其并发症、或其组合。
  3. 如权利要求1所述的用途，其特征在于，当式I中
    Figure PCTCN2022078851-appb-100006
    为单键，所述活性成分为如式II所示化合物或其溶剂化物或其前药或其组合物：
    Figure PCTCN2022078851-appb-100007
    式中，R 1、R 2如上定义。
  4. 如权利要求1所述的用途，其特征在于，所述的活性成分选自下组：
    Figure PCTCN2022078851-appb-100008
    A7 A1-A6的组合。
  5. 如权利要求1所述的用途，其特征在于，所述的制剂包括：口服制剂和非口服制剂。
  6. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (a1)第一活性成分，所述第一活性成分为式I所示化合物或其溶剂化物或其前药或其组合物；
    (a2)任选的第二活性成分，所述的第二活性成分为抗病毒药物，其选自下组：干扰素、RNA依赖的RNA聚合酶抑制剂(如Remdesivir(瑞德西韦或GS-5734)、法匹拉韦(favipiravir)、Galidesivir、GS-441524)；3CL蛋白酶抑制剂(如GC-376)、洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、奈非那韦(Nelfinavir)；氯喹(Chloroquine,Sigma-C6628)、羟氯喹(hydroxychloroquine)、环孢菌素(cyclosporine)、可利霉素(Carrimycin)、黄芩苷(baicalin)、黄芩素(baicalein)、 萘酚喹(Naphthoquine)、环索奈德(Ciclesonide)、利巴韦林(Ribavirin)、喷昔洛韦(Penciclovir)、来氟米特(Leflunomide)、特立氟胺(Teriflunomide)、萘莫司他(nafamostat)、硝唑尼特(nitazoxanide)、达芦那韦(Darunavir)、阿比多尔(Arbidol)、卡莫司他(Camostat)、氯硝柳胺(Niclosamide)、巴瑞替尼(baricitinib)、芦可替尼(Ruxolitinib)、达沙替尼(Dasatinib)、沙奎那韦(Saquinavir)、Beclabuvir、司美匹韦(Simeprevir)、或其药学上可接受的盐、或其组合；
    和/或所述的第二活性成分选自下组：锌(Zinc)、芬戈莫德(Fingolimod)、维生素C(Vitamin C)、奥美沙坦酯(Olmesartan Medoxomil)、缬沙坦(valsartan)、氯沙坦(Losartan)、沙利度胺(Thalidomide)、甘草酸(glycyrrhizic acid)、青蒿素(Artemisinin)、双氢青蒿素(dihydroartemisinin)、青蒿琥酯(Artesunate)、青蒿酮(Artemisone)、阿奇霉素(Azithromycin)、七叶皂苷(Escin)、萘普生(Naproxen)、或其组合；
    及(b)药学上可接受的载体。
  7. 一种权利要求6所述的药物组合物的用途，其特征在于，用于制备(a)抑制新型冠状病毒3CL蛋白酶抑制剂；和/或(b)治疗和/或预防、缓解由新型冠状病毒感染引起的相关疾病的药物。
  8. 一种活性成分，其特征在于，所述活性成分为式II所示的化合物或其溶剂化物或其前药或其组合物，其中式II如上定义。
  9. 一种体外的抑制新型冠状病毒3CL蛋白酶的方法，其特征在于，包括步骤：
    将第一活性成分或含所述第一活性成分的制剂与冠状病毒(SARS-CoV-2)接触，抑制所述冠状病毒3CL蛋白酶的活性，从而抑制冠状病毒的复制；
    其中，所述的第一活性成分为杨梅素磷酸酯类化合物或其溶剂化物或其组合物。
  10. 如权利要求9所述的方法，其特征在于，所述的第一活性成分选自下组：
    Figure PCTCN2022078851-appb-100009
    Figure PCTCN2022078851-appb-100010
    A7 A1-A6的组合。
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