WO2025201575A2 - 一类抗新型冠状病毒的芳基萘木脂素类化合物及其制备方法和应用 - Google Patents

一类抗新型冠状病毒的芳基萘木脂素类化合物及其制备方法和应用

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WO2025201575A2
WO2025201575A2 PCT/CN2025/096691 CN2025096691W WO2025201575A2 WO 2025201575 A2 WO2025201575 A2 WO 2025201575A2 CN 2025096691 W CN2025096691 W CN 2025096691W WO 2025201575 A2 WO2025201575 A2 WO 2025201575A2
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group
anl
substituted
cov
independently selected
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French (fr)
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WO2025201575A3 (zh
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胡英杰
张宏杰
郑永唐
朱轩
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Guangzhou University Of Chinese Medicine Guangzhou Institute Of Chinese Medicine
Kunming Institute of Zoology of CAS
University of Hong Kong HKU
Hong Kong Baptist University HKBU
Centre for Virology Vaccinology and Therapeutics Ltd
Original Assignee
Guangzhou University Of Chinese Medicine Guangzhou Institute Of Chinese Medicine
Kunming Institute of Zoology of CAS
University of Hong Kong HKU
Hong Kong Baptist University HKBU
Centre for Virology Vaccinology and Therapeutics Ltd
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Publication of WO2025201575A2 publication Critical patent/WO2025201575A2/zh
Publication of WO2025201575A3 publication Critical patent/WO2025201575A3/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
    • A61K31/365—Lactones
    • 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
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C07H15/252—Naphthacene radicals, e.g. daunomycins, adriamycins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms

Definitions

  • the present invention relates to a class of inhibitors of the new coronavirus, and specifically to a class of arylnaphthyl lignan compounds with anti-new coronavirus activity, as well as a preparation method and application thereof.
  • Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses belonging to the families Coronaviridae, Arteriviridae, and Roniviridae.
  • SARS-CoV-2 the virus that causes the current COVID-19 pandemic, is a beta-coronavirus.
  • the possibility of mutating SARS-CoV-2 into a more pathogenic variant cannot be ruled out. Therefore, highly effective viral inhibitors are urgently needed to combat coronaviruses.
  • oral small molecule drugs offer advantages such as low cost, ease of production, convenient transportation, and ease of use. They are a key focus in the development of therapeutics against the novel coronavirus, which is likely to persist in humans for a long time.
  • the main targets include the pathogen's spike protein (S protein), E protein (envelope protein), M membrane protein, N nucleocapsid protein, and various enzymes involved in viral replication; there are also host targets such as ACE2.
  • Remdesivir is the first small molecule drug to treat COVID-19, approved by the US FDA in October 2020.
  • Remdesivir is a nucleoside analog prodrug that is metabolized to its triphosphate form in the body and specifically inhibits the RNA-dependent RNA polymerase (RdRp) to exert its antiviral activity.
  • RdRp RNA-dependent RNA polymerase
  • Merck's Monovaccin and Pfizer's Paxlovid received emergency authorization from the FDA for the treatment of COVID-19 in December 2021.
  • Molnupiravir is a small molecule RdRp inhibitor with a chemical structure similar to that of remdesivir. It is effective against gamma and delta variants of the new coronavirus and can effectively reduce the mortality rate.
  • Paxlovid is composed of two small molecules, one is the M pro inhibitor Nirmatrelvir (PF-07321332), and the other is ritonavir, an oral bioavailability enhancer of Nirmatrelvir.
  • the drug contains 20 tablets of 150mg Nirmatrelvir and 10 tablets of 100mg ritonavir. It has quickly become a first-line prescription drug worldwide, replacing the use of remdesivir, but the price is higher.
  • Azvudine (2'-deoxy-2'- ⁇ -fluoro-4'-azidocytidine;FNC; C 9 H 11 FN 6 O 4 ), developed by Professor Chang Junbiao and others at Zhengzhou University in China, is a small molecule RdRp inhibitor. Azvudine was approved by the National Medical Products Administration (NMPA) on July 25, 2022, becoming China's first oral medication approved for emergency conditional treatment of COVID-19. Azvudine, a nucleoside analog, was recently approved by the NMPA as a new anti-HIV-1 drug (in July 2021) with specific dual inhibitory effects (dual targeting) on HIV-1 reverse transcriptase (RT) and accessory protein (Vif).
  • NMPA National Medical Products Administration
  • the object of the present invention is to provide a class of aromatic naphthyl lignan compounds that are resistant to the new coronavirus, as well as a preparation method and application thereof.
  • the present invention discloses a novel class of arylnaphthalene analogs, the preparation of related compounds and novel intermediates, and their use in treating novel coronavirus infections such as SARS-CoV-2.
  • the first aspect of the present invention is an aryl naphthyl lignan compound or a pharmaceutically acceptable salt or prodrug thereof for treating, preventing, or delaying the progression of infection with a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV), wherein the compound has formula (I):
  • X is oxygen or sulfur
  • R 1 is R 15 , -OR 15 , -C(O)R 15 , or -C(O)OR 15 ;
  • R 2 , R 5 , R 6 , R 10 , R 13 and R 14 are each hydrogen or halogen;
  • R 7 , R 8 and R 9 are each independently selected from -OR 15 and -OC(O)R 15 ; or R 7 and R 8 , together with the carbon atoms to which they are attached, form a 5-6 membered heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 ; or R 8 and R 9 , together with the carbon atoms to which they are attached, form a 5-6 membered heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 ;
  • R 11 and R 12 together form an oxo group; or when one of R 11 and R 12 is hydrogen or halogen, the other of R 11 and R 12 is selected from R 15 , -OR 15 , -C(O)R 15 and -C(O)OR 15 ;
  • R 15 is, at each occurrence, independently selected from hydrogen, alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, heteroaryl, -OR 17 , -C(O)R 18 , -C(O)N(R 17 )R 18 , -C(O)OR 17 , -OC(O)R 17 , -S(O) 2 R 17 , -S(O) 2 N(R 17 )R 18 , -N ⁇ C(R 17 )R 18 , -N(R 17 )R 18 , -N(R 17 )N(R 17 )R 18 , -N(R 17 )C(O)R 18 , -N(R 17 )S(O) 2 R 18 , 1,3,2-dioxaborolane optionally substituted by 1, 2, 3 or 4 groups independently selected from alkyl, a glycoside group, an alkynyl group optionally substituted by a trial
  • R 17 and R 18 are independently at each occurrence hydrogen, alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or are selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted with 1, 2, 3, 4, or 5 groups independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl, and C 1-6 alkoxy;
  • the glycosidic groups are typically carbohydrates, particularly monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or polysaccharides, and may exist in various isomeric forms, such as ⁇ -D, ⁇ -L, ⁇ -D, or ⁇ -L.
  • the carbohydrate groups may optionally be substituted with other types of substituents or even additional glycosidic groups. However, the total number of monosaccharide groups and substituted monosaccharide groups contained in the chemical structure of the compound may not exceed 10.
  • the glycosidic group may be a group of formula (i) or (ii):
  • R 19 and R 20 may together form an oxo group; or when one of R 19 and R 20 is hydrogen or halogen, the other of R 19 and R 20 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group, a substituted disaccharide group, a trisaccharide group, a substituted trisaccharide group, a tetrasaccharide group, and a substituted tetrasaccharide group;
  • R 21 and R 22 may together form an oxo group; or when one of R 21 and R 22 is hydrogen or halogen, the other of R 21 and R 22 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group, a substituted disaccharide group, a trisaccharide group, a substituted trisaccharide group, a tetrasaccharide group, and a substituted tetrasaccharide group;
  • R 23 and R 24 may together form an oxo group; or when one of R 23 and R 24 is hydrogen or halogen, the other of R 23 and R 24 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group, a substituted disaccharide group, a trisaccharide group, a substituted trisaccharide group, a tetrasaccharide group, and a substituted tetrasaccharide group;
  • R 25 and R 26 may together form an oxo group; or when one of R 25 and R 26 is hydrogen or halogen, the other of R 25 and R 26 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , -CH 2 R 27 and -C(O)R 27 ;
  • R 27 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, alkyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 , -(CH 2 ) k -heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 , -OR 17 , -C( O ) R 18 , -C(O)N(R 17 )R 18 , -C(O)OR 17 , -OC(O)R 17 , -S(O) 2 R 17 , -S(O) 2 N (R 17 )R 18 , -N(R 17 )R 18 , -N(R 17 )N(R 17 )R 18 , -N(R 17 )C(O)R 18 , -N(R 17 )S(O) 2 R 18 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group
  • the second aspect of the present invention is a method for preparing ANL-2, and the preparation reaction formula is as follows:
  • the preparation method of ANL-2 comprises the following steps:
  • acetal (2) is prepared by ethylene glycol protection, (2) reacts with piperonal to obtain compound (3), compound (3) is rapidly converted into isobenzofuran by heating in the presence of acetic acid, and then a diester (4) is obtained by Diels-Alder reaction, which is reduced to obtain salvinol (5); salvinol (5) is then reacted with trifluoromethanesulfonic anhydride to obtain product (6); product (6) reacts with trimethylethynylsilane to obtain product (7); product (7) is reacted in the presence of potassium carbonate to obtain the target product ANL-2.
  • the specific steps include:
  • Ethylene glycol and p-toluenesulfonic acid are added to a toluene solution of 2-bromo-4,5-dimethoxybenzaldehyde (1), and the mixture is heated to 140°C overnight; the mixture is cooled to room temperature, and the toluene is removed by evaporation under reduced pressure; the reaction mixture is dissolved in ethyl acetate, and extracted with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution in sequence, and the organic phase is dried over anhydrous sodium sulfate; the crude product is evaporated to dryness under reduced pressure, and then subjected to silica gel column chromatography to obtain the acetal intermediate (2);
  • the molar ratio of bromo-4,5-dimethoxybenzaldehyde, ethylene glycol, and p-toluenesulfonic acid is 1 ⁇ 0.5:2 ⁇ 1:0.2 ⁇ 0.1; further 1:2:0.2;
  • the cooling is cooling to -70°C to -80°C; further cooling to -78°C;
  • the stirring time is 10-60 minutes; further 30 minutes;
  • the molar ratio of the intermediate (3) to dimethyl butynedioate is 1 ⁇ 0.5:1 ⁇ 0.5; further 1:1;
  • the reaction conditions are reflux at 100°C-160°C for 6-20 hours; further reflux at 140°C for 12 hours.
  • the molar ratio of the diester compound intermediate (4) to sodium borohydride is 1 ⁇ 0.5:5 ⁇ 2.5; further 1:5;
  • the heating reflux condition is heating reflux at 60°C-100°C for 6-20 hours; further heating reflux at 80°C for 12 hours;
  • the hydrochloric acid is 1-5M hydrochloric acid; further 2M hydrochloric acid;
  • the molar ratio of the schizofiarin (5), 4-dimethylaminopyridine and trifluoromethanesulfonic anhydride is 1 ⁇ 0.5:2 ⁇ 1:1.5 ⁇ 0.5; further 1:2:1.2;
  • the cooling is cooling to -5°C to 5°C; further to 0°C;
  • the stirring time is 2-8 hours; further 4 hours.
  • the heating and stirring conditions are stirring at 50°C-100°C for 5-16 hours; further stirring at 80°C for 10 hours.
  • the molar ratio of ANL-4 to allyl chloroformate is 1 ⁇ 0.1:2.5 ⁇ 0.5; further 1:2.
  • the treatment conditions are to use triethylamine Et3N and dimethylaminopyridine DMAP as catalysts.
  • the reaction temperature is -5°C to 40°C; further, 0°C to room temperature.
  • the room temperature refers to 20 to 30°C.
  • the seventh aspect of the present invention is a pharmaceutical preparation comprising an alkynylaryl naphthyl lignan compound or a pharmaceutically acceptable salt or prodrug thereof, for treating, preventing, or delaying the progression of a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV) infection in a patient.
  • a novel coronavirus including SARS-CoV-1, SARS-CoV-2, or MERS-CoV
  • the ninth aspect of the present invention is a pharmaceutical preparation comprising a schizofiarin analogue or a pharmaceutically acceptable salt or prodrug thereof, for treating, preventing, or delaying the progression of a novel coronavirus (including SARS-CoV-1, SARS-CoV-2, or MERS-CoV) infection in a patient.
  • a novel coronavirus including SARS-CoV-1, SARS-CoV-2, or MERS-CoV
  • the tenth aspect of the present invention is a pharmaceutical formulation comprising an ANL-2 analogue or a pharmaceutically acceptable salt or prodrug thereof for use in treating, preventing or delaying the progression of a viral infection in a patient.
  • the novel coronavirus described in the present invention refers, in a broad sense, to a coronavirus that can cause severe symptoms, is lethal, and is contagious, including but not limited to at least one of the viruses that cause severe acute respiratory syndrome, Middle East respiratory syndrome, and the novel coronavirus disease COVID-19.
  • the novel coronavirus includes but is not limited to at least one of SARS-CoV-1 or SARS-CoV-2 or MERS-CoV or SARS-CoV-2 Omicron strain.
  • aspects of the present invention relate to providing methods for synthesizing ANL-2, ANL-3, ANL-4, ANL-5, ANL-6 or ANL-7 compounds and intermediate compounds during the synthesis.
  • the present invention relates to intermediate compounds that can be used to prepare the compounds of the present invention.
  • the degree of protection includes counterfeit or fraudulent products that contain or purport to contain compounds of the invention, regardless of whether they actually contain such compounds and regardless of whether any such compounds are contained in a therapeutically effective amount.
  • packages that include descriptions or instructions indicating that the package contains a species or pharmaceutical formulation of the present invention, as well as products that are or contain, or purport to be or contain, such formulations or species.
  • packages may, but are not necessarily, counterfeit or false.
  • FIG2 is a high-resolution mass spectrum (HR-ESIMS) of ANL-2 provided in Example 2 of the present invention
  • FIG4 is a 13 C nuclear magnetic resonance ( 13 C NMR) spectrum of ANL-2 provided in Example 2 of the present invention.
  • FIG5 is a 1 H nuclear magnetic resonance ( 1 H NMR) spectrum of ANL-3 provided in Example 3 of the present invention.
  • FIG8 is a 13 C nuclear magnetic resonance ( 13 C NMR) spectrum of ANL-4 provided in Example 4 of the present invention.
  • FIG9 is a 1 H nuclear magnetic resonance ( 1 H NMR) spectrum of ANL-5 provided in Example 5 of the present invention.
  • FIG11 is a 1 H nuclear magnetic resonance ( 1 H NMR) spectrum of ANL-6 provided in Example 5 of the present invention.
  • FIG12 is a 13 C nuclear magnetic resonance ( 13 C NMR) spectrum of ANL-6 provided in Example 5 of the present invention.
  • FIG13 is a 1 H nuclear magnetic resonance ( 1 H NMR) spectrum of ANL-7 provided in Example 5 of the present invention.
  • FIG14 is a 13 C nuclear magnetic resonance ( 13 C NMR) spectrum of ANL-7 provided in Example 5 of the present invention.
  • FIG15 shows the effect of ANL-2 on Caco2 cell viability provided in Example 6 of the present invention
  • Figure 16 shows the effect of ANL-2 provided in Example 6 of the present invention on viral gene copies of Caco2 cells infected with SARS-CoV-1, MERS-CoV, SARS-CoV-2 wild type or BA.5.2 variant;
  • Figure 17 shows the RdRp gene copy number in the lung and nasal turbinate tissues of hamsters infected with SARS-CoV-2 WT virus as provided in Example 6 of the present invention
  • FIG21 is a blood drug concentration-time curve of ANL-2 fat emulsion administered by tail vein to three groups of rats provided in Example 9 of the present invention.
  • FIG22 is a blood drug concentration-time curve of three groups of rats orally administered with ANL-2 fat emulsion provided in Example 9 of the present invention.
  • subject refers to an individual.
  • subjects may include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, hamsters, etc.), and birds.
  • Subjects may also include mammals, such as primates or humans.
  • reduce is meant a decrease in an event or characteristic (e.g., tumor growth). It will be understood that this is usually relative to some standard or expected value, in other words, it is relative, but does not always need to refer to that standard or relative value.
  • reduced tumor growth means a decrease in the rate of tumor growth relative to a standard or control.
  • treating is meant administering a composition or performing a method to reduce, prevent, inhibit, or eliminate a particular characteristic or event (eg, tumor growth or survival).
  • controlling is used synonymously with the term “treating.”
  • terapéuticaally effective means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only requires reduction or modification, not elimination.
  • Such salts include: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxyphenyl)-1-ol benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,
  • salts include sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids such as hydrohalides (e.g., hydrochloride and hydrobromide), sulfates, phosphates, sulfamates, nitrates, acetates, trifluoroacetates, trichloroacetates, propionates, hexanoates, cyclopentylpropionates, glycolates, glutarates, pyruvates, lactates, malonates, succinates, sorbates, ascorbates, malate, maleates; fumarates, tartrates, citrates, benzoates, 3-(4-hydroxybenzoyl)benzoate, picrates, cinnamates, mandelates, phthalates.
  • hydrohalides e.g., hydrochloride and hydrobromid
  • NNL aryl naphthalene lignan
  • arylnaphthalene lignan compounds are interchangeable.
  • aryl naphthalene lignan or “arylnaphthalene lignan” or “ANL” includes reference to compounds containing the base structure of 2,3-dimethyl-1-phenyl-naphthalene as shown below:
  • carbon numbering of arylnaphthyl lignan molecules includes reference to compounds comprising the numbering system shown below:
  • the carbon numbering of the arylnaphthyl lignan side chains includes reference to compounds comprising the numbering system shown below:
  • alkenyl includes reference to straight or branched chain alkyl moieties having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and additionally having at least one double bond, and, where applicable, E or Z stereochemistry.
  • the term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl, and 3-hexenyl.
  • alkynyl includes references to straight or branched chain alkyl moieties having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and additionally having at least one triple bond.
  • the term includes references to groups such as ethynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, and 3-hexynyl.
  • alkoxy and C 1-6 alkoxy include references to -O-alkyl groups, wherein the alkyl group is straight or branched and contains 1, 2, 3, 4, 5, or 6 carbon atoms. In one class of embodiments, the alkoxy group has 1, 2, 3, or 4 carbon atoms.
  • the term includes references to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.
  • cycloalkyl includes references to alicyclic moieties having 3, 4, 5, 6, 7, or 8 carbon atoms. Such groups may be bridged or polycyclic ring systems. More common cycloalkyl groups are monocyclic. The term includes references to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and bicyclo[2.2.2]octyl.
  • aryl includes reference to aromatic ring systems containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring carbon atoms.
  • Aryl is typically phenyl, but can be a polycyclic ring system having two or more rings, at least one of which is aromatic.
  • the term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthracenyl, and the like.
  • Cyclic group means a ring or ring system which may be unsaturated or partially unsaturated, but is typically saturated, and typically contains 5 to 13 ring atoms, such as a 5- or 6-membered ring. It includes carbocyclyl and heterocyclyl moieties.
  • carbocyclyl includes reference to saturated (e.g., cycloalkyl) or unsaturated (e.g., aryl) ring moieties having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon ring atoms.
  • carbocyclyl includes 3- to 10-membered rings or ring systems, particularly 5- or 6-membered rings, which may be saturated or unsaturated.
  • the carbocyclyl moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthracenyl, and the like.
  • heterocyclyl includes reference to saturated (e.g., heterocycloalkyl) or unsaturated (e.g., heteroaryl) heterocyclic moieties having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, at least one of which is selected from boron, nitrogen, oxygen, phosphorus, silicon, and sulfur.
  • heterocyclyl includes 3- to 10-membered rings or ring systems, more particularly 5- or 6-membered rings, which may be saturated or unsaturated.
  • the heterocyclic moiety is selected, for example, from oxiranyl, azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furanyl, tetrahydrofuranyl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl
  • heterocycloalkyl includes reference to saturated heterocyclic moieties having 3, 4, 5, 6, or 7 ring carbon atoms and 1, 2, 3, 4, or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur.
  • the group can be a polycyclic ring system, but is more typically a monocyclic ring.
  • heteroaryl includes references to aromatic heterocyclic ring systems having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, and sulfur.
  • the group may be a polycyclic ring system having two or more rings, at least one of which is aromatic, but is more typically a monocyclic ring.
  • the term includes references to groups such as pyrimidinyl, furanyl, benzothienyl, thienyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridyl, benzofuranyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolyl, quinazolinyl, pteridinyl, and the like.
  • halogen includes reference to F, Cl, Br, or I.
  • halogen-containing moiety includes reference to a moiety containing from 1 to 30 polyvalent atoms selected from carbon, nitrogen, oxygen, and sulfur, which moiety includes at least one halogen.
  • the moiety may be a hydrocarbon group, such as a C 1-6 alkyl group or a C 1-6 alkoxy group, or a carbocyclic group, such as an aryl group.
  • substituted with reference to a moiety means that one or more, in particular up to 5, more particularly 1, 2 or 3 hydrogen atoms in the moiety are replaced independently of one another by the corresponding number of substituents described.
  • optionally substituted means substituted or unsubstituted.
  • substituents are only in chemically possible positions, and a person skilled in the art will be able to determine (experimentally or theoretically) whether a particular substitution is possible without undue effort.
  • enantiomer means one of two stereoisomers that are mirror images of one another.
  • stereoisomer refers to a class of isomeric molecules that have the same molecular formula and sequence of bonded atoms but different three-dimensional orientations of their atoms in space.
  • tautomer refers to isomeric molecules that are readily interconvertible via chemical reactions.
  • the reaction typically results in the migration of a hydrogen atom, which results in the switching of a single bond and an adjacent double bond.
  • a prodrug is a drug that is administered as an inactive (or less than fully active) chemical derivative that is subsequently converted in vivo to the active agent, typically by normal metabolic processes.
  • CC50 is a measure of cytotoxicity at the concentration of the test drug that inhibits cell growth by 50%.
  • EC50 or IC50 is a measure of antiviral activity at the effective concentration of the test drug that inhibits 50% of viral growth.
  • SI selectivity index
  • the symbol “ ⁇ ” in a chemical structure indicates that the bond is attached above (or in front of) the plane of the paper or screen.
  • the symbol “ ⁇ ” in a chemical structure indicates that the bond is attached below (or behind) the plane of the paper or screen.
  • a solid wedge in a chemical structure indicates that the bond is above (or in front of) the plane of the paper or screen toward the viewer.
  • a dashed (or broken) wedge in a chemical structure indicates that the bond is below (or behind) the plane of the paper or screen away from the viewer.
  • a compound and its use in the manufacture of a medicament for treating a viral infection as well as a method of using the compound to treat a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound, wherein the compound has formula (I):
  • X is oxygen or sulfur
  • R 1 is R 15 , -OR 15 , -C(O)R 15 , or -C(O)OR 15 ;
  • R 2 , R 5 , R 6 , R 10 , R 13 and R 14 are each independently hydrogen or halogen;
  • R 3 and R 4 are each independently selected from -OR 15 and -OC(O)R 15 ; or R 3 and R 4 together with the carbon atoms to which they are attached form a 5-6 membered heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 ;
  • R 7 , R 8 and R 9 are each independently selected from -OR 15 and -OC(O)R 15 ; or R 7 and R 8 , together with the carbon atoms to which they are attached, form a 5-6 membered heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 ; or R 8 and R 9 , together with the carbon atoms to which they are attached, form a 5-6 membered heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 ;
  • R 11 and R 12 together form an oxo group; or when one of R 11 and R 12 is hydrogen or halogen, the other of R 11 and R 12 is selected from R 15 , -OR 15 , -C(O)R 15 and -C(O)OR 15 ;
  • R 15 is, at each occurrence, independently selected from hydrogen, alkynyl, halogen, trichloromethyl, trifluoromethyl, cyano, nitro, heteroaryl, -OR 17 , -C(O)R 18 , -C(O)N(R 17 )R 18 , -C(O)OR 17 , -OC(O)R 17 , -S(O) 2 R 17 , -S(O) 2 N(R 17 )R 18 , -N ⁇ C(R 17 )R 18 , -N(R 17 )R 18 , -N(R 17 )N(R 17 )R 18 , -N(R 17 )C(O)R 18 , -N(R 17 )S(O) 2 R 18 , 1,3,2-dioxaborolane optionally substituted by 1, 2, 3, or 4 groups independently selected from alkyl, a glycoside group, an alkynyl group optionally substituted by a trial
  • R 17 and R 18 are independently at each occurrence hydrogen, alkyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or are selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from halogen, trichloromethyl, trifluoromethyl, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy.
  • the glycosidic groups are typically carbohydrates, particularly monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or polysaccharides, and may exist in various isomeric forms, such as ⁇ -D, ⁇ -L, ⁇ -D, or ⁇ -L.
  • the carbohydrate groups may optionally be substituted with other types of substituents or even additional glycosidic groups. However, the total number of monosaccharide groups and substituted monosaccharide groups contained in the chemical structure of the compound may not exceed 10.
  • the glycosidic group may be a group of formula (i) or (ii):
  • R 19 and R 20 may together form an oxo group; or when one of R 19 and R 20 is hydrogen or halogen, the other of R 19 and R 20 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group, a substituted disaccharide group, a trisaccharide group, a substituted trisaccharide group, a tetrasaccharide group, and a substituted tetrasaccharide group;
  • R 23 and R 24 may together form an oxo group; or when one of R 23 and R 24 is hydrogen or halogen, the other of R 23 and R 24 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group, a substituted disaccharide group, a trisaccharide group, a substituted trisaccharide group, a tetrasaccharide group, and a substituted tetrasaccharide group;
  • R 27 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, alkyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 , -(CH 2 ) k -heterocyclyl optionally substituted by 1, 2, 3, 4 or 5 groups independently selected from R 16 , -OR 17 , -C( O ) R 18 , -C(O)N(R 17 )R 18 , -C(O)OR 17 , -OC(O)R 17 , -S(O) 2 R 17 , -S(O) 2 N (R 17 )R 18 , -N(R 17 )R 18 , -N(R 17 )N(R 17 )R 18 , -N(R 17 )C(O)R 18 , -N(R 17 )S(O) 2 R 18 , a monosaccharide group, a substituted monosaccharide group, a disaccharide group
  • the compound of formula (I) is a compound selected from ANL-2:
  • the compound of formula (I) is a compound selected from ANL-3, ANL-4, ANL-5, ANL-6, or ANL-7:
  • R 1 is hydrogen, alkyl, aryl, heteroaryl (such as furan, thiophene, pyridine, etc.), -N(R 17 )R 18 , -N ⁇ C(R 17 )(R 18 ), alkynyl optionally substituted with a trialkylsilane, a glycoside group, 1,3,2-dioxaborolane optionally substituted with 1, 2, 3, or 4 groups independently selected from alkyl, or -O-(CR 2 ) m -R 16 , wherein m is an integer selected from 1-4, 1-3, or 1-2; R is independently hydrogen, alkyl, cycloalkyl, or aryl at each occurrence; and R 16 is alkynyl, cyano, -OR 17 , -N(R 17 )R 18 , -C(O)N(R 17 )R 18 , or -C(O)OR 17 .
  • R 1 is —O—CH 2 —R 16 , wherein R 16 is alkynyl, cyano, —OR 17 , —N(R 17 )R 18 , —C(O)N(R 17 )R 18 , or —C(O)OR 17 .
  • each of R3 and R4 is independently selected from -OR15 and -OC(O) R15 , wherein R15 is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; or R3 and R4 , together with the carbon atoms to which they are attached, form a 5-6 membered heterocyclyl.
  • R3 and R4 are -OR15 , wherein R15 is C1 - C6 alkyl, C1 - C4 alkyl, or C1 - C2 alkyl.
  • each of R and R is independently selected from -OR and -OC(O) R , wherein R is hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; or R and R together with the carbon atoms to which they are attached form a 5-6 membered heterocyclyl. In certain embodiments, R and R together with the carbon atoms to which they are attached form a methylenedioxy ring.
  • R 11 and R 12 are taken together to form oxo.
  • each of R 3 and R 4 is -O-alkyl; R 8 and R 9 , together with the carbon atom to which they are attached, form a methylenedioxy ring;
  • R 1 is heteroaryl, -OR 15 , -C(O)R 15 , -N(R 17 )R 18 , -N(R 17 )C(O)R 18 , -N ⁇ C(R 17 )R 18 , pinacol boroyl, -OS(O) 2 CF 3 , a glycosidic group, a heterocyclyl optionally substituted with 1 or 2 groups independently selected from R 16 , or an alkynyl optionally substituted with a trialkylsilane; or R 1 is -OCH 2 -cyano, -OCH 2 -C ⁇ CH, -OCH 2 -C(O)N(R 17 )R 18 , or -C(O)OR 17 ; and R 11 and R 12 together form an o
  • R 19 and R 20 when one of R 19 and R 20 is hydrogen, the other of R 19 and R 20 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 .
  • R 21 and R 22 when one of R 21 and R 22 is hydrogen, the other of R 21 and R 22 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 .
  • R 23 and R 24 when one of R 23 and R 24 is hydrogen, the other of R 23 and R 24 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 .
  • R 25 and R 26 when one of R 25 and R 26 is hydrogen, the other of R 25 and R 26 is selected from R 15 , -OR 15 , -C(O)R 15 , -C(O)OR 15 , -CH 2 R 27 , and -C(O)R 27 .
  • each compound may be in the form of a free compound, an acid or base addition salt, or a prodrug, where appropriate.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound described herein and at least one pharmaceutically acceptable excipient (such as an excipient); the pharmaceutical composition can be prepared according to a pharmaceutically described preparation method.
  • the compounds described herein and their pharmaceutically acceptable salts can be administered to a subject alone or in combination with a pharmaceutically acceptable excipient, carrier, and/or diluent as a pharmaceutical composition according to standard pharmaceutical practice.
  • the compounds can be administered orally or parenterally.
  • Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous, and topical administration, with intravenous and topical administration being preferred.
  • the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more compounds described herein formulated together with one or more pharmaceutically acceptable excipients, carriers (additives) and/or diluents.
  • the pharmaceutical compositions of the present invention can be formulated in particular for administration in solid or liquid form, including those suitable for: (1) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, such as, for example, sterile solutions or suspensions, or sustained release formulations; and (2) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual and systemic absorption), boluses, powders, granules, pastes for application to the tongue.
  • parenteral administration e.g., by subcutaneous, intramuscular, intravenous or epidural injection, such as, for example, sterile solutions or suspensions, or sustained release formulations
  • some embodiments of the compound described herein may contain a basic functional group, such as an amino group, and therefore can form a pharmaceutically acceptable salt with a pharmaceutically acceptable acid.
  • pharmaceutically acceptable salt refers to the relatively nontoxic inorganic and organic acid addition salts of the compound of the present invention. These salts can be prepared in situ during administration of a vehicle or dosage form manufacturing process, or by reacting the purified compound of the present invention in its free base form with a suitable organic or inorganic acid individually, and separating the salt thus formed during subsequent purification to prepare.
  • Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, glucoheptonate, lactobionate and laurylsulfonate etc.
  • compositions of the compounds of the present invention include conventional non-toxic salts or quaternary ammonium salts of the compounds, for example, from non-toxic organic or inorganic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, ox
  • the compounds described herein may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salt refers to relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can also be prepared in situ during the preparation of a dosing vehicle or dosage form, or by reacting a purified compound in its free acid form with a suitable base (such as a pharmaceutically acceptable hydroxide, carbonate or bicarbonate of a metal cation), with ammonia, or with a pharmaceutically acceptable organic primary amine, secondary amine or tertiary amine alone.
  • a suitable base such as a pharmaceutically acceptable hydroxide, carbonate or bicarbonate of a metal cation
  • Representative alkali metal or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts, etc.
  • Representative organic amines that can be used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc.
  • the reaction was refluxed at 140 ° C for 12 hours.
  • the reaction solution was cooled to room temperature and diluted with water.
  • the reaction solution was extracted with dichloromethane.
  • the organic phases were combined, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain a yellow solid (4) (30 g, 62%).
  • the above diester compound intermediate (4) (5 mmol) was dissolved in anhydrous tetrahydrofuran (70 mL), sodium borohydride (0.945 g, 25 mmol) was slowly added, and the mixture was heated under reflux at 80°C for 12 hours.
  • the reaction solution was cooled to room temperature and acidified dropwise with 2M hydrochloric acid to a pH value close to 2.
  • schizoferrin (5) (3.8 g, 10 mmol), 4-dimethylaminopyridine (2.44 g, 20 mmol) were dissolved in 100 mL of ultra-dry dichloromethane, cooled to 0°C, and trifluoromethanesulfonic anhydride (2.5 mL, 12 mmol) was added dropwise. The mixture was stirred at room temperature for 4 hours. Thin layer chromatography showed that the reaction was complete and a new spot was generated. The reaction solution was dried to obtain a light yellow solid, which was washed with ethanol and purified to obtain product (6) (4.61 g, 90%).
  • This product was used directly in the next step without further purification by dissolving it in CHCl (5 mL), cooling it to 0°C, and then adding hydrogen bromide (HBr, 33% AcOH, 1.5 mL) dropwise. The reaction was warmed to room temperature and stirred for an additional 4 hours. The reaction was then quenched with water, and the aqueous layer was extracted with CHCl . The organic layers were combined, washed with water, 10% NaHCO , and saturated brine, dried over NaSO , and concentrated under reduced pressure to obtain the glycosyl bromide. The crude glycosyl bromide was concentrated and used directly in the next step without further purification.
  • Vero E6 cells were seeded at 2 ⁇ 104 cells/well in a 96-well plate and cultured overnight at 37°C, 5% CO2. When the monolayer of cells reached approximately 70% growth, different concentrations of the drug to be tested (seven types of horticultural horticultural glycoside derivatives ANL-1-ANL-7, or the positive control drug remdesivir) were added at 100 ⁇ L/well, with three replicate wells. A negative control well containing no drug was also set up. The cells were cultured at 37°C, 5% CO2 for 72 hours, and cell viability was assessed using a CCK8 assay.
  • OD values were measured using a Bio-Tek Synergy 2 multi-function microplate reader at a wavelength of 450 nm and a reference wavelength of 630 nm.
  • the CC50 value (50% Cytotoxic Concentration) was calculated, which is the drug concentration that produces toxicity to 50% of Vero E6 cells.
  • Vero E6 cells (common commercial products) were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum. The cells were passaged once one day before the experiment to keep the cells in the logarithmic growth phase. SARS-CoV-2 was amplified in Vero E6 cells, and the culture medium was collected, filtered through a 0.22 ⁇ m filter, and aliquoted into 0.5mL tubes and stored at -80°C. Vero E6 cells were seeded into 96-well plates at 2 ⁇ 104 /well and cultured overnight at 37°C, 5% CO2 . When the monolayer cells grew to about 70%, they were transferred to the P3 laboratory for use.
  • Drug dilution 6 concentration gradients were set, with 3 replicate wells for each gradient.
  • 50 ⁇ L of pre-prepared drugs 7 types of salvia miltiorrhiza or its glycoside derivatives ANL-1 to ANL-7, or the positive control drug remdesivir
  • 50 ⁇ L of virus dilution supernatant 4
  • Negative controls without drug or virus, and positive controls without hornwort or its glycoside derivatives but containing remdesivir were set up.
  • Cells were cultured at 37°C, 5% CO2 for 72 hours.
  • IC50 value refers to the drug concentration that inhibits 50% of Vero E6 growth.
  • CC 50 The half-maximal inhibitory concentration of the sample on Vero E6 cells
  • IC 50 The concentration of the sample when half of the Vero E6 cells die of SARS-CoV-2
  • Therapeutic index TI CC 50 /IC 50 .
  • the in vitro anti-COVID-19 activity of the phosphate-active structure of monoclavir and namatevir in the Vero E6 cell model is 1.2 times and 0.4 times that of remdesivir, respectively, measured by TI values (Fiaschi L, et al. Viruses 2022; 14: 1374.); the IC 50 of azithromycin (phosphate-active structure) is 4.3 ⁇ M, and its antiviral activity is lower than that of remdesivir (Zhang JL, et al. Signal Transduct Target Ther. 2021; 6: 414).
  • the in vitro anti-COVID-19 activity of these marketed drugs is much lower than that of ANL-7, ANL-2, and ANL-6.
  • SARS-CoV-1 GZ50 GenBank: AY304495
  • MERS-CoV EMC/2012, GenBank: JX869059
  • SARS-CoV-2 wild-type WT, HKU-001a, GenBank: MT230904
  • Omicron BA 5.2 GISAID: EPI_ISL_13777658
  • SARS-CoV-1 and MERS-CoV were propagated in Vero E6 cells. All variants of SARS-CoV-2 were cultured in Vero E6-TMPRSS2 cells and titrated by plaque assay. All live virus culture, in vivo, and in vitro experiments were performed in a Biosafety Level 3 laboratory at the University of Hong Kong, following strict protocols.
  • Caco2 cells were obtained from ATCC (ATCC HTB-37) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Amarillo, Texas, USA) according to the supplier's guidelines.
  • VeroE6-TMPRSS2 cells were obtained from the Japan Research Center for Bioresources (JCRB) Cell Bank (JCRB1819) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Amarillo, Texas, USA) according to the manufacturer's instructions. All cell lines used in this study were routinely tested for mycoplasma and maintained in a mycoplasma-free environment.
  • Cell viability was quantified using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, USA). Caco2 cells were incubated with various concentrations of ANL-2 (0–10 ⁇ M) for 24 h and then processed according to the manufacturer's instructions. Luminescent signals were detected using a Victor X3 2030 Multi-Purpose Microplate Analyzer (Perkin Elmer, USA).
  • SARS-CoV-2 WT SARS-CoV-2 WT was inoculated intranasally in the hamsters.
  • the SARS-CoV-2 WT stock solution was diluted with PBS to a concentration of 3 ⁇ 10 3 PFU per hamster.
  • each hamster was intraperitoneally injected with ANL-2 (50 mg/kg or 25 mg/kg) or 35% PEG400 aqueous solution in a final volume of 1000 ⁇ L.
  • ANL-2 50 mg/kg or 25 mg/kg of ANL-2 was administered by dissolving ANL-2 in 35% PEG400 aqueous solution.
  • Method 2 We also studied and evaluated the effect of ANL-2 in inhibiting the replication of the new coronavirus in host Caco2 cells (Method 2). First, the effect of ANL-2 on the viability of host Caco2 cells was determined using a luminescent cell viability assay kit. It was found that ANL-2 had no effect on Caco2 cell viability within its detection concentration range (0-10 ⁇ M), and the CC 50 value was greater than 10 ⁇ M ( Figure 15).
  • Caco2 cells were infected with various coronaviruses (including SARS-CoV-1, MERS-CoV, SARS-CoV-2WT and SARS-CoV-2BA.5.2) and added with different concentrations of ANL-2 (0-10 ⁇ M). The cells were incubated for 24 hours for RNA extraction and qRT-PCR to quantify RdRp, NP or sgRNA gene copies.
  • coronaviruses including SARS-CoV-1, MERS-CoV, SARS-CoV-2WT and SARS-CoV-2BA.5.2
  • ANL-2 25 mg/kg significantly reduced the viral load in the lung tissue of hamsters infected with SARS-CoV-2 WT, but had no effect on the viral load in the nasal concha.
  • the high-dose group (50 mg/kg) of ANL-2 had no significant effect on the viral load in the lung/nasal concha of hamsters infected with SARS-CoV-2 WT. This may be due to the lower solubility of ANL-2 in the injection solution of the high-dose group (50 mg/kg) than that of the low-dose group (25 mg/kg).
  • a 0.5% sodium carboxymethylcellulose (CMC-Na) aqueous solution was used to prepare an ANL-2 suspension as test solution 1, with doses of 500 mg/kg, 2000 mg/kg, and 5000 mg/kg.
  • a 0.3% (w/v) ANL-2 fat emulsion was prepared as test solution 2.
  • the preparation methods of the two test solutions are as follows:
  • ANL-2 suspension Weigh 3.00 g of ANL-2, add a certain volume of 0.5% CMC-Na aqueous solution, vortex and ultrasonically disperse it evenly, and dilute it with 0.5% CMC-Na aqueous solution to prepare ANL-2 suspensions with concentrations of 50.00 mg/mL, 66.67 mg/mL and 166.67 mg/mL, respectively, which is test solution 1.
  • the colostrum was homogenized in a high-pressure homogenizer for 15 min with the parameter set to 1000 Bar to obtain a 3.00 mg/mL C180A-P1 fat emulsion, i.e., a 3.00 mg/mL ANL-2 fat emulsion, which was test solution 2.
  • the ANL-2 fat emulsion was observed and photographed under a transmission electron microscope, as shown in Figure 19.
  • the droplets were uniform in size, ranging from 10 nm to 160 nm in diameter, and spherical in shape.
  • the present invention uses the median lethal dose (LD50) Bliss method specified by the State Food and Drug Administration to evaluate the acute toxicity of the samples.
  • ICR mice that have passed the adaptability observation were selected and randomly divided into 5 groups based on weight and gender, with 10 animals in each group, half male and half female.
  • mice were orally administered 1 to 3 times within 8 hours, with a volume of 10 mL/kg each time and a cumulative dose of 500 mg/kg, 2000 mg/kg, and 5000 mg/kg.
  • ANL-2 test solution 2 An acute toxicity test of ANL-2 test solution 2 was conducted on mice by intravenous administration, intraperitoneal injection or oral administration. ICR mice were administered intravenously, intraperitoneally or orally three times within 8 hours, with a dosage volume of 16.7 mL/kg each time. ANL-2 test solution 2 at a concentration of 3.00 mg/mL was used, and the cumulative dose for three times was 150 mg/kg.
  • the present invention uses SPF-grade SD rats, male, 220-260g, animal certificate: No. 44005800013136.
  • the above animals were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Experimental Animal Production License No.: SCXK (Guangdong) 2018-0034), and the use of experimental animals was approved by the Ethics Committee (Approval No.: 20211012004).
  • the animals were raised in the Experimental Animal Center of Guangzhou University of Chinese Medicine (Experimental Animal Use License No.: SYXK (Guangdong) 2018-0001), with a temperature of 22 ⁇ 3°C, a relative humidity of 30-70%, and a light-dark cycle of 12 hours.
  • a 3.00 mg/mL ANL-2 fat emulsion was diluted with normal saline injection to 0.2 mg/mL, 0.4 mg/mL, and 0.8 mg/mL, and directly used for intravenous injection; a single tail vein injection of 2 mg/kg, 4 mg/kg, and 8 mg/kg was given with a dosing volume of 10 mL/1 kg.
  • 200 ⁇ L of blood was collected before administration (0 h) and 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, and 12 h after administration; blood was collected in prefabricated EP tubes containing EDTA ⁇ 2K, centrifuged at 3000 rpm for 15 min, plasma was separated, and frozen at -80°C.
  • a 3.00 mg/mL ANL-2 fat emulsion was diluted with normal saline injection to 1.5 mg/mL, 2.5 mg/mL, and 3 mg/mL for oral administration.
  • a single oral gavage of 15 mg/kg, 25 mg/kg, and 50 mg/kg was administered at a volume of 10 mL/1 kg (the 50 mg/kg dose group had a volume of 16.67 mL/1 kg).
  • 200 ⁇ L of blood was collected before administration (0 h) and 10 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, 24 h, and 36 h after administration.
  • Blood was collected in prefabricated EP tubes containing EDTA ⁇ 2K, centrifuged at 3000 rpm for 15 min, and plasma was separated and stored frozen at -80°C.
  • ANL-2 concentrations in rat plasma following administration were determined using an established UPLC-MS/MS method. These values, along with corresponding time points, were entered into Excel for statistical analysis. Mean ⁇ SD values were calculated for each group, and plasma concentration-time curves were plotted. All data were formatted and imported into DAS 3.0 statistical software. A non-compartmental model was used for data analysis of each dose group, and key pharmacokinetic parameters, including AUC, Cmax , tmax , t1 ⁇ 2, MRT, Vz/F, Clz/F, and C0 , were calculated. These parameters were statistically analyzed using Excel, and oral bioavailability was calculated to evaluate the results.
  • the compounds described herein can exhibit activity against SARS-CoV-2.
  • a compound of formula (I) is administered to a patient to inhibit the replication of SARS-CoV-2 or reduce the cytopathic effect of the virus.
  • the compounds, pharmaceutical compositions, and treatment methods described herein can be used to prevent, treat, or ameliorate infections caused by coronaviruses, including but not limited to SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

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Abstract

本发明公开一类抗新型冠状病毒的芳基萘木脂素类化合物及其制备方法和应用。结果显示，ANL-1、ANL-2、ANL-4～ANL-7对Vero E6细胞无毒，ANL-3对Vero E6细胞低毒；在Vero E6细胞模型的体外抗新型冠状病毒活性以TI值计，ANL-7和ANL-2是分别瑞德西韦的19倍和16倍，高于上市药物的体外抗新冠病毒活性；ANL-2对新冠病毒的RNA依赖性的RdRp活性有显著的抑制作用；ANL-2还有效限制了多种致病性冠状病毒在Caco2细胞或仓鼠体内的复制。可见，芳基萘木脂素类化合物具有良好的抗冠状病毒活性，可用于在患者中治疗、预防新型冠状病毒感染或者延缓新型冠状病毒感染的进展。

Description

一类抗新型冠状病毒的芳基萘木脂素类化合物及其制备方法和应用 技术领域
本发明涉及一类新型冠状病毒的抑制剂，具体而言，涉及一类具有抗新型冠状病毒活性的芳基萘木脂素类化合物及其制备方法和应用。
背景技术
冠状病毒(CoV)是有包膜的单链正向RNA病毒，包括冠状病毒科(Coronaviridae)、动脉炎病毒科(Arteriviridae)和杆套病毒科(Roniviridae)。引起当前COVID-19大流行的SARS-CoV-2是β-冠状病毒。目前，很多努力都集中在从老药和新化合物中筛选针对病毒生命周期的关键酶的抑制剂。但从科学的角度看，新冠病毒通过变异再出现强致病力变异毒株的可能性是不能排除的。因此，迫切需要高度有效的病毒抑制剂来对抗冠状病毒。
与疫苗、抗体相比，口服小分子化学药物具有成本低、易生产、方便运输、易于使用等优点，是开发对抗可能与人类长期并存的新冠病毒治疗药物的焦点。在小分子药物研究领域，大部分药学家都在寻找针对新冠病毒的靶向特异性药物，主要靶标有针对病原的：棘蛋白(S蛋白)、E蛋白(包膜蛋白)、M膜蛋白、N核衣壳蛋白，以及与病毒复制有关的各种酶；也有针对宿主的靶标如ACE2。瑞德西韦是第1个治疗COVID-19小分子药物，2020年10月获美国FDA批准。瑞德西韦(Remdesivir)是一种核苷类似物前药，在体内代谢为三磷酸形式，靶向抑制NA依赖性RNA聚合酶(RdRp)，发挥抗病毒活性。由于只能经由静脉给药，限制了早期和大范围用药。默沙东公司的莫诺拉韦和辉瑞公司的Paxlovid在2021年12月相继获得FDA的COVID-19治疗紧急授权。莫诺拉韦(Molnupiravir)是一种化学结构与瑞德西韦相似的小分子RdRp抑制剂，对新冠病毒的伽马、德尔塔等株变异毒株都有效，能够有效降低病死率；由于可口服给药，可用于感染早期轻、中症患者的治疗，避免其向重症转化。Paxlovid由两种小分子组合而成，一种是Mpro抑制剂奈玛特韦(Nirmatrelvir；PF-07321332)，另一种是奈玛特韦的口服生物利用度促进剂利托那韦。该药含150mg规格奈玛特韦20片、100mg规格利托那韦10片；已很快成为全球一线处方用药，替代了瑞德西韦的应用，但价格较高。我国郑州大学常俊标教授等创制的阿兹夫定(Azvudine；2'-去氧-2'-β-氟代-4'-叠氮基胞嘧啶核苷；FNC；C9H11FN6O4)是一种小分子RdRp抑制剂。阿兹夫定已在2022年7月25日通过国家药监局(NMPA)审批，成为中国首个“应急附条件”批准治疗COVID-19的口服用药。阿兹夫定作为一种核苷类似物药物，较早前(2021年7月)才被NMPA批准为抗HIV-1新药，对HIV-1逆转录酶(RT)和辅助蛋白(Vif)具有特异性的双重抑制作用(双靶)。
在现代药物发现中，天然产物已成为发现先导化合物的丰富来源。通过筛选超过3,500种植物提取物，我们确定野靛棵(Justicia cf.patentiflora)有有效的抗HIV活性。该植物的茎和皮的甲醇提取物的生物活性指导下的分级导向分离产生了三种ANL(芳基萘)糖苷化合物。所述化合物展示出针对多种病毒的有效抑制活性。它们还显示对耐药性HIV毒株的显著抑制效应。文献中已报道了一些芳基萘木脂素具有抗病毒活性。虽然这些化合物中的一些显示出针对各种病毒株的显著抗病毒活性，但由于它们的低选择性指数(SI)，它们不被认为是潜在的抗病毒候选药物。
因此，需要开发出能够满足至少一些前述需求的改进的抗病毒剂。
发明内容
为了克服现有技术的缺点与不足，本发明的目的在于提供一类抗新型冠状病毒的芳基萘木脂素类化合物及其制备方法和应用。
本发明的目的通过下述技术方案实现：
本发明公开涉及一类新的芳基萘类似物、相关化合物和新中间体的制备，以及它们用于治疗如SARS-CoV-2等新型冠状病毒感染的用途。
因此，本发明的第一方面是芳基萘木脂素类化合物或其药物上可接受的盐或前药用于治疗、预防新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染或延缓新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染的进展，其中化合物具有式(I)：
或其药学上可接受的盐或前药，其中
X为氧或硫；
R1为R15、-OR15、-C(O)R15、或-C(O)OR15；
R2、R5、R6、R10、R13和R14各自为氢或卤素；
R3和R4各自独立地选自-OR15和-OC(O)R15；或者R3和R4与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；
R7、R8和R9各自独立地选自-OR15和-OC(O)R15；或者R7和R8与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；或者R8和R9与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；
R11和R12一起形成氧代基；或者在R11和R12中的一者为氢或卤素时，R11和R12中的另一者选自R15、-OR15、-C(O)R15和-C(O)OR15；
R15在每次出现时独立地选自氢、炔基、卤素、三氯甲基、三氟甲基、氰基、硝基、杂芳基、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N＝C(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18、-N(R17)S(O)2R18、任选地被1、2、3或4个独立地选自烷基的基团取代的1,3,2-二氧杂环戊硼烷、糖苷基团、任选地被三烷基硅烷取代的炔基、任选地被1、2、3、4或5个独立地选自R16的基团取代的烃基、任选地被1、2、3、4或5个独立地选自R16的基团取代的杂环基，以及任选地被1、2、3、4或5个独立地选自R16的基团取代的-(CH2)k-杂环基，其中k为1-6的整数；
R16在每次出现时独立地选自炔基、卤素、三氯甲基、三氟甲基、氰基、硝基、氧代基、＝NR17、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18和-N(R17)S(O)2R18；并且
R17和R18在每次出现时独立地为氢、烷基、炔基、环烷基、芳基、或杂芳基，或者选自烃基和杂环基，其任一者任选地被1、2、3、4或5个独立地选自卤素、氰基、氨基、羟基、C1-6烷基和C1-6烷氧基的基团取代；
所述的糖苷基团通常为碳水化合物，尤其是单糖基、二糖基、三糖基、四糖基或多糖基团，并且可以各种异构体形式存在，例如α-D、α-L、β-D或β-L形式。所述碳水化合物基团可任选地经其它类型的取代基或甚至额外糖苷基取代。然而，化合物的化学结构中所含单糖基和经取代单糖基的总数不可超过10。
糖苷基可为结构式(i)或(ii)的基团：
其中
R19和R20可一起形成氧代基；或者当R19和R20中的一个为氢或卤素时，R19和R20中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
R21和R22可一起形成氧代基；或者当R21和R22中的一个为氢或卤素时，R21和R22中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
R23和R24可一起形成氧代基；或者当R23和R24中的一个为氢或卤素时，R23和R24中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
R25和R26可一起形成氧代基；或者当R25和R26中的一个为氢或卤素时，R25和R26中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、-CH2R27和-C(O)R27；
R27独立地选自氢、卤素、三氟甲基、氰基、硝基、任选地被1个、2个、3个、4个或5个独立地选自R16的基团取代的烃基、任选地被1个、2个、3个、4个或5个独立地选自R16的基团取代的-(CH2)k-杂环基、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18、-N(R17)S(O)2R18、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基。
特别地，式(I)所述的化合物为：
特别地，式(I)所述的化合物为：
本发明的第二方面是所述ANL-2的制备方法，其制备反应式如下：
所述ANL-2的制备方法，包括以下步骤：
以2-溴-4,5-二甲氧基苯甲醛(1)为原料，通过乙二醇保护制备缩醛(2)，(2)与胡椒醛反应得到化合物(3)，化合物(3)在乙酸存在下通过加热迅速转化为异苯并呋喃，再通过Diels-Alder反应得到双酯(4)，将其还原得到山荷叶素(5)；然后将山荷叶素(5)与三氟甲磺酸酐反应得到产物(6)；产物(6)与三甲基乙炔基硅反应得到产物(7)；产物(7)在碳酸钾存在下得到目标产物ANL-2。
具体包括以下步骤：
(1)在2-溴-4,5-二甲氧基苯甲醛(1)的甲苯溶液中加入乙二醇、对甲苯磺酸，加热至140℃过夜；冷却至室温，减压蒸除甲苯；用乙酸乙酯溶解反应混合物，并依次用饱和碳酸氢钠溶液、水、饱和氯化钠溶液萃取，并用无水硫酸钠干燥有机相；减压蒸干得到的粗产物，再经硅胶柱层析得到缩醛中间体(2)；
优选的，步骤(1)中，所述-溴-4,5-二甲氧基苯甲醛、乙二醇、对甲苯磺酸的摩尔比为1±0.5:2±1:0.2±0.1；进一步为1:2:0.2；
(2)将上述缩醛中间体(2)溶于无水四氢呋喃中，冷却，逐滴加入正丁基锂溶液，搅拌，逐滴加入胡椒醛的四氢呋喃溶液并缓慢升至室温；将反应液用水淬灭，并用乙酸乙酯萃取；合并的有机相分别用水和饱和氯化钠溶液萃取，并用无水硫酸钠干燥，减压蒸馏得到淡黄色油状液体中间体(3)并迅速用于下一步Diels-Alde反应；
优选的，步骤(2)中，所述缩醛中间体(2)、正丁基锂、胡椒醛的摩尔比为1±0.5:1.2±0.5:1±0.5；进一步为1:1.2:1；
优选的，步骤(2)中，所述的冷却为冷却至-70℃至-80℃；进一步为冷却至-78℃；
优选的，步骤(2)中，所述搅拌的时间为10-60分钟；进一步为30分钟；
(3)将上述中间体(3)溶于二氯甲烷中，加入冰醋酸和丁炔二酸二甲酯；反应；反应液冷却至室温后用水稀释，反应液用二氯甲烷萃取；合并有机相，用无水硫酸钠干燥，并经硅胶柱层析分离得到二酯化合物中间体(4)；
优选的，步骤(3)中，所述中间体(3)和丁炔二酸二甲酯的摩尔比为1±0.5:1±0.5；进一步为1:1；
优选的，步骤(3)中，所述的反应的条件为于100℃-160℃回流6-20小时；进一步为于140℃回流12小时。
(4)将上述二酯化合物中间体(4)溶于无水四氢呋喃中，缓慢加入硼氢化钠并加热回流；反应液冷却至室温，并用盐酸逐滴酸化至pH值接近2；酸化后，反应液经乙酸乙酯萃取，合并有机相，并经水和饱和氯化钠洗涤，无水硫酸钠干燥，硅胶柱层析得到淡黄色固体(5)(山荷叶素)；
优选的，步骤(4)中，所述二酯化合物中间体(4)和硼氢化钠的摩尔比为1±0.5:5±2.5；进一步为1:5；
优选的，步骤(4)中，所述加热回流的条件为于60℃-100℃加热回流6-20小时；进一步为于80℃回流12小时；
优选的，步骤(4)中，所述盐酸为1-5M盐酸；进一步为2M盐酸；
(5)在氮气保护下，山荷叶素(5)，4-二甲氨基吡啶溶于超干二氯甲烷中，冷却，逐滴加入三氟甲磺酸酐，于室温下搅拌；薄层色谱显示反应完成并生成新点；反应液旋干得到淡黄色的固体，并用乙醇洗涤纯化得到中间体(6)；
优选的，步骤(5)中，所述山荷叶素(5)、4-二甲氨基吡啶和三氟甲磺酸酐的摩尔比为1±0.5:2±1:1.5±0.5；进一步为1:2:1.2；
优选的，步骤(5)中，所述冷却为冷却至-5℃至5℃；进一步为0℃；
优选的，步骤(5)中，所述搅拌的时间为2-8小时；进一步为4小时。
(6)在反应容器中加入二氯双(三苯基膦)钯，碘化铜，中间体(6)；在氮气保护下在上述反应液中逐滴加入三甲基乙炔基硅和三乙胺的二甲基甲酰胺溶液；反应液加热并搅拌；冷却至室温后，反应液经水淬灭并用二氯甲烷萃取；有机相经无水硫酸钠干燥后，经硅胶柱层析分离纯化得到中间体(7)；
优选的，步骤(6)中，所述中间体(6)、二氯双(三苯基膦)钯、碘化铜、三甲基乙炔基硅和三乙胺的摩尔比为1±0.5:0.05±0.01:0.15±0.06:2±1:3±1；进一步为1:0.05:0.15:2:3；
优选的，步骤(6)中，所述加热并搅拌的条件为于50℃-100℃搅拌5-16小时；进一步为于80℃搅拌10小时。
(7)中间体(7)溶于甲醇中，并加入碳酸钾，于室温下搅拌；反应混合物经硅胶柱层析分离纯化得到目标产物ANL-2。
优选的，步骤(7)中，所述中间体(7)和碳酸钾的摩尔比为1±0.5:2±1；进一步为1:2。
优选的，步骤(7)中，所述搅拌的时间为4-20小时；进一步为12小时。
本发明的第三方面是所述ANL-3的制备方法，包括以下步骤：
将山荷叶素(5)与溴乙腈反应得到产物ANL-3；
具体包括以下步骤：
(8)将上述步骤(4)的山荷叶素(5)和溴乙腈溶于丙酮中，缓慢加入Cs2CO3，搅拌，TLC显示形成一个极性较低的点；混合物经纯化得到所需产物ANL-3。
优选的，步骤(8)中，所述山荷叶素(5)、溴乙腈和Cs2CO3的摩尔比为1±0.5:2±1:2±1；进一步为1:2:2；
优选的，步骤(8)中，所述搅拌的时间为5至20小时，进一步为12小时。
本发明的第四方面是所述ANL-4的制备方法，包括以下步骤：
以D-木糖为原料，通过与乙酸酐、溴化氢反应，得到糖基溴；取山荷叶素(ANL-1)在TBAB存在下与糖基溴反应，得到目标产物ANL-4。
具体包括以下步骤：
(9)取D-木糖溶于吡啶中，加入乙酸酐室温下搅拌过夜；反应结束后，加CH2Cl2稀释，用HCl溶液洗涤；合并有机相，用NaHCO3饱和溶液萃取，取有机相，经无水Na2SO4干燥，浓缩得全乙酰化木糖粗产物，无需进一步纯化，直接用于下一步；即溶解在CH2Cl2中，冷却，然后滴入溴化氢；将反应加热至室温，再搅拌；然后用水淬灭反应，用CH2Cl2萃取水层；合并有机层，用水、NaHCO3和饱和食盐水洗涤，用Na2SO4干燥，在减压下浓缩以得到糖基溴；糖基溴粗产物浓缩后直接用于下一步，无需进一步纯化；山荷叶素(5)溶解于CHCl3和氢氧化钠水溶液中，加入四丁基溴化铵TBAB，反应加热，加入糖基溴，搅拌；冷却至室温后，用CHCl3萃取水层，结合的有机层用饱和食盐水洗涤，Na2SO4干燥，减压浓缩；用硅胶色谱柱(正己烷/乙酸乙酯＝1/1)分离反应混合物，得到目标产物ANL-4；
优选的，所述D-木糖与乙酸酐的摩尔比为1±0.5:8±2；进一步为1:7；
优选的，用1％至15％ HCl溶液洗涤；进一步为用10％ HCl溶液洗涤；
优选的，所述冷却为冷却至-5℃至5℃；进一步为0℃；
优选的，所述再搅拌的时间为1至10小时；进一步为4小时；
优选的，所述山荷叶素与糖基溴的摩尔比为1±0.5:1±0.5；进一步为1:1；
优选的，所述氢氧化钠水溶液的浓度为(0.1±0.05)M；进一步为0.1M；
优选的，所述反应加热为加热至30℃至60℃，进一步为加热至40℃；保持10–60min；进一步为保持10min。
本发明的第五方面是所述ANL-5、ANL-6和ANL-7的制备方法，具体包括以下步骤：
(10)将ANL-4的糖基部分的-O-乙酰基脱去，得到山荷叶素7-O-木糖苷(8)；
(11)山荷叶素7-O-木糖苷(8)通过乙酸酐Ac2O和乙酸四丁基铵TBAOAc反应，得到木糖3″-乙酰化糖苷衍生物ANL-5。
具体地，山荷叶素7-O-木糖苷(8)溶于干乙腈中，加入乙酸酐和乙酸四丁基铵，搅拌；反应完成后，将溶剂用旋转蒸发仪浓缩后通过硅胶柱层析(以体积比为1.5:1的石油醚/乙酸乙酯洗脱)分离纯化，得到主产物ANL-5。
(12)ANL-5用氯甲酸丙烯酯处理，产物经硅胶柱层析(以体积比为2:1的石油醚/乙酸乙酯洗脱)分离，得到山荷叶素木糖苷的糖基酰化产物ANL-6(2″,4″-双-O-甲酰丙烯基-3″-O-乙酰基山荷叶素木糖苷)和ANL-7(4″-O-甲酰丙烯基-3″-O-乙酰基山荷叶素木糖苷)。
优选的，步骤(10)中，采用碱性溶液将ANL-4的糖基部分的-O-乙酰基脱去，所述碱性溶液为氢氧化钠水溶液和氢氧化钾水溶液中的至少一种；
所述ANL-4与氢氧化钠或氢氧化钾的摩尔比为1±0.1:1.5±0.4；进一步为1:1.2；
优选的，步骤(11)中，所述山荷叶素7-O-木糖苷(8)、乙酸酐、乙酸四丁基铵的摩尔比为1±0.1:1±0.2:0.5±0.2；进一步为1:1.11:0.3。
优选的，步骤(11)中，所述反应时间为2至24h；进一步为12h。
优选的，步骤(11)中，所述反应温度为室温至60℃；进一步为40℃。
优选的，步骤(12)中，所述ANL-4、氯甲酸丙烯酯的摩尔比为1±0.1:2.5±0.5；进一步为1:2。
优选的，步骤(12)中，所述处理条件为使用三乙胺Et3N和二甲氨基吡啶DMAP为催化剂。
优选的，步骤(12)中，所述处理时间为2至24h；进一步为15h。
优选的，步骤(12)中，所述反应温度为-5℃至40℃；进一步为0℃至室温。
本发明中，所述室温是指20～30℃。
本发明的第六方面是一种包含芳基萘木脂素类化合物或其药学上可接受的盐或前药的药物制剂，以用于在患者中治疗、预防新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染或者延缓新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染的进展。
本发明的第七方面是一种包含炔基芳基萘木脂素类化合物或其药学上可接受的盐或前药的药物制剂，以用于在患者中治疗、预防新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染或者延缓新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染的进展。
本发明的第八方面是一种包含山荷叶素类似物或其药学上可接受的盐或前药的药物制剂，以用于在患者中治疗、预防新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染或者延缓新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染的进展。
本发明的第九方面是一种包含山荷叶素苷类似物或其药学上可接受的盐或前药的药物制剂，以用于在患者中治疗、预防新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染或者延缓新型冠状病毒(包括SARS-CoV-1或SARS-CoV-2或MERS-CoV)感染的进展。
本发明的第十方面是一种包含ANL-2类似物或其药学上可接受的盐或前药的药物制剂，以用于在患者中治疗、预防病毒感染或者延缓病毒感染的进展。
本发明中所述的新型冠状病毒从广义来讲是指能引物严重症状、具有致死性、传染性等特点的冠状病毒，包括但不限于引起严重急性呼吸综合征、中东呼吸综合征和新型冠状病毒疾病COVID-19的病毒中的至少一种。
进一步优选的，所述新型冠状病毒包括但不限于SARS-CoV-1或SARS-CoV-2或MERS-CoV或SARS-CoV-2奥米克戎株中的至少一种。
本发明的其他方面涉及提供合成ANL-2、ANL-3、ANL-4、ANL-5、ANL-6或ANL-7化合物以及合成期间的中间体化合物的方法。此外，本发明涉及可用于制备本发明的化合物的中间体化合物。
本发明的化合物能够以不同的形式存在，如游离酸、游离碱、酯以及其他前药、盐和互变异构体，并且本公开包括这些化合物的所有变体形式。
保护的程度包括含有或声称含有本发明的化合物的假冒或虚假产品，而不管它们实际上是否含有这样的化合物以及不管任何这样的化合物是否以治疗有效量被含有。
包括在保护范围内的是包括描述或说明书的包装，所述描述或说明书指示该包装含有本发明的物类或药物制剂以及产品，所述产品是或包含，或者声称是或包含这样的制剂或物类。这样的包装可能是但不一定是假冒或虚假的。
结合本发明的特定方面、实施方案或实施例描述的特性、整数、特征、化合物、化学部分或基团应理解为适用于本文所述的任何其他方面、实施方案或实施例，除非与其不相容。
本发明相对于现有技术具有如下的优点及效果：
为了验证芳基萘木脂素类化合物(式I)的抗冠状病毒活性，我们在本发明展示合成的系列芳基萘木脂素类化合物ANL-1、ANL-2、ANL-3、ANL-4、ANL-5、ANL-6或ANL-7。通过实验结果显示，ANL-1、ANL-2、ANL-4、ANL-5、ANL-6或ANL-7对Vero E6细胞无毒，ANL-3对Vero E6细胞低毒；在Vero E6细胞模型的体外抗新型冠状病毒(SARS-CoV-2)活性以TI值计，ANL-7和ANL-2是分别瑞德西韦的19倍和16倍，远高于上市药物的体外抗新冠病毒活性；ANL-4、ANL-5、ANL-6的体外抗新冠病毒活性以TI值计均远高于瑞德西韦，ANL-3的体外抗新冠病毒活性以TI值计接近瑞德西韦；ANL-2对新冠病毒的RNA依赖性的RNA聚合酶(RNA-dependent RNA polymerase；RdRp)的活性具有显著的抑制作用；ANL-2还有效限制了多种致病性冠状病毒在Caco2细胞或仓鼠体内的复制。可见，以ANL-2和ANL-7为代表的芳基萘木脂素类化合物具有良好的抗冠状病毒活性，可用于在患者中治疗、预防新型冠状病毒(比如SARS-CoV-2等)感染或者延缓新型冠状病毒(比如SARS-CoV-2等)感染的进展。
附图说明
图1为本发明实施例2提供的ANL-2的高效液相色谱(HPLC)图；
图2为本发明实施例2提供的ANL-2的高分辨质谱(HR-ESIMS)图；
图3为本发明实施例2提供的ANL-2的1H核磁共振(1H NMR)图谱；
图4为本发明实施例2提供的ANL-2的13C核磁共振(13C NMR)图谱；
图5为本发明实施例3提供的ANL-3的1H核磁共振(1H NMR)图谱；
图6为本发明实施例3提供的ANL-3的13C核磁共振(13C NMR)图谱；
图7为本发明实施例4提供的ANL-4的1H核磁共振(1H NMR)图谱；
图8为本发明实施例4提供的ANL-4的13C核磁共振(13C NMR)图谱；
图9为本发明实施例5提供的ANL-5的1H核磁共振(1H NMR)图谱；
图10为本发明实施例5提供的ANL-5的13C核磁共振(13C NMR)图谱；
图11为本发明实施例5提供的ANL-6的1H核磁共振(1H NMR)图谱；
图12为本发明实施例5提供的ANL-6的13C核磁共振(13C NMR)图谱；
图13为本发明实施例5提供的ANL-7的1H核磁共振(1H NMR)图谱；
图14为本发明实施例5提供的ANL-7的13C核磁共振(13C NMR)图谱；
图15为本发明实施例6提供的ANL-2对Caco2细胞活力的影响；
图16为本发明实施例6提供的ANL-2对被SARS-CoV-1、MERS-CoV、SARS-CoV-2野生型或BA.5.2变体感染的Caco2细胞的病毒基因拷贝的影响；
图17为本发明实施例6提供的被SARS-CoV-2WT病毒感染后的仓鼠肺和鼻甲组织中的RdRp基因拷贝数；
图18为本发明实施例7提供的ANL-2脂肪乳剂经粒度分析仪测得的粒径分布图；
图19为本发明实施例7提供的ANL-2脂肪乳剂经透射电镜测得的粒径状态；
图20为本发明实施例8提供的小鼠口服给予ANL-2混悬液和脂肪乳剂后体重变化曲线；
图21为本发明实施例9提供的三组大鼠尾静脉给药ANL-2脂肪乳剂的血药浓度-时间曲线；
图22为本发明实施例9提供的三组大鼠口服给药ANL-2脂肪乳剂的血药浓度-时间曲线。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
贯穿本说明书的描述和权利要求，词语“包含”意指包括但不限于，并且不旨在排除例如其他添加剂、组分、整数或步骤。
如在描述和所附权利要求中所用，单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文另有明确规定。因此，例如，提及到的“一种组合物”包括两种或更多种这样的组合物的混合物，提及到的“所述化合物”包括两种或更多种这样的化合物的混合物，提及到的“一种试剂”包括两种或更多种这样的试剂的混合物等。
“任选的”或“任选地”意指随后描述的事件或情形可以或不可发生，并且该描述包括事件或情形发生的情况以及不发生的情况。
如本文所用，“受试者”意指个体。因此，“受试者”可包括驯养动物(例如猫、狗等)、家畜(例如牛、马、猪、绵羊、山羊等)、实验室动物(例如小鼠、兔、大鼠、仓鼠等)和鸟类。“受试者”还可包括哺乳动物，例如灵长类或人类。
所谓的“减少”意指降低事件或特征(例如肿瘤生长)。应当理解，这通常与某些标准或预期值有关，换言之，它是相对的，但并不总是需要提及该标准或相对值。例如，“减少肿瘤生长”意指相对于标准或对照而言减少肿瘤生长率。
所谓的“预防”或该词语其他形式，如“预防”意指停止特定事件或特征，稳定或延缓特定事件或特征的发展或进展，或者使特定事件或特征发生的机会最小化。预防不需要与对照比较，因为它通常比例如减少更绝对。如本文所用，某物可被减少但不被预防，但是减少的某物也可被预防。同样，某物可被预防但不被减少，但预防的某物也可被减少。应当理解，在使用减少或预防的情况下，除非另外特别指出，否则其他词语的使用也明确地公开。
所谓的“治疗”意指给药组合物或者执行方法以减少、预防、抑制、或消除特定特征或事件(例如肿瘤生长或存活)。术语“控制”与术语“治疗”同义使用。
术语“抗病毒”是指抑制特定病毒复制，抑制病毒传播，或者预防病毒在其宿主体内建立自身，以及改善或减轻病毒感染引起的疾病的症状的能力。如果病毒载量减少、死亡率和/或发病率减小，则认为该治疗是治疗性的。
术语“治疗有效”意指所用组合物的量是足以改善疾病或障碍的一种或多种病因或症状的量。这样的改善仅需要减少或改变，而不必消除。
如本文所用，术语“药学上可接受的盐”是指本发明的化合物的任何盐，其保留其生物学性质，并且对于药物用途没有毒性或者不是其他方面不期望的。这样的盐可衍生自本领域公知的多种有机和无机抗衡离子并且包括它们。这样的盐包括：(1)与有机或无机酸形成的酸加成盐，所述有机或无机酸如盐酸、氢溴酸、硫酸、硝酸、磷酸、氨基磺酸、乙酸、三氟乙酸、三氯乙酸、丙酸、己酸、环戊基丙酸、乙醇酸、戊二酸、丙酮酸、乳酸、丙二酸、琥珀酸、山梨酸、抗坏血酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、3-(4-羟基苯甲酰基)苯甲酸、苦味酸、肉桂酸、扁桃酸、邻苯二甲酸、月桂酸、甲磺酸、乙磺酸、1,2-乙烷-二磺酸、2-羟基乙磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4-甲苯磺酸、樟脑酸、樟脑磺酸、4-甲基二环[2.2.2]-辛-2-烯-1-羧酸、葡庚糖酸、3-苯基丙酸、三甲基乙酸、叔丁基乙酸、月桂基硫酸、葡糖酸、苯甲酸、谷氨酸、羟基萘酸、水杨酸、硬脂酸、环己基氨基磺酸、奎宁酸、粘康酸等酸；或者(2)当存在于母体化合物中的酸性质子(a)被金属离子(例如，碱金属离子、碱土金属离子或铝离子)、或碱金属或碱土金属氢氧化物(例如，钠、钾、钙、镁、铝、锂、锌和钡氢氧化物)、氨替代，或者(b)与有机碱配位时形成的盐，所述有机碱如脂族、脂环族、或芳族有机胺，如氨、甲胺、二甲胺、二乙胺、甲基吡啶、乙醇胺、二乙醇胺、三乙醇胺、乙二胺、赖氨酸、精氨酸、鸟氨酸、胆碱、N,N’-二苄基乙二胺、氯普鲁卡因、二乙醇胺、普鲁卡因、N-苄基苯乙胺、N-葡甲胺哌嗪、三(羟甲基)-氨基甲烷、四甲基氢氧化铵等。此外，盐的实例包括钠、钾、钙、镁、铵、四烷基铵等，并且当化合物含有碱性官能团时包括无毒有机或无机酸的盐，如氢卤化物(例如，盐酸盐和氢溴酸盐)、硫酸盐、磷酸盐、氨基磺酸盐、硝酸盐、乙酸盐、三氟乙酸盐、三氯乙酸盐、丙酸盐、己酸盐、环戊基丙酸盐、乙醇酸盐、戊二酸盐、丙酮酸盐、乳酸盐、丙二酸盐、琥珀酸盐、山梨酸盐、抗坏血酸盐、苹果酸盐、马来酸盐；富马酸盐、酒石酸盐、柠檬酸盐、苯甲酸盐、3-(4-羟基苯甲酰基)苯甲酸盐、苦味酸盐、肉桂酸盐、扁桃酸盐、邻苯二甲酸盐、月桂酸盐、甲烷磺酸盐(甲磺酸盐)、乙烷磺酸盐、1,2-乙烷-二磺酸盐、2-羟乙基磺酸盐、苯磺酸盐(benzenesulfonate)(苯磺酸盐(besylate))、4-氯苯磺酸盐、2-萘磺酸盐、4-甲苯磺酸盐、樟脑酸盐、樟脑磺酸盐、4-甲基二环[2.2.2]-辛-2-烯-1-羧酸盐、葡庚糖酸盐、3-苯基丙酸盐、三甲基乙酸盐、叔丁乙酸盐、月桂基硫酸盐、葡糖酸盐、苯甲酸盐、谷氨酸盐、羟萘甲酸盐、水杨酸盐、硬脂酸盐、环己基氨基磺酸盐、奎尼酸盐、粘康酸盐等。
如本文所用，术语“糖苷”或“糖苷的”化合物是可互换的，并且包括提及的水解时产生糖和糖苷配基的任何类别的化合物。
如本文所用，术语“ANL”或“芳基萘木脂素(aryl naphthalene lignan)”或“芳基萘木脂素(arylnaphthalene lignan)”化合物是可互换的。
如本文所用，术语“芳基萘木脂素(aryl naphthalene lignan)”或“芳基萘木脂素(arylnaphthalene lignan)”或“ANL”包括提及的包含如下所示的2,3-二甲基-1-苯基-萘的基础结构的化合物：
如本文所用，芳基萘木脂素分子的碳编号包括提及的包含如下所示的编号系统的化合物：
在一类芳基萘化合物的核心结构中，两个甲基形成γ-内酯环以变成如下所示的芳基萘并呋喃-2-酮木脂素或芳基萘并呋喃-3-酮木脂素：
如本文所用，芳基萘木脂素侧链的碳编号包括提及的包含如下所示的编号系统的化合物：
如本文所用，术语“烃基”包括提及的仅由氢和碳原子组成的部分；这样的部分可包含脂族和/或芳族部分。所述部分可包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个碳原子。烃基的实例包括C1-6烷基(例如，C1、C2、C3或C4烷基，例如甲基、乙基、丙基、异丙基、正丁基、仲丁基或叔丁基)；被芳基(例如苄基)或者被环烷基(例如环丙基甲基)取代的C1-6烷基；环烷基(例如，环丙基、环丁基、环戊基或环己基)；芳基(例如，苯基、萘基或芴基)；C1-6烯基(例如，乙烯基、2-丙烯基或3-丁烯基)；C1-6炔基(例如，乙炔基、2-丙炔基或3-丁炔基)等。
如本文所用，术语“烷基”和“C1-6烷基”包括提及的具有1、2、3、4、5或6个碳原子的直链或支链烷基部分。该术语包括提及的如甲基、乙基、丙基(正丙基或异丙基)、丁基(正丁基、仲丁基或叔丁基)、戊基、己基等基团。特别地，烷基部分可具有1、2、3或4个碳原子。
如本文所用，术语“烯基”包括提及的具有2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个碳原子并且另外具有至少一个双键的直链或支链烷基部分，并且在适用的情况下为E或Z立体化学。该术语包括提及的如乙烯基、2-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、1-戊烯基、2-戊烯基、3-戊烯基、1-己烯基、2-己烯基和3-己烯基等基团。
如本文所用，术语“炔基”包括提及的具有2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个碳原子并且另外具有至少一个三键的直链或支链烷基部分。该术语包括提及的如乙炔基、2-丙炔基、1-丁炔基、2-丁炔基、3-丁炔基、1-戊炔基、2-戊炔基、3-戊炔基、1-己炔基、2-己炔基和3-己炔基等基团。
如本文所用，术语“烷氧基”和“C1-6烷氧基”包括提及的-O-烷基，其中烷基是直链或支链的并且包含1、2、3、4、5或6个碳原子。在一类实施方案中，烷氧基具有1、2、3或4个碳原子。该术语包括提及的如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、叔丁氧基、戊氧基、己氧基等基团。
如本文所用，术语“环烷基”包括提及的具有3、4、5、6、7或8个碳原子的脂环族部分。该基团可为桥联或多环环系。更常见的环烷基是单环的。该术语包括提及的如环丙基、环丁基、环戊基、环己基、降冰片基、双环[2.2.2]辛基等基团。
如本文所用，术语“芳基”包括提及的包含6、7、8、9、10、11、12、13、14、15或16个环碳原子的芳族环系。芳基通常为苯基，但可为具有两个或更多个环的多环环系，所述环中的至少一者为芳族。该术语包括提及的如苯基、萘基、芴基、薁基、茚基、蒽基等基团。
“环状基团”意指环或环系，其可为不饱和或部分不饱和的，但通常是饱和的，通常含有5至13个成环原子，例如5-或6-元环。其包括碳环基和杂环基部分。
如本文所用，术语“碳环基”包括提及的具有3、4、5、6、7、8、9、10、11、12、13、14、15或16个碳环原子的饱和的(例如环烷基)或不饱和的(例如芳基)环部分。特别地，碳环基包括3-至10-元环或环系，特别是5-或-6元环，其可为饱和的或不饱和的。碳环部分例如选自环丙基、环丁基、环戊基、环己基、降冰片基、双环[2.2.2]辛基、苯基、萘基、芴基、薁基、茚基、蒽基等。
如本文所用，术语“杂环基”包括提及的具有3、4、5、6、7、8、9、10、11、12、13、14、15或16个环原子的饱和的(例如杂环烷基)或不饱和的(例如杂芳基)杂环部分，所述环原子中的至少一者选自硼、氮、氧、磷、硅和硫。特别地，杂环基包括3-至10-元环或环系，更特别地5-或-6元环，其可为饱和的或不饱和的。
杂环部分选自例如环氧乙烷基、吖丙因基、1.2-氧硫杂环戊烷基、咪唑基、噻吩基、呋喃基、四氢呋喃基、吡喃基、噻喃基、噻蒽基、异苯并呋喃基、苯并呋喃基、色烯基、2H-吡咯基、吡咯基、吡咯啉基、吡咯烷基、吡咯里西啶基、咪唑基、咪唑烷基、苯并咪唑基、吡唑基、吡嗪基、吡唑烷基、噻唑基、异噻唑基、二噻唑基、噁唑基、异噁唑基、吡啶基、吡嗪基、嘧啶基、哌啶基、哌嗪基、哒嗪基、吗啉基、硫吗啉基，尤其是硫代吗啉基、吲哚嗪基、异吲哚基、3H-吲哚基、吲哚基、苯并咪唑基、香豆素基(cumaryl)、吲唑基、三唑基、四唑基、嘌呤基、4N-喹嗪基、异喹啉基、喹啉基、四氢喹啉基、四氢异喹啉基、十氢喹啉基、八氢异喹啉基、苯并呋喃基、二苯并呋喃基、苯并噻吩基、二苯并噻吩基、酞嗪基、萘啶基、喹喔啉基、喹唑啉基、喹唑啉基、噌啉基、蝶啶基、咔唑基、B-咔啉基、菲啶基、吖啶基、咟啶基、菲咯啉基、呋咕基、吩嗪基、吩噻嗪基、吩噁嗪基、色烯基、异色满基、色满基、1,3,2-二氧杂环戊硼烷等。
如本文所用，术语“杂环烷基”包括提及的具有3、4、5、6或7个环碳原子以及1、2、3、4或5个选自氮、氧、磷和硫的环杂原子的饱和杂环部分。该基团可为多环环系，但更通常为单环。该术语包括如氮杂环丁烷基、吡咯烷基、四氢呋喃基、哌啶基、环氧乙烷基、吡唑烷基、咪唑基、吲哚里西啶基、哌嗪基、四氢噻唑基、吗啉基、硫吗啉基、喹嗪基等基团。
如本文所用，术语“杂芳基”包括提及的具有5、6、7、8、9、10、11、12、13、14、15或16个环原子的芳族杂环环统，所述环原子中的至少一者选自氮、氧和硫。该基团可为具有两个或更多个环的多环环系，所述环中的至少一者为芳族，但更通常为单环。该术语包括提及的如嘧啶基、呋喃基、苯并噻吩基、噻吩基、吡咯基、咪唑基、吡咯烷基、吡啶基、苯并呋喃基、吡嗪基、嘌呤基、吲哚基、苯并咪唑基、喹啉基、吩噻嗪基、三嗪基、酞嗪基、2H-色烯基、噁唑基、异噁唑基、噻唑基、异吲哚基、吲唑基、嘌呤基、异喹啉基、喹唑啉基、蝶啶基等基团。
本文所用，术语“卤素”包括提及的F、Cl、Br或I。
如本文所用，表述“含卤素部分”包括提及的包含1至30个选自碳、氮、氧和硫的多价原子的部分，该部分包括至少一种卤素。该部分可为烃基，例如C1-6烷基或C1-6烷氧基，或者碳环基，例如芳基。
如本文所用，提及到部分的术语“取代的”意指所述部分中的一个或多个，尤其是多至5个，更尤其是1、2或3个氢原子彼此独立地被相应数量的所描述取代基替代。如本文所用，术语“任选取代的”意指取代的或未取代的。当然，应当理解，取代基仅处于化学上可能的位置，本领域的技术人员无需不适当的努力就能够决定(实验上或理论上)特定的取代是否可能。
当两个或更多个部分被描述为“各自独立地”选自原子或基团的列表时，这意味着所述部分可以相同或不同。因此，每个部分的特性独立于一个或多个其他部分的特性。
如本文所用，术语“对映体”意指彼此具有镜像的两种立体异构体之一。
如本文所用，术语“立体异构体”意指这样一类异构分子：其具有相同的分子式以及键合原子顺序，但它们的原子在空间上具有不同的三维取向。
术语“互变异构体”意指易于通过化学反应相互转化的异构分子。该反应通常导致氢原子迁移，这导致单键和相邻双键的转换。
前药是作为非活性(或小于全活性)化学衍生物给药的药物，其随后在体内通常通过正常代谢过程转化为活性药剂。
CC50是测试药物抑制50％细胞生长的浓度的细胞毒性量度。
EC50或IC50是测试药物抑制50％病毒生长的有效浓度的抗病毒活性量度。
术语“选择指数”或“SI”意指通过将给定CC50值除以测试药物的EC50或IC50值(CC50/EC50或CC50/IC50)来测量细胞毒性与抗病毒活性之间的窗口的比率。更高的SI比率意指测试药物在体外实验中对于给定的病毒感染将更有效且更安全。
化学结构中的符号表示指定化学结构与另一化学结构键合的位置。
化学结构中的符号“β”指示键连接在纸或屏幕平面的上方(或前方)。化学结构中的符号“α”指示键连接在纸或屏幕平面的下方(或后方)。
化学结构中的实心楔形物指示该键在朝向观察者的纸或屏幕平面的上方(或前方)。化学结构中的虚线(或断线)楔形物指示键连接在逐渐远离观察者的纸或屏幕平面的下方(或后方)。
本文提供了一种化合物和该化合物在制造用于治疗病毒感染的药物中的用途，以及使用该化合物在受试者中治疗病毒感染的方法，该方法包括向受试者给药治疗有效量的化合物的步骤，其中化合物具有式(I)：
或其药学上可接受的盐或前药，其中
X为氧或硫；
R1为R15、-OR15、-C(O)R15、或-C(O)OR15；
R2、R5、R6、R10、R13和R14各自独立地为氢或卤素；
R3和R4各自独立地选自-OR15和-OC(O)R15；或者R3和R4与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；
R7、R8和R9各自独立地选自-OR15和-OC(O)R15；或者R7和R8与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；或者R8和R9与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；
R11和R12一起形成氧代基；或者在R11和R12中的一者为氢或卤素时，R11和R12中的另一者选自R15、-OR15、-C(O)R15和-C(O)OR15；
R15在每次出现时独立地选自氢、炔基、卤素、三氯甲基、三氟甲基、氰基、硝基、杂芳基、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N＝C(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18、-N(R17)S(O)2R18、任选地被1、2、3、或4个独立地选自烷基的基团取代的1,3,2-二氧杂环戊硼烷、糖苷基团、任选地被三烷基硅烷取代的炔基、任选地被1、2、3、4或5个独立地选自R16的基团取代的烃基、任选地被1、2、3、4或5个独立地选自R16的基团取代的杂环基，以及任选地被1、2、3、4或5个独立地选自R16的基团取代的-(CH2)k-杂环基，其中k为1-6的整数(例如1、2、3、4或5)；
R16在每次出现时独立地选自炔基、卤素、三氯甲基、三氟甲基、氰基、硝基、氧代基、＝NR17、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18和-N(R17)S(O)2R18；并且
R17和R18在每次出现时独立地为氢、烷基、炔基、环烷基、芳基、或杂芳基，或者选自烃基和杂环基，其任一者任选地被1、2、3、4或5个独立地选自卤素、三氯甲基、三氟甲基、氰基、氨基、羟基、C1-6烷基和C1-6烷氧基的基团取代。
所述的糖苷基团通常为碳水化合物，尤其是单糖基、二糖基、三糖基、四糖基或多糖基团，并且可以各种异构体形式存在，例如α-D、α-L、β-D或β-L形式。所述碳水化合物基团可任选地经其它类型的取代基或甚至额外糖苷基取代。然而，化合物的化学结构中所含单糖基和经取代单糖基的总数不可超过10。
糖苷基可为结构式(i)或(ii)的基团：
其中
R19和R20可一起形成氧代基；或者当R19和R20中的一个为氢或卤素时，R19和R20中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
R21和R22可一起形成氧代基；或者当R21和R22中的一个为氢或卤素时，R21和R22中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
R23和R24可一起形成氧代基；或者当R23和R24中的一个为氢或卤素时，R23和R24中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
R25和R26可一起形成氧代基；或者当R25和R26中的一个为氢或卤素时，R25和R26中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、-CH2R27和-C(O)R27；
R27独立地选自氢、卤素、三氟甲基、氰基、硝基、任选地被1个、2个、3个、4个或5个独立地选自R16的基团取代的烃基、任选地被1个、2个、3个、4个或5个独立地选自R16的基团取代的-(CH2)k-杂环基、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18、-N(R17)S(O)2R18、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基。
在某些实施方案中，式(I)的化合物为选自ANL-2的化合物：
在某些实施方案中，式(I)的化合物为选自ANL-3、ANL-4、ANL-5、ANL-6或ANL-7的化合物：
在某些实施方案中，R1为氢、烷基、芳基、杂芳基(如呋喃、噻吩、吡啶等)、-N(R17)R18、-N＝C(R17)(R18)、任选地被三烷基硅烷取代的炔基、糖苷基团、任选地被1、2、3、或4个独立地选自烷基的基团取代的1,3,2-二氧杂环戊硼烷、或-O-(CR2)m-R16，其中m为选自1-4、1-3、或1-2的整数；R在每次出现时独立地为氢、烷基、环烷基、或芳基；并且R16为炔基、氰基、-OR17、-N(R17)R18、-C(O)N(R17)R18、或-C(O)OR17。
在某些实施方案中，R1为-O-CH2-R16，其中R16为炔基、氰基、-OR17、-N(R17)R18、-C(O)N(R17)R18、或-C(O)OR17。
在某些实施方案中，R3和R4中的每者独立地选自-OR15和-OC(O)R15，其中R15为氢、烷基、环烷基、杂环基、芳基和杂芳基；或者R3和R4与它们所连接的碳原子一起形成5-6元杂环基。在某些实施方案中，R3和R4为-OR15，其中R15为C1-C6烷基、C1-C4烷基、或C1-C2烷基。
在某些实施方案中，R8和R9中的每者独立地选自-OR15和-OC(O)R15，其中R15为氢、烷基、环烷基、杂环基、芳基和杂芳基；或者R8和R9与它们所连接的碳原子一起形成5-6元杂环基。在某些实施方案中，R8和R9与它们所连接的碳原子一起形成亚甲二氧基环。
在某些实施方案中，R11和R12一起形成氧代基。
在某些实施方案中，R3和R4中的每者为-O-烷基；R8和R9与它们所连接的碳原子一起形成亚甲二氧基环；R1为杂芳基、-OR15、-C(O)R15、-N(R17)R18、-N(R17)C(O)R18、-N＝C(R17)R18、频哪醇硼基、-OS(O)2CF3、糖苷基团、任选地被1或2个独立地选自R16的基团取代的杂环基、或任选地被三烷基硅烷取代的炔基；或者R1为-OCH2-氰基、-OCH2-C≡CH、-OCH2-C(O)N(R17)R18、或-C(O)OR17；并且R11和R12一起形成氧代基。
在某些实施方案中，当R19和R20中的一个为氢时，R19和R20中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15。
在某些实施方案中，当R21和R22中的一个为氢时，R21和R22中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15。
在某些实施方案中，当R23和R24中的一个为氢时，R23和R24中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15。
在某些实施方案中，当R25和R26中的一个为氢时，R25和R26中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、-CH2R27和-C(O)R27。
本发明的化合物的实例包括以下所示的那些。当然，应当理解，在适当的情况下，每种化合物可为游离化合物、酸或碱加成盐、或前药的形式。
本发明还提供了一种药物组合物，该药物组合物包含至少一种本文所述的化合物以及至少一种药学上可接受的辅料(比如赋形剂)；可按照药学上记载的制剂制备方法制成制剂。
根据标准药学实践，本文所述的化合物及其药学上可接受的盐可以单独地或者与药学上可接受的赋形剂、载体和/或稀释剂组合地以药物组合物给药于受试者。化合物可以口服或胃肠外给药。肠胃外给药包括静脉内、肌内、腹膜内、皮下和局部，优选的方法是静脉内和局部给药。
因此，本发明提供了药学上可接受的组合物，该药学上可接受的组合物包含治疗有效量的一种或多种本文所述的化合物，所述化合物与一种或多种药学上可接受的赋形剂、载体(添加剂)和/或稀释剂一起配制。本发明的药物组合物可以尤其配制用于以固体或液体形式给药，包括适于以下的那些：(1)肠胃外给药，例如，通过皮下、肌内、静脉内或硬膜外注射，作为例子如无菌溶液或混悬剂、或持续释放制剂；以及(2)口服给药，例如，灌服剂(水性或非水性溶液或混悬剂)、片剂(例如，针对颊面、舌下和全身吸收的那些)、大丸剂、散剂、颗粒剂、用于施用于舌的糊剂。
如本文所陈述，本文所述的化合物的某些实施方案可含有碱性官能团，如氨基，并因此能够与药学上可接受的酸形成药学上可接受的盐。在这方面，术语“药学上可接受的盐”是指本发明的化合物的相对无毒的无机和有机酸加成盐。这些盐可在给药媒介物或剂型制造过程中原位制备，或者通过使经纯化的呈其游离碱形式的本发明化合物与合适的有机或无机酸单独地反应，并且在后续纯化期间分离由此形成的盐来制备。代表性的盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、硝酸盐、乙酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、苯甲酸盐、乳酸盐、磷酸盐、甲苯磺酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、萘酸盐、甲磺酸盐、葡庚糖酸盐、乳糖酸盐和月桂基磺酸盐等。
本发明的化合物的药学上可接受的盐包括化合物的常规无毒盐或季铵盐，例如来自无毒有机或无机酸。例如，这样的常规无毒盐包括衍生自无机酸的那些，所述无机酸如盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等；以及由有机酸制备的盐，所述有机酸如乙酸、丙酸、琥珀酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、柠檬酸、抗坏血酸、棕榈酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、磺胺酸、2-乙酰氧基苯甲酸、富马酸、甲苯磺酸、甲磺酸、乙烷二磺酸、草酸、异硫羰酸等。
在其他情况下，本文所述的化合物可以含有一个或多个酸性官能团，并因此能够与药学上可接受的碱形成药学上可接受的盐。在这些情况下，术语“药学上可接受的盐”是指本发明的化合物的相对无毒的无机和有机碱加成盐。这些盐同样可在给药媒介物或剂型制备过程中原位制备，或者通过使经纯化的呈其游离酸形式的化合物与合适的碱(如药学上可接受的金属阳离子的氢氧化物、碳酸盐或碳酸氢盐)，与氨，或者与药学上可接受的有机伯胺、仲胺或叔胺单独地反应来制备。代表性的碱金属或碱土金属盐包括锂、钠、钾、钙、镁和铝盐等。可用于形成碱加成盐的代表性有机胺包括乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、哌嗪等。
润湿剂、乳化剂和润滑剂(如月桂基硫酸钠和硬脂酸镁)，以及着色剂、释放剂、包衣剂、甜味剂、矫味剂和芳香剂、防腐剂、增溶剂、缓冲剂和抗氧化剂也可存在于组合物中。
制备这些制剂或化合物的方法包括使本文所述的化合物与载体或赋形剂以及任选地一种或多种辅助成分结合的步骤。通常，通过将本公开的化合物与液体载体(液体制剂)、液体载体继之以冻干(用于经无菌水等重构的粉末制剂)、或细粒固体载体、或这两者均匀且紧密地结合，然后根据需要使产物成形或包装来制备制剂。
适用于肠胃外给药的本发明的药物组合物包含一种或多种本文所述的化合物，该化合物组合有一种或多种药学上可接受的无菌等渗水性或非水性溶液、分散体、混悬剂或乳剂、或者可在临用前重构为无菌可注射溶液或分散体的无菌粉末，其可含有糖、醇、抗氧化剂、缓冲剂、抑菌剂、螯合剂、使得制剂与预期受体的血液等渗的溶质或悬浮剂或增稠剂。
可用于本发明的药物组合物中的合适的水性和非水性载体的实例包括水、乙醇、多元醇(如甘油、丙二醇、聚乙二醇等)以及它们的合适的混合物、植物油(如橄榄油)和可注射有机酯(如油酸乙酯)。适当的流动性可例如通过使用如卵磷脂的包衣材料，通过在分散体的情况下维持所需的粒度，以及通过使用表面活性剂来维持。
这些组合物还可以含有佐剂，如防腐剂、润湿剂、乳化剂和分散剂。可以通过包含各种抗细菌剂和抗真菌剂，例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸等来确保防止微生物对本发明的化合物的作用。还可期望在组合物中包括等渗剂，如糖、氯化钠等。此外，可注射药物形式的延长吸收可以通过包含延缓吸收的试剂如单硬脂酸铝和明胶来实现。
本发明提供了具有抗SARS-CoV-2芳基萘类似物以及它们的合成。合成本发明的化合物并且评价其的抗SARS-CoV-2活性、在小鼠体内的毒性以及药物代谢动力学研究。
实施例
下面将对本发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的其他实施例，都属于本发明保护的范围。除非另外说明，份为重量份，温度为℃或者为环境温度，并且压力等于或接近大气压。存在反应条件的多种变型形式和组合，例如组分浓度、温度、压力，以及可用于优化由所描述过程获得的产物纯度和收率的其他反应范围和条件。仅需要合理和常规的实验来优化这样的过程条件。本发明的化合物的实例包括以下所示的那些。当然，应当理解，在适当的情况下，每种化合物可为游离化合物、酸或碱加成盐、或前药的形式。
实施例1山荷叶素(代号ANL-1或2.15)的全合成
以2-溴-4,5-二甲氧基苯甲醛(1)为原料，通过乙二醇保护制备缩醛(2)，(2)与胡椒醛反应得到化合物(3)，(3)在乙酸存在下通过加热迅速转化为异苯并呋喃，再通过Diels-Alder反应得到双酯(4)，将其还原得到山荷叶素(5)，即ANL-1或2.15。
在2-溴-4,5-二甲氧基苯甲醛(1)(30g，125mmol)的甲苯溶液(1L)中加入乙二醇(14mL，250mmol)、对甲苯磺酸(5.2g，25mmol)，安装分水器并加热至140℃过夜。冷却至室温，用旋转蒸发仪减压蒸除甲苯。用乙酸乙酯溶解反应混合物，并依次用饱和碳酸氢钠溶液、水、饱和氯化钠溶液萃取，并用无水硫酸钠干燥有机相。减压蒸干得到的粗产物再经硅胶柱层析得到缩醛2-(2-溴-4,5-二甲氧基)-1,3-二氧戊环(2)(33.5g，95％)。将上述缩醛2-(2-溴-4,5-二甲氧基)-1,3-二氧戊环(2)(30g，104mmol)溶于1L无水四氢呋喃中，冷却至-78℃，逐滴加入正丁基锂溶液(1.8M，69.3mL，124.8mmol)，搅拌30分钟，逐滴加入胡椒醛(15.6g，104mmol)的四氢呋喃溶液并缓慢升至室温。2小时后，将反应液用水淬灭，并用乙酸乙酯萃取。合并的有机相分别用水和饱和氯化钠溶液萃取，并用无水硫酸钠干燥，减压蒸馏得到淡黄色油状液体(3)并迅速用于下一步Diels-Alde反应。将上述中间体3溶于二氯甲烷(100m L)中，加入冰醋酸(35mL)和丁炔二酸二甲酯(16.7mL，104mmol)。反应于140℃回流12小时。反应液冷却至室温后用水稀释，反应液用二氯甲烷萃取。合并有机相，用无水硫酸钠干燥，并经硅胶柱层析分离得到黄色固体(4)(30g，62％)。将上述二酯化合物中间体(4)(5mmol)溶于无水四氢呋喃(70mL)中，缓慢加入硼氢化钠(0.945g，25mmol)并于80℃加热回流12小时。反应液冷却至室温，并用2M盐酸逐滴酸化至pH值接近2。酸化后，反应液经乙酸乙酯萃取，合并有机相，并经水和饱和氯化钠洗涤，无水硫酸钠干燥，硅胶柱层析得到淡黄色固体(5)，即ANL-1或2.15(山荷叶素，1.8g，95％)，为一种已知有机化合物。HR-EIMS:m/z[M+H]+381.0914(calcd 381.0912；M＝C21H16O7).1H NMR(400MHz,acetone-d6)δ7.69(1H,s,H-6),7.09(1H,s,H-3),6.96(1H,d,J＝7.9Hz,H-5'),6.85(1H,d,J＝1.6Hz,H-2'),6.91(1H,dd,J＝7.9,1.7Hz,H-6'),6.09(1H,br s,OCH2O),6.07(1H,br s,OCH2O),5.37(2H,s,H-9),3.99(3H,s,5-OMe),3.73(3H,s,4-OMe)。
实施例2 7-去氧-7-乙炔基山荷叶素(ANL-2)的全合成
在氮气保护下，山荷叶素(5)(3.8g，10mmol)，4-二甲氨基吡啶(2.44g，20mmol)溶于100mL超干二氯甲烷中，冷却至0℃，逐滴加入三氟甲磺酸酐(2.5mL，12mmol)，于室温下搅拌4小时。薄层色谱显示反应完成并生成新点。反应液旋干得到淡黄色的固体，并用乙醇洗涤纯化得到产物(6)(4.61g，90％)。在100mL圆底烧瓶中加入二氯双(三苯基膦)钯(70mg，0.1mmol)，碘化铜(57.2mg，0.3mmol)，化合物(6)(1g，2mmol)。在氮气保护下在上述反应液中逐滴加入三甲基乙炔基硅(0.4g，4mmol)和三乙胺(0.6g，6mmol)的二甲基甲酰胺溶液。反应液加热于80℃搅拌10小时。冷却至室温后，反应液经水淬灭并用二氯甲烷萃取。有机相经无水硫酸钠干燥后，经硅胶柱层析分离纯化得到产物(7)(0.8g，90％)。化合物(7)(240mg，0.52mmol)溶于25mL甲醇中，并加入碳酸钾(138mg，1.04mmol)，于室温下搅拌2小时。反应混合物经硅胶柱层析分离纯化得到目标产物ANL-2(122mg，70％)。纯度99.12％(HPLC)(图1)。HR-EIMS:m/z[M+H]+389.1015(calcd.389.1020；M＝C23H16O6)；[M+Na]+411.0833(calcd.411.0839)(图2).1H NMR(400MHz,CDCl3)δ7.68(1H,s,H-6),7.14(1H,s,H-3),6.97(1H,d,J＝8.0Hz,H-5′),6.85(1H,d,J＝1.2Hz,H-2′),6.84(1H,dd,J＝8.0,4.4Hz,H-6′),6.10(1H,d,J＝1.2Hz,-OCH2O-),6.06(1H,d,J＝1.2Hz,-OCH2O-),5.43(2H,d,J＝5.2Hz,H-9),4.11(3H,s,5-OMe),3.82(3H,s,4-OMe),3.802(1H,s,H-2″)(图3).13C NMR(100MHz,CDCl3)δ169.6(C-9'),152.7(C-5),150.3(C-4),147.8(C-3'),147.6(C-4'),143.8(C-7),140.4(C-7'),133.3(C-2),128.8(C-1'),127.9(C-1),123.5(C-6'),121.4(C-8'),118.4(C-8),110.5(C-2'),108.3(C-5'),106.4(C-3),104.1(C-6),101.4(-OCH2O-),87.7(C-2″),77.7(C-1″),68.2(C-9),56.2(5-OMe),55.9(4-OMe)(图4)。
实施例3 7-氧-乙腈基山荷叶素(ANL-3)的全合成
将山荷叶素5(38mg,0.1mmol)和溴乙腈(24mg,0.2mmol)溶于1mL的丙酮中，缓慢加入Cs2CO3(65mg,0.2mmol)。搅拌12小时，TLC显示形成一个极性较低的点。混合物经制备硅胶板纯化得到所需产物ANL-3(40mg,95％)。HR-EIMS m/z[M+H]+420.1061(calcd.420.1079；M＝C23H17NO7).1H NMR(400MHz,DMSO-d6)δ7.52(1H,s,H-6),7.05(1H,d,J＝7.9Hz,H-5'),7.01(1H,s,H-3),6.91(1H,d,J＝1.6Hz,H-2'),6.79(1H,dd,J＝1.7,7.9Hz,H-6'),6.13(2H,s,-OCH2O-),5.60(2H,s,H-1″),5.34(2H,s,H-9),3.99(3H,s,5-OCH3),3.68(3H,s,4-OCH3)(图5)。13C NMR(100MHz,DMSO-d6)δ168.8(C-9'),151.8(C-5),150.2(C-4),147.1(C-3'),147.0(C-4'),144.7(C-7),135.5(C-7'),129.8(C-2),128.4(C-1'),127.9(C-8'),126.0(C-1),123.6(C-6'),118.8(C-8),117.1(C-2″,C≡N),110.8(C-2'),108.1(C-5'),105.8(C-3),101.2(C-6),100.3(-OCH2O-),66.1(C-9),58.0(C-1″,CH2),55.9(5-OCH3),55.3(4-OCH3)(图6)。
实施例4山荷叶素糖苷衍生物ANL-4的全合成
取D-木糖(91.6mg，0.61mmol)溶于吡啶(2mL)溶液中加入乙酸酐(Ac2O，0.4mL，4.21mmol)室温下搅拌过夜。反应结束后，加CH2Cl2稀释，用10％ HCl溶液洗涤，合并有机相，用NaHCO3饱和溶液萃取，取有机相，无水Na2SO4干燥，浓缩得全乙酰化木糖粗产物，无需进一步纯化，直接用于下一步，即溶解在CH2Cl2(5mL)中，冷却至0℃，然后滴入溴化氢(HBr，33％ AcOH，1.5mL)。将反应加热至室温，再搅拌4小时。然后用水淬灭反应，用CH2Cl2萃取水层。合并有机层，用水、10％NaHCO3和饱和食盐水洗涤，用Na2SO4干燥，在减压下浓缩以得到糖基溴。糖基溴粗产物浓缩后直接用于下一步，无需进一步纯化。山荷叶素(ANL-1，215mg，0.61mmol)溶解于CHCl3(15mL)和氢氧化钠水溶液(NaOH，0.1M，20mL)中，加入四丁基溴化铵TBAB(306mg，0.95mmol)，反应加热至40℃保持10min，加入糖基溴(0.61mmol)，40℃搅拌12h。冷却至室温后，用CHCl3萃取水层，结合的有机层用饱和食盐水洗涤，Na2SO4干燥，减压浓缩。用硅胶色谱柱(正己烷/乙酸乙酯＝1/1)分离反应混合物，得到所需的产物山荷叶素-2″,3″,4″-三乙酰氧基-7-O-β-D-木吡喃糖苷ANL-4(233.7mg，三步产率60％)。HR-EIMS m/z[M+H]+639.1684(calcd.639.1708；M＝C32H30O14).1H NMR(400MHz,CDCl3)δ7.51(1H,d,J＝1.0Hz,H-6),7.07(1H,br s,H-3),6.95(1H,d,J＝7.6Hz,H-5'),6.82(1H,m,H-6'),6.80(H,br s,H-2'),6.09和6.05(各1H,m,OCH2O),5.48–5.38(5H,m,H-2″,H-3″,H-4″,OCH2O),5.32(1H,d,J＝8.8Hz,H-1″),5.12(2H,m,H-9),4.13(1H,dd,J＝7.2,4.8Hz,Hb-5″),4.07(3H,s,5-OCH3),3.80(3H,s,4-OCH3),3.41(1H,m,Ha-5″),2.12(3H,s,Ac),2.10(3H,s,Ac),2.07(3H,s,Ac)(图7)。13C NMR(100MHz,CDCl3)δ170.2(Ac),170.0(Ac),169.6(Ac),169.5(C-9'),152.1(C-5),150.5(C-4),149.6(C-3'),147.7(C-4'),144.1(C-7),136.9(C-7'),136.5(C-1),130.9(C-2),128.3(C-1'),127.9(C-6'),126.5(C-8),123.7(C-6),119.4(C-3),110.8(C-2'),108.4(C-8'),106.4(C-5'),101.6(C-1″),101.4(-OCH2O-),80.8(C-2″),79.9(C-4″),71.8(C-3″),71.5(C-5″),68.8(C-9),56.4(5-OMe),56.0(4-OMe),21.0(Ac),20.9(Ac×2)(图8)。
实施例5山荷叶素糖苷衍生物ANL-5、ANL-6和ANL-7的全合成
先在室温下将ANL-4经NaOH水溶液(ANL-4与NaOH的摩尔比为1:1.2)处理，将其糖基部分的三个O-乙酰基脱去，产物转移到二氯甲烷中，得到山荷叶素7-O-木糖苷(8)。再将化合物8(150mg，0.3mmol)溶于干乙腈(1.5mL)中，加入乙酸酐(32μL，0.32mmol)和乙酸四丁基铵(27mg，0.09mmol)，反应液在40℃下搅拌12h。反应完成后，将溶剂用旋转蒸发仪浓缩后通过硅胶柱层析(石油醚/乙酸乙酯＝1.5/1，等度洗脱)分离纯化，得到产物山荷叶素-3″-乙酰氧基-β-D-木吡喃糖苷ANL-5(99.8mg，得率60％)。在三乙胺/4-二甲氨基吡啶(Et3N/DMAP)存在下，将ANL-5用氯甲酸丙烯酯(ANL-5与氯甲酸丙烯酯的摩尔比为1:2)0℃至室温处理15h，产物经硅胶柱层析分离，得到山荷叶素木糖苷的烯丙氧羰氧基(O-alloc)和氧乙酰基(O-Ac)取代产物，包括山荷叶素-3″-乙酰氧基-2″,4″-二烯丙氧羰氧基-β-D-木吡喃糖苷ANL-6，以及3″-乙酰氧基-4″-烯丙氧羰氧基-β-D-木吡喃糖苷4-β-D-木吡喃糖苷ANL-7(ANL-6，5mg，得率20％；ANL-7，9.5mg，得率41％)。
ANL-5：HR-EIMS m/z[M+H]+555.1471(calcd.555.1497；M＝C28H24O12).1H NMR(400MHz,CDCl3)δ7.86(1H,s,H-6),6.98(1H,m,H-3),6.87(1H,m,H-5'),6.75(1H,m,H-6'),6.68(H,m,H-2'),6.03和5.99(各1H,br s,OCH2O),5.44和5.34(各1H,br d,J＝15.2Hz,H-9),4.88(1H,t,J＝8.8Hz,H-3″),4.82(1H,m,H-1″),4.04和4.01(各1H,br d,J＝4.8Hz,H-5),3.97(3H,s,5-OCH3),3.74(3H,s,4-OCH3),2.04(3H,s,Ac)(图9)。13C NMR(100MHz,CDCl3)δ173.3(Ac),170.2(C-9'),152.1(C-5),150.3(C-4),147.6(C-3'),147.6(C-4'),144.4(C-7),136.7(C-7'),131.0(C-1),130.8(C-2),128.3(C-1'),128.3(C-6'),119.1(C-8),110.8(C-6),110.8(C-3),108.3.8(C-2'),106.3(C-8'),105.4(C-5'),101.4(-OCH2O-),101.0(C-1″),79.2(C-4″),72.3(C-2″),68.6(C-3″),67.6(C-9),65.7(C-5″),56.4(5-OMe),55.9(4-OMe),21.2(Ac)(图10)。
ANL-6：HR-EIMS:m/z[M+H]+723.1897(calcd.723.1920；M＝C36H34O16).1H NMR(400MHz,CDCl3)δ7.55(1H,s,H-6),7.10(1H,s,H-3),6.97(1H,d,J＝8.0Hz,H-5'),6.83-6.79(2H,m,H-2',6'),6.10-6.05(2H,m,-OCH2O-),5.93和5.88(各1H,m,H-9),5.50-5.22(8H,m,O-Alloc,H-2″,H-3″),5.06(1H,d,J＝8.0Hz,H-1″),5.00(1H,br td,J＝9.2,5.6Hz,H-4″),4.74-4.62(4H,m,O-Alloc),4.24(1H,dd,J＝11.2,5.6Hz,Ha-5″),4.06(3H,s,5-OMe),3.81(3H,s,4-OMe),3.36(1H,br t,J＝11.2Hz,Hb-5″),2.11(3H,s,OAc)(图11)。13C NMR(100MHz,CDCl3)δ170.1(Ac),169.7(C-9'),154.3(Alloc),154.0(Alloc),152.4(C-5),150.4(C-4),147.7(C-3'),147.7(C-4'),137.1(C-7),131.2(Alloc),131.1(Alloc),131.0(C-7'),130.9(C-1),128.2(C-2),126.7(C-1'),123.7(C-6'),120.1(C-8),119.7(Alloc),119.4(Alloc),110.8(C-2'),110.7(C-8'),108.4(C-5',C-6),106.5(C-3),102.3(C-1″),101.4(-OCH2O-),75.6(C-3″),72.2(C-2″),71.9(C-4″),69.6(Alloc),69.3(Alloc),67.2(C-9),63.0(C-5″),56.7(5-OMe),56.0(4-OMe),20.9(Ac)(图12)。
ANL-7：HR-EIMS:m/z[M+H]+639.1680(calcd.639.1708；M＝C32H30O14).1H NMR(400MHz,CDCl3)δ7.90(1H,s,H-6),δ7.07(1H,d,s,H-3),δ6.94(1H,br d,J＝8.0Hz,H-5'),6.82-6.76(2H,m,H-2',6'),6.09和6.04(各1H,br s,-OCH2O-),5.96–5.87(1H,m,Alloc烯氢),5.50-5.28(5H,m,Ha-9,Alloc烯氢,H-3″,H-4″),5.13(1H,t,J＝8.0Hz,H-1″),4.95(1H,m,Hb-9),4.88(1H,d,J＝8Hz,H-2″),4.65-4.63(2H,m,Alloc烷氢),4.24(1H,m,Ha-5″),4.02(3H,s,5-OMe),3.80(3H,s,4-OMe),3.35(1H,m,Hb-5″),2.18(3H,s,OAc)(图13)。13C NMR(100MHz,CDCl3)δ172.1(Ac),169.9(C-9'),154.1(Alloc),152.2(C-5),150.4(C-4),147.7(C-3'),147.6(C-4'),144.2(C-7),137.0(C-7'),131.2(C-6),131.1(Alloc),130.9(C-8),128.3(C-1),127.2(C-2),123.7(C-1'),119.7(Alloc),119.3(C-3),110.8(C-2'),108.3(C-5'),106.4(C-6'),105.5(C-1″),101.4(-OCH2O-),100.9(C-8'),75.4(C-3″),73.1(C-4″),72.1(C-2″),69.3(Alloc),67.4(C-9),62.8(C-5″),56.5(5-OMe),56.0(4-OMe),21.1(Ac)(图14)。
实施例6
抗-SARS-CoV-2体外活性的测定(方法1)
(一)ANL-1、ANL-2、ANL-3、ANL-4、ANL-5、ANL-6或ANL-7等7种芳基萘木脂素对Vero E6细胞毒性评价(方法1)
96孔板内，2×104个/孔接种Vero E6细胞，37℃，5％ CO2培养过夜，待单层细胞长至70％左右时，加入不同浓度的待测药物(7种山荷叶素或其糖苷衍生物ANL-1-ANL-7，或，阳性对照药瑞德西韦)，100μL/孔，设3个重复孔。同时设置不含药物的阴性对照孔。37℃，5％ CO2培养72h，采用CCK8试剂盒检测细胞存活率。Bio-Tek Synergy 2多功能微孔板检测仪测定OD值，测定波长为450nm，参考波长为630nm。计算CC50值(50％ Cytotoxic Concentration)，即对50％的Vero E6产生毒性时的药物浓度。
(二)ANL-1、ANL-2、ANL-3、ANL-4、ANL-5、ANL-6或ANL-7等7种芳基萘木脂素抗SARS-CoV-2体外活性的测定(方法1)
Vero E6细胞(常规市售产品)均以含10％胎牛血清的DMEM高糖完全培养基进行培养。在实验前1天将细胞传代一次，使所用细胞处于对数生长期。SARS-CoV-2，Vero E6细胞内扩增，收取培养液，0.22μm滤膜过滤，0.5mL/支分装，-80℃保存。96孔板内接种Vero E6细胞，2×104个/孔，37℃，5％ CO2培养过夜，待单层细胞长至70％左右时，转移至P3实验室待用。药物稀释：设6个浓度梯度，每个梯度3个重复孔。P3实验室内，在细胞培养板内，每孔同时加入预先配制的药物(7种山荷叶素或其糖苷衍生物ANL-1～ANL-7，或，阳性对照药瑞德西韦)50μL和50μL病毒稀释上清(MOI＝0.1)。设置不含药物和病毒的阴性对照、不含山荷叶素或其糖苷衍生物但是含有瑞德西韦的阳性对照。37℃，5％ CO2培养72h，采用CCK8试剂盒检测细胞存活率，Bio-Tek EON微孔板检测仪测定OD值，测定波长为450nm，参考波长为630nm。计算药物对病毒复制的抑制率和IC50。IC50值(50％ Inhibitory Concentration)是指能够抑制50％的Vero E6生长时的药物浓度。
方法1实验结果：
山荷叶素或其糖苷衍生物ANL-7、ANL-2和ANL-6的体外抗新冠病毒(SARS-CoV-2)活性最强，半数抑制浓度IC50分别是0.23、0.44和0.38μM；治疗指数TI值分别为>1375、>1160和>730；相比之下，阳性对照药瑞德西韦(Remdesivir)的体外抗新冠病毒活性较低，IC50是2.75μM，TI值为72.81。化合物ANL-7、ANL-2和ANL-6的体外抗新冠病毒活性以TI值计，分别是瑞德西韦的19倍、16倍和10倍。见表1。
表1.7种芳基萘木脂素化合物及瑞德西韦的体外抗新冠病毒活性(方法1)

注：CC50：样品对Vero E6细胞的半数抑制浓度；IC50：样品对SARS-CoV-2致半数Vero E6细胞死亡时的浓度；治疗指数
TI＝CC50/IC50。
据文献报道，莫诺拉韦的磷酸酯活性结构和奈玛特韦在Vero E6细胞模型的体外抗新冠病毒活性以TI值计，分别为瑞德西韦的1.2倍和0.4倍(Fiaschi L,et al.Viruses 2022；14:1374.)；阿兹夫定(磷酸酯活性结构)的IC50为4.3μM，抗病毒活性低于瑞德西韦(Zhang JL,et al.Signal Transduct Target Ther.2021；6:414)。以治疗指数计，这些上市药物的体外抗新冠病毒活性均远低于ANL-7、ANL-2和ANL-6。
(三)抗冠状病毒体内外活性的测定(方法2)
病毒与生物安全：SARS-CoV-1GZ50(GenBank：AY304495)、MERS-CoV(EMC/2012，GenBank：JX869059)、SARS-CoV-2野生型(WT，HKU-001a，GenBank：MT230904)和奥米克戎BA 5.2(OmicronBA.5.2，GISAID:EPI_ISL_13777658)病毒来源于香港大学(HKU)微生物学系。SARS-CoV-1和MERS-CoV在Vero E6细胞中繁殖。SARS-CoV-2的所有变体均使用VeroE6-TMPRSS2细胞行培养和通过斑点测定法进行滴定。所有活病毒的培养、体内和体外实验是在香港大学生物安全三级实验室严格按照操作规范进行。
细胞培养：Caco2细胞来自ATCC(ATCC HTB-37)，并根据供应商的指南在Dulbecco改良的Eagle培养基(DMEM)(Gibco，Amarillo，Texas，USA)中培养。VeroE6-TMPRSS2细胞来自日本生物资源研究中心(JCRB)细胞库(JCRB1819)，并根据说明在Dulbecco改良的Eagle培养基(DMEM)(Gibco，Amarillo，Texas，USA)中培养。本研究中使用的所有细胞系定期进行支原体检测，并在无支原体的环境中培养。
ANL-2体外抗冠状病毒半抑制浓度测定：
把SARS-CoV-1、MERS-CoV、SARS-CoV-2WT或SARS-CoV-2BA.5.2(MOI＝0.1)加到Caco2细胞。感染后2小时，去除病毒接种物，用磷酸盐缓冲盐水(PBS)洗涤细胞3次，然后加入不同浓度ANL-2(0～10μM)。感染后24小时利用QIAsymphony RNA试剂盒(Qiagen，德国)进行RNA提取和qRT-PCR以量化RdRp、NP或sgRNA基因拷贝，然后使用GraphPad Prism 6计算IC50。
细胞活力通过CellTiter-Glo发光细胞活力测定试剂盒(Promega，美国)对细胞活力进行量化。加入不同浓度的ANL-2(0～10μM)到Caco2细胞并孵育24小时，然后按照制造商的说明进行处理使用Victor X3 2030多功能微量盘分析仪(Perkin Elmer，美国)检测发光信号。
抗SARS-CoV-2体内活性的测定：
动物实验得到香港大学Committee on the Use of Live Animals in Teaching and Research(CULATR)的批准。6-8周大的雄性和雌性叙利亚仓鼠是通过香港大学比较医学研究中心(CCMR)从香港中文大学实验动物服务中心取得。仓鼠保持在65％的湿度和21～23℃的环境温度下，自由饮食饮水，光暗周期为12小时。
仓鼠被腹腔注射氯胺酮(200mg/kg)和甲苯噻嗪(10mg/kg)麻醉后，在仓鼠鼻内接种每只仓鼠50μLSARS-CoV-2WT。SARS-CoV-2WT储备液利用PBS稀释，浓度为每只仓鼠3×103PFU。在感染后6小时，每只仓鼠用ANL-2(50mg/kg或25mg/kg)或35％ PEG400水溶液在1000μL最终体积下进行腹腔注射。受感染的仓鼠随后在感染后第1、2和3天腹腔注射ANL-2(50mg/kg或25mg/kg)或35％ PEG400水溶液，总共4剂。所有仓鼠在感染后第4天被处死，用于病毒学评估。收集仓鼠肺和鼻甲组织后，使用RNeasy Mini试剂盒(Qiagen，德国)从仓鼠肺组织中提取RNA，并使用QuantiNova Probe RT-PCR试剂盒(Qiagen，德国)通过RNA依赖性RNA聚合酶(RdRp)对SARS-CoV-2的病毒基因拷贝进行了量化。
其中，50mg/kg或25mg/kg的ANL-2是将ANL-2溶解于35％ PEG400水溶液中给药。
我们对ANL-2在宿主Caco2细胞内抑制新型冠状病毒复制的作用亦进行了研究评估(方法2)。首先，ANL-2对宿主Caco2细胞活力的作用以发光细胞活力测定试剂盒测定，发现ANL-2在其检测浓度范围内(0～10μM)对Caco2细胞活力没有影响，CC50值大于10μM(图15)。在ANL-2体外抗冠状病毒半抑制浓度的测定中，Caco2细胞分别被各种冠状病毒感染后(包括SARS-CoV-1、MERS-CoV、SARS-CoV-2WT和SARS-CoV-2BA.5.2)加入不同浓度的ANL-2(0～10μM)，孵育24小时进行RNA提取和qRT-PCR以量化RdRp、NP或sgRNA基因拷贝。根据图16的结果，ANL-2有效地减少了SARS-CoV-1(IC50＝2.69nM)，MERS-CoV(IC50＝1.49nM)，SARS-CoV-2野生型(IC50<0.64nM)和变体BA.5.2(IC50＝9.06～16.3nM)在Caco2细胞内的复制。
叙利亚仓鼠动物模型被用于评估ANL-2的体内抗SARS-CoV-2活性。由图17可见，ANL-2(25mg/kg)可显著降低受SARS-CoV-2WT感染的仓鼠肺组织的病毒量，却对鼻甲的病毒量没有影响。此外，ANL-2高剂量组(50mg/kg)对受SARS-CoV-2WT感染的仓鼠肺/鼻甲组织的病毒量没有显著，这可能是由于ANL-2在高剂量组(50mg/kg)注射液的溶解度较低剂量组(25mg/kg)低而引致。
实施例7用于动物体内安全性和药物代谢动力学评价的ANL-2测试液的制备
以0.5％羧甲基纤维素钠(CMC-Na)水溶液配制ANL-2混悬液作为测试液1，给药剂量为500mg/kg、2000mg/kg和5000mg/kg；配制0.3％(w/v)浓度的ANL-2脂肪乳剂作为测试液2。两种测试液的配制方法如下：
ANL-2混悬液：称取3.00g的ANL-2，加入一定体积的0.5％CMC-Na水溶液，涡旋并超声分散均匀，以0.5％ CMC-Na水溶液稀释，分别制成浓度50.00mg/mL、66.67mg/mL和166.67mg/mL的ANL-2混悬液，即为测试液1。
ANL-2脂肪乳剂：
(1)称取4.50g的ANL-2，加入300.00g大豆油，1.50g油酸，搅拌并超声使药物分散均匀，得油相。
(2)称取30.00g大豆卵磷脂，15.00g泊洛沙姆P-68，33.75g甘油，0.75g维生素E，加入1100mL超纯水，在75℃分散均匀，得水相。
(3)油相与水相分别预热至75℃，将油相缓慢滴加至水相中，边滴加边搅拌，然后用超纯水定容至1500mL，继续搅拌10min，在超声细胞破碎仪，以强度600W超声5min，得到初乳。
(4)将初乳在高压均质仪中均质15min，参数设置为1000Bar，即得3.00mg/mL的C180A-P1脂肪乳剂，即3.00mg/mL的ANL-2脂肪乳剂，即为测试液2。
(5)取ANL-2脂肪乳剂20μL，加生理盐水注射液稀释至1mL，用粒度分析仪测定粒径，其粒径为(145.0±82.4)nm，结果见图18。乳剂粒径符合用注射用脂肪乳剂粒径要求。
(6)取ANL-2脂肪乳剂，置透射电镜下观察并拍照，照片见图19。可见乳滴大小较均匀，直径为10nm～160nm，形态为类球形。
实施例8小鼠中ANL-2的急性毒性评价
本发明按照国家药品监督管理局规定的半数致死量Bliss法来评价样品的急性毒性。选用经适应性观察合格的ICR小鼠，按体重和性别随机均衡分为5组，每组10只动物，雌雄各半。
使用ANL-2测试液1进行小鼠口服给药急性毒性试验，在8h内对ICR小鼠口服给药1～3次，每次的给药体积为10mL/kg，累计给药剂量为500mg/kg、2000mg/kg和5000mg/kg。
对ANL-2测试液2进行小鼠静脉给药、腹腔注射给药或口服给药急性毒性试验，在8h内分别对ICR小鼠进行静脉给药、腹腔注射给药或口服给药3次，每次给药体积为16.7mL/kg，使用3.00mg/mL浓度的ANL-2测试液2，3次累计剂量为150mg/kg。
各组动物在给药后连续观察4小时，以后每天观察1～2次，持续观察14天。观察内容：外观体征、行为活动、腺体分泌、呼吸、排泄物性状、饮食情况、中毒反应和死亡情况等。给药后详细记录所有动物的死亡情况、中毒症状及中毒反应的起始时间、严重程度、持续时间、是否可逆等。中毒症状观察内容参照《药物单次给药毒性研究技术指导原则》。同时按计划进行体重、摄食量检查。试验期间如发现动物死亡或处于濒死状态，及时进行大体解剖检查，并固定异常脏器，进行组织病理学检查。存活动物按计划进行大体解剖检查，异常脏器固定后进行组织病理学检查。
ANL-2的体内急性毒性实验结果(图20)，表明ANL-2脂肪乳剂在150mg/kg剂量下对小鼠口服灌胃无明显不良反应；ANL-2的CMC-NA混悬液在5000mg/kg剂量下对小鼠口服灌胃无明显不良反应。因此，可认为ANL-2脂肪乳剂对小鼠静脉给药、腹腔注射给药或口服给药的未见不良反应剂量水平NOAEL＞150mg/kg；ANL-2的CMC-NA混悬液口服给药的NOAEL＞5000mg/kg，ANL-2的CMC-NA混悬液对小鼠不具有口服急性毒性。
实施例9ANL-2在大鼠体内的药物代谢动力学评价
本发明选用SPF级SD大鼠，雄性，220～260g，动物合格证：No.44005800013136。以上动物均购买于广州中医药大学实验动物中心(实验动物生产许可证号：SCXK(粤)2018-0034)，实验动物的使用获得伦理委员会的批准(批准文号：20211012004)。动物饲养于广州中医药大学实验动物中心(实验动物使用许可证号：SYXK(粤)2018-0001)，温度22±3℃，相对湿度30～70％，光暗周期为12小时。自由饮食饮水，饲料由广州中医药大学实验动物中提供。36只健康SD大鼠，雄性，体重(220～260g)，SPF级环境适应性饲养一周。随机分成6组，分别为尾静脉注射给药3个剂量组：2mg/kg、4mg/kg和8mg/kg和口服给药3个剂量组：15mg/kg、25mg/kg和50mg/kg。每组6只。采血前禁食12h，不禁水。其中，尾静脉注射给药和口服给药的剂型均是0.3％浓度的ANL-2脂肪乳剂。
(1)大鼠静脉注射给药与血样采集
取3.00mg/mL的ANL-2脂肪乳剂加生理盐水注射液稀释至0.2mg/mL、0.4mg/mL和0.8mg/mL，直接用于静脉注射；单次尾静脉注射2mg/kg、4mg/kg和8mg/kg剂量，给药体积为10mL/1kg，于给药前(0h)和给药后5min、15min、30min、1h、2h、3h、5h、8h、12h各采血200μL；血液均采集于含EDTA·2K的预制EP管中，3000rpm离心15min，分离血浆，-80℃冷冻保存。
(2)大鼠口服给药与血样采集
取3.00mg/mL的ANL-2脂肪乳剂加生理盐水注射液稀释至1.5mg/mL、2.5mg/mL和3mg/mL，直接用于口服；单次灌胃15mg/kg、25mg/kg和50mg/kg剂量，给药体积为10mL/1kg(50mg/kg剂量组给药体积为16.67mL/1kg)，于给药前(0h)和给药后10min、30min、1h、2h、3h、5h、8h、12h、24h、36h各采血200μL；血液均采集于含EDTA·2K的预制EP管中，3000rpm离心15min，分离血浆，-80℃冷冻保存。
(3)大鼠空白血浆的采集：健康SD大鼠6只，雄性，6～8周龄，体重(220～260g)，2％戊巴比妥钠麻醉，剂量为40～50mg/kg(0.2mL/100g)，腹主动脉取血于含EDTA·2K的真空管中，3000rpm离心15min，分离血浆，用离心管分装好，封口，-80℃冷冻保存。
采用建立的UPLC-MS/MS法测定给药后大鼠含药血浆中ANL-2的浓度，将其与对应时间点输入EXCEL进行统计分析，各组计量数据均计算Mean±SD，绘制血药浓度-时间曲线图。将所有数据按格式整理好数据表，导入DAS 3.0统计软件，采用非房室模型对每个剂量组进行数据处理，计算AUC、Cmax、tmax、t1/2、MRT、Vz/F、Clz/F、C0等主要的药代动力学参数指标。并使用Excel统计软件对以上参数进行统计分析，计算口服生物利用度，以此对试验结果进行评估。
根据血药浓度-时间曲线(图21和图22)和药代动力学参数(表2～4)可知，口服给药ANL-2脂肪乳剂后口度吸收较差，达峰浓度低，达峰时间较长，消除较慢，药时曲线下面积较低。表明该化合物口服生物利用度低，但在体内滞留时间较长。
表2.大鼠尾静脉给药ANL-2脂肪乳剂的药代动力学参数(n＝6)
表3.大鼠口服给药ANL-2脂肪乳剂的药代动力学参数(n＝6)
表4.ANL-2脂肪乳剂口服生物利用度
本文所述的化合物可表现出抗SARS-CoV-2的活性。根据本发明的方法，将式(I)的化合物给药于患者以抑制SARS-CoV-2的复制或者减少病毒的细胞病变效应。
本文所述的化合物、药物组合物和治疗方法可用于预防、治疗、或改善由冠状病毒(包括但不限于SARS-CoV-1、SARS-CoV-2和MERS-CoV)引起的感染。
为了清楚理解的目的，现在已经通过例示和实施例的方式在一些细节上充分描述了本发明，对于本领域的普通技术人员显而易见的是，在不影响本发明或其任何具体实施方案的范围的情况下，同样可以通过在条件、制剂及其他参数的较宽和等同范围内修改或改变本发明来执行，并且这样的修改或改变旨在涵盖在所附权利要求的范围内。所采用的术语和表达被作为描述而非限制性的术语使用，并且并非旨在使用这样的术语和表达排除所示出和描述的特性的任何等同物或其部分，但是应当认识到，在所要求保护的本发明的范围之内，各种修改均是可能的。因此，应当理解，尽管本发明已经由优选的实施方案和可选的特性具体地公开，但是本领域技术人员可采用本文所公开的概念的修改和变化，并且认为这样的修改和变化在本发明的范围内。
如本文所用，“包含”与“包括”同义。“含有”或“特征在于”是包括性或开放性的，并且不排除额外未列举的要素或方法步骤。如本文所用，“由……组成”排除权利要求要素中未规定的任何要素、步骤、或成分。如本文所用，“基本上由……组成”不排除实质上不影响权利要求的基本和新颖特征的材料或步骤。在本文的每种情况下，术语“包含”、“基本上由……组成”和“由……组成”中的任一者可以被其他两个术语中的任一者替代。
当在本文中公开一组材料、组合物、组分或化合物时，应理解那些组的所有各个成员及其所有亚组单独地公开。当在本文中使用马库什组或其他分组时，该组的所有各个成员以及该组的所有可能的组合和子组合旨在单独地包括在本发明中。除非另行指出，本文描述或例示的组分的每一种制剂或组合可用于实践本发明。无论何时在说明书中给出范围，例如温度范围、时间范围、或组合物范围，所有中间范围和子范围，以及包括在给出范围中的所有各个值均旨在包括在本发明中。在本发明和权利要求中，“和/或”意指除此之外或另选地。此外，任何使用的单数形式的术语也涵盖复数形式。
本文引用的所有参考文献据此全文以引用方式并入，其程度与本说明书的公开内容不存在不一致。本文所提供的一些参考文献以引用方式并入，以提供关于本发明的起始材料来源、额外的起始材料、额外的试剂、额外的合成方法、额外的分析方法、额外的生物材料、额外的细胞以及额外的用途的细节。本文使用的所有标题仅是为了方便。说明书中所提及的所有专利和出版物指示了本发明所属领域的技术人员的技术水平，并且以引用方式并入本文，其程度如同各个的出版物、专利或专利申请被具体地和单独地指示以引用方式并入。本文引用的参考文献全文以引用方式并入本文，以指示如其公开或申请日的技术状态，并且旨在该信息可根据需要在本文中采用以排除现有技术中的具体实施方案。例如，当要求保护物质组合物时，应理解在申请人的发明之前本领域已知和可获得的化合物(包括在本文引用的参考文献中提供的允许公开的化合物)不旨在包括在本文的物质组合物权利要求中。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 芳基萘木脂素类化合物或其药物上可接受的盐或前药的应用，其特征在于：为下述应用中的至少一种：
    (1)在制备用于治疗、预防新型冠状病毒感染或延缓新型冠状病毒感染的进展的药物中的应用；
    (2)在制备新型冠状病毒抑制剂中的应用；
    其中，所述新型冠状病毒包括引起严重急性呼吸综合征、中东呼吸综合征和新型冠状病毒疾病COVID-19的病毒中的至少一种；
    所述芳基萘木脂素类化合物具有式(I)：
    其中
    X为氧或硫；
    R1为R15、-OR15、-C(O)R15、或-C(O)OR15；
    R2、R5、R6、R10、R13和R14各自为氢或卤素；
    R3和R4各自独立地选自-OR15和-OC(O)R15；或者R3和R4与它们所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；
    R7、R8和R9各自独立地选自-OR15和-OC(O)R15；或者R7和R8与其所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；或者R8和R9与其所连接的碳原子一起形成任选地被1、2、3、4或5个独立地选自R16的基团取代的5-6元杂环基；
    R11和R12一起形成氧代基；或者在R11和R12中的一者为氢或卤素时，R11和R12中的另一者选自R15、-OR15、-C(O)R15和-C(O)OR15；
    R15在每次出现时独立地选自氢、炔基、卤素、三氯甲基、三氟甲基、氰基、硝基、杂芳基、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N＝C(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18、-N(R17)S(O)2R18、任选地被1、2、3或4个独立地选自烷基的基团取代的1,3,2-二氧杂环戊硼烷、糖苷基团、任选地被三烷基硅烷取代的炔基、任选地被1、2、3、4或5个独立地选自R16的基团取代的烃基、任选地被1、2、3、4或5个独立地选自R16的基团取代的杂环基，或任选地被1、2、3、4或5个独立地选自R16的基团取代的-(CH2)k-杂环基，其中k为1-6的整数；
    R16在每次出现时独立地选自炔基、卤素、三氯甲基、三氟甲基、氰基、硝基、氧代基、＝NR17、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18和-N(R17)S(O)2R18；并且
    R17和R18在每次出现时独立地为氢、烷基、炔基、环烷基、芳基、或杂芳基，或者选自烃基和杂环基，其任一者任选地被1、2、3、4或5个独立地选自卤素、氰基、氨基、羟基、C1-6烷基和C1-6烷氧基的基团取代。
  2. 根据权利要求1所述的应用，其特征在于：
    所述糖苷基团为单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基或经取代的四糖基。
  3. 根据权利要求1所述的应用，其特征在于：
    所述糖苷基团选自结构式(i)或(ii)的基团：
    其中
    R19和R20可一起形成氧代基；或者当R19和R20中的一个为氢或卤素时，R19和R20中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
    R21和R22可一起形成氧代基；或者当R21和R22中的一个为氢或卤素时，R21和R22中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
    R23和R24可一起形成氧代基；或者当R23和R24中的一个为氢或卤素时，R23和R24中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基；
    R25和R26可一起形成氧代基；或者当R25和R26中的一个为氢或卤素时，R25和R26中的另一个选自R15、-OR15、-C(O)R15、-C(O)OR15、-CH2R27和-C(O)R27；
    R27独立地选自氢、卤素、三氟甲基、氰基、硝基、任选地被1个、2个、3个、4个或5个独立地选自R16的基团取代的烃基、任选地被1个、2个、3个、4个或5个独立地选自R16的基团取代的-(CH2)k-杂环基、-OR17、-C(O)R18、-C(O)N(R17)R18、-C(O)OR17、-OC(O)R17、-S(O)2R17、-S(O)2N(R17)R18、-N(R17)R18、-N(R17)N(R17)R18、-N(R17)C(O)R18、-N(R17)S(O)2R18、单糖基、经取代的单糖基、二糖基、经取代的二糖基、三糖基、经取代的三糖基、四糖基和经取代的四糖基。
  4. 根据权利要求1所述的应用，其特征在于：
    所述芳基萘木脂素类化合物选自ANL-2～ANL-7中的至少一种：

  5. 根据权利要求1～4任一项所述的应用，其特征在于：
    所述新型冠状病毒包括但不限于SARS-CoV-1、SARS-CoV-2、MERS-CoV和SARS-CoV-2奥米克戎株中的至少一种。
  6. 权利要求1～5任一项中所述的芳基萘木脂素类化合物，其特征在于，所述芳基萘木脂素类化合物不为山荷叶素。
  7. 一种药物组合物，其特征在于，包含至少一种权利要求6所述的芳基萘木脂素类化合物或其药学上可接受的盐或前药。
  8. 根据权利要求7所述的药物组合物，其特征在于，还包含至少一种药学上可接受的辅料。
  9. 权利要求7或8所述的药物组合物的应用，其特征在于：为下述应用中的至少一种：
    1)在制备用于治疗、预防新型冠状病毒感染或延缓新型冠状病毒感染的进展的药物中的应用；
    2)在制备新型冠状病毒抑制剂中的应用。
  10. 权利要求6所述的芳基萘木脂素类化合物的制备方法，其特征在于：
    所述ANL-2的制备方法，包括以下步骤：
    以2-溴-4,5-二甲氧基苯甲醛(1)为原料，通过乙二醇保护制备缩醛(2)，(2)与胡椒醛反应得到化合物(3)，化合物(3)在乙酸存在下通过加热迅速转化为异苯并呋喃，再通过Diels-Alder反应得到双酯(4)，将其还原得到山荷叶素(5)；然后将山荷叶素(5)与三氟甲磺酸酐反应得到产物(6)；产物(6)与三甲基乙炔基硅反应得到产物(7)；产物(7)在碳酸钾存在下得到目标产物ANL-2；
    所述ANL-2的制备反应式如下：

    所述ANL-3的制备方法，包括以下步骤：
    将山荷叶素(5)与溴乙腈反应得到产物ANL-3；
    所述ANL-4的制备方法，包括以下步骤：
    以D-木糖为原料，通过与乙酸酐、溴化氢反应，得到糖基溴；山荷叶素(5)在TBAB存在下与糖基溴反应，得到目标产物ANL-4；
    所述ANL-5、ANL-6和ANL-7的制备方法，包括以下步骤：
    将ANL-4的糖基部分的-O-乙酰基脱去，得到山荷叶素7-O-木糖苷；山荷叶素7-O-木糖苷通过乙酸酐Ac2O和乙酸四丁基铵TBAOAc反应，得到木糖3″-乙酰化糖苷衍生物ANL-5；将ANL-5用氯甲酸丙烯酯处理得到山荷叶素木糖苷不同的酰化产物ANL-6和ANL-7。
PCT/CN2025/096691 2024-03-27 2025-05-22 一类抗新型冠状病毒的芳基萘木脂素类化合物及其制备方法和应用 Pending WO2025201575A2 (zh)

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