WO2023160586A1 - 具有抗乙肝病毒和冠状病毒作用的苯并二氢吡喃类化合物 - Google Patents

具有抗乙肝病毒和冠状病毒作用的苯并二氢吡喃类化合物 Download PDF

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WO2023160586A1
WO2023160586A1 PCT/CN2023/077694 CN2023077694W WO2023160586A1 WO 2023160586 A1 WO2023160586 A1 WO 2023160586A1 CN 2023077694 W CN2023077694 W CN 2023077694W WO 2023160586 A1 WO2023160586 A1 WO 2023160586A1
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methoxychroman
heptyl
formula
compound
hydrochloride
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PCT/CN2023/077694
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English (en)
French (fr)
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黄相中
白溪山
李育晓
李艳红
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云南民族大学
云南恒益生物科技有限公司
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Publication of WO2023160586A1 publication Critical patent/WO2023160586A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/74—Benzo[b]pyrans, hydrogenated in the carbocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to a chroman compound having anti-hepatitis B virus and coronavirus, in particular to the use of the chroman compound in the preparation of an anti-hepatitis B virus or anti-SARS-CoV-2 drug.
  • Viruses are small, simple-structured, non-cellular organisms that use DNA or RNA as their genetic material and must parasitize and multiply in living cells (Woolhouse M, Scott F, Hudson Z, et al. Human viruses: discovery and emergence[J].Philos Trans R Soc Lond B Biol Sci,2012,367:2864-2871). Viral infections can be divided into acute viral infections caused by influenza virus, novel coronavirus (SARS-CoV-2), etc., and acute viral infections caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), etc. according to the time the virus stays in the body. persistent viral infection. The life cycle of a virus can be roughly divided into stages of invasion, biosynthesis, assembly, and release.
  • HBV is a hepatropic DNA virus that can cause acute or chronic hepatitis B in the body. It can continuously replicate in the human body and cause liver lesions. At present, about 3.5% of the world's population is infected with chronic hepatitis B virus. If chronic hepatitis B is not treated in time, it will easily develop into liver cirrhosis, liver failure, and even liver cancer, which seriously threatens human health. About 1 million patients die of chronic hepatitis B every year. Hepatitis-related diseases (Yuen MF, Chen DS, Dusheiko GM, et al. Hepatitis B virus infection[J]. Nat Rev Dis Primers, 2018, 4:18035).
  • interferon As a natural defense of host cells against external stimuli such as viral pathogens, interferon (IFN) is a group of cytokines released from host cells that can effectively promote cccDNA clearance or silencing.
  • IFN interferon
  • nucleoside analogs directly act on viral replication by inhibiting the reverse transcription process of the virus; long-term nucleoside analog therapy can effectively improve liver disease, reduce the incidence and mortality of liver cancer, but this type of drug has shown a very low Hepatitis B surface antigen (HBsAg) clearance rate, and can only functionally inhibit chronic hepatitis B, it is difficult to realize the functional cure of hepatitis B virus.
  • HsAg Hepatitis B surface antigen
  • Coronavirus belongs to Nidoviridae, Coronaviridae, and Coronaviridae in classification, and its genome length is about 25000nt to 30000nt.
  • Antiviral drugs currently used for the treatment of COVID-19 mainly include small molecule drugs (such as protease inhibitors, polymerase inhibitors), biological macromolecular drugs (such as high-titer immunoglobulins, convalescent plasma, monoclonal antibodies), etc.
  • small molecule drugs have the advantages of easy absorption, small molecular size, ability to penetrate cell membranes, and easy large-scale industrial production, and are the focus of drug research and development.
  • protease inhibitors such as paclovir
  • RNA polymerase inhibitors that rely on ribonucleic acid (RNA) (such as Monaprevir)
  • Owen DR Allerton CMN
  • Monaprevir ribonucleic acid
  • Molnupiravir promotes SARS -CoV-2 mutagenesis via the RNA template[J].J Biol Chem,2021,297(1):100770).
  • antiviral drugs available to fight against SARS-CoV-2 infection.
  • effective and safe antiviral drugs have been developed and widely used It is an important measure to reduce the harm of diseases.
  • the present invention provides a chroman compound, a preparation method of a chroman compound, and its use in preventing, treating or alleviating viral infectious diseases , anti-hepatitis B virus or anti-coronavirus, especially SARS-CoV-2 medicine or pharmaceutical composition and preparation method thereof, as well as for preventing, treating or alleviating hepatitis B virus infectious diseases and coronavirus infectious diseases.
  • the present invention provides chroman compounds represented by formula I, their isomers or pharmaceutically acceptable salts.
  • the structural formula of formula I is as follows:
  • R1 and R2 are independently selected from H, OH, OMe, halogen;
  • R3 is selected from H, C1-C11 alkyl, C3-C6 cycloalkyl, phenyl, benzyl, substituted benzyl, substituted benzene
  • R4 is selected from C1-C11 alkyl;
  • X is independently selected from O, S and NH;
  • the substituted benzyl is selected from any one of the following: benzyl substituted with one or more halogen atoms, benzyl substituted with a trifluoromethyl group, benzyl substituted with a trifluoromethyl group and a halogen atom group, containing a cyano-substituted benzyl group;
  • the substituted phenyl is selected from any one of the following: phenyl substituted with one or more halogen atoms, phenyl substituted with a trifluoromethyl group, phenyl substituted with a trifluoromethyl group and a halogen atom group, containing a cyano-substituted phenyl group.
  • the present invention also provides chroman compounds represented by formula I, their isomers or pharmaceutically acceptable salts, characterized in that the compound is selected from: 2-propyl-4-hydroxybenzo Dihydropyran, 2-butyl-4-hydroxychroman, 2-pentyl-4-hydroxychroman, 2-hexyl-4-hydroxychroman, 2 -Heptyl-4-hydroxychroman, 2-octyl-4-hydroxychroman, 2-nonyl-4-hydroxychroman, 2-undecyl -4-hydroxychroman, 2-propyl-4-hydroxy-7-methoxychroman, 2-butyl-4-hydroxy-7-methoxychroman Pyran, 2-pentyl-4-hydroxy-7-methoxychroman, 2-hexyl-4-hydroxy-7-methoxychroman, 2-heptyl-4 -Hydroxy-7-methoxychroman, 2-octyl-4-hydroxy-7-methoxychroman, 2-nonyl-4-hydroxy-7-methoxy chroman, 2-undec
  • the present invention also provides chroman compounds represented by formula I, their isomers or pharmaceutically acceptable salts, characterized in that the compounds form addition salts with acids, and the acids are independently selected from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, lactic acid, acetic acid, tartaric acid, p-toluenesulfonic acid, maleic acid, salicylic acid, citric acid, succinic acid.
  • the present invention also provides chroman compounds represented by formula I, its isomers or pharmaceutically acceptable salts, characterized in that the pharmaceutically acceptable salts are selected from: 2-heptyl- 4-Isobutylamino-7-methoxychroman hydrochloride, 2-heptyl-4-(4-chlorobenzylamino)-7-methoxychroman hydrochloride Salt, 2-heptyl-4-(3-fluorobenzylamino)-7-methoxychroman hydrochloride, 2-heptyl-4-(3-chlorobenzylamino)-7 -Methoxychroman hydrochloride, 2-heptyl-4-(3-trifluoromethylbenzylamino)-7-methoxychroman hydrochloride, 2- Heptyl-4-(4-fluorobenzylamino)-7-methoxychroman hydrochloride, 2-heptyl-4-benzylamino-7-methoxychroman Pyranyl hydrochloride, 2-
  • the present invention also provides the preparation method of the chroman compound represented by formula I, its isomer or pharmaceutically acceptable salt, including the preparation method of the compound represented by formula II or the compound represented by formula III, wherein
  • the preparation method of the compound shown in the formula II the compound shown in the formula IV is used as a raw material, and the brominated product shown in the formula V is obtained through a bromination reaction, and then first reacted with triphenylphosphine, and then reacted with sodium hydroxide
  • the compound shown in formula VI is obtained; the compound shown in formula VI reacts with aldehyde to obtain the compound shown in formula VII; the compound shown in formula VII can be ring-closed under alkaline conditions to obtain the compound shown in formula VIII;
  • formula VIII Compounds shown with primary amines or salts Acid hydroxylamine carries out reductive amination reaction and obtains the compound shown in formula II, and the synthetic reaction step of adopting is as follows:
  • the preparation method of the compound shown in the formula III using the compound shown in the formula VIII as a raw material, reacting with a hydrogenation reagent to obtain the compound shown in the formula IX, and then reacting with a halogenated hydrocarbon compound to obtain the compound shown in the formula III.
  • the synthetic reaction steps are as follows:
  • the present invention also provides a pharmaceutical composition, comprising the chroman compound represented by formula I, its isomer or pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or auxiliary material.
  • the pharmaceutical composition of the present invention is characterized in that the pharmaceutical composition is selected from tablets, capsules, pills, and injections; and the pharmaceutical composition is selected from sustained-release preparations or controlled-release preparations.
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for humans or a pharmaceutical composition for animals, wherein the animals include pigs, bovines, canines, equines, ovines, felines, rodents , primates such as humans, cows, pigs or horses.
  • the present invention also provides chroman compounds represented by formula I, its isomers or pharmaceutically acceptable salts, including chroman compounds represented by formula I, its isomers or Use of the pharmaceutical composition of a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier or auxiliary material in the preparation of drugs for preventing, treating or alleviating hepatitis B virus infectious diseases and coronavirus infectious diseases.
  • the use of the compound or pharmaceutical composition of the present invention is characterized in that it is used to prevent, treat or alleviate novel coronavirus (SARS-CoV-2) infectious diseases.
  • SARS-CoV-2 novel coronavirus
  • the compound of formula I of the present invention exhibits a good inhibitory effect on hepatitis B virus and coronavirus, especially SARS-CoV-2, suggesting that it has good antiviral activity, and can be used as an antiviral active ingredient or lead compound, and has great Good application prospects.
  • the synthesis method of the compound of formula I of the present invention has few steps, easy acquisition of raw materials, low cost, mild reaction conditions, and subsequent industrial production is easy to realize.
  • the compound of formula I of the present invention is a small molecular compound, which can be obtained by crystallization or conventional column chromatography during the preparation process.
  • the compound preparation process is simple and the obtained compound has high purity.
  • the present invention finds for the first time that the salt of the compound of formula I has a good inhibitory effect on hepatitis B virus and coronavirus, especially SARS-CoV-2, suggesting that it has good antiviral activity.
  • the conventional acid-base neutralization preparation method can be adopted, the compound preparation operation process is simple, and the obtained compound has high purity.
  • Fig. 1 is before and after the compound 50a is administered to the influence figure of shelduck duck body weight
  • Fig. 2 is 2-heptyl-4-(3-trifluoromethylbenzylamino)-7-methoxychroman hydrochloride (43), 2-heptyl-4-(3, 4-Difluorobenzylamino)-7-methoxychroman hydrochloride (48), cis-2-heptyl-4-(3-chloro-4-fluorobenzylamino)-7 -Methoxychroman hydrochloride (50a) and 2-heptyl-4-(2,4-difluorobenzylamino)-7-methoxychroman hydrochloride (51) Dose-effect curve for inhibiting SARS-CoV-2.
  • the chroman compound represented by formula I of the present invention, its isomer or pharmaceutically acceptable salt, the formula I structural formula is as follows:
  • R1 and R2 are independently selected from H, OH, OMe, halogen;
  • R3 is selected from H, C1-C11 alkyl, C3-C6 cycloalkyl, phenyl, benzyl, substituted benzyl, substituted benzene
  • R4 is selected from C1-C11 alkyl;
  • X is independently selected from O, S and NH;
  • the substituted benzyl is selected from any one of the following: benzyl substituted with one or more halogen atoms, benzyl substituted with a trifluoromethyl group, benzyl substituted with a trifluoromethyl group and a halogen atom group, containing a cyano-substituted benzyl group;
  • the substituted phenyl is selected from any one of the following: phenyl substituted with one or more halogen atoms, phenyl substituted with a trifluoromethyl group, phenyl substituted with a trifluoromethyl group and a halogen atom group, containing a cyano-substituted phenyl group.
  • the C1-C11 alkyl group includes but not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl , 1-methylbutyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl, 3-methyl Pentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, n-heptyl, isoheptyl, n-octyl, 6-methylheptyl Base, n-nonyl, 7-methyloctyl, n-decyl, 8-methylnonyl, n-undecyl, 9-methyldecy
  • the C3-C6 cycloalkyl group includes, but is not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and its isomers, wherein the cycloalkyl group can contain heteroatoms (O, S, N, etc. ).
  • the R3 is preferably selected from any one of the following groups: hydrogen, propyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-heptyl, cyclopentyl, cyclohexyl, phenyl, 4- Fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 3,4-difluorophenyl, 3-chloro-4-fluorophenyl, 4-trifluoromethylphenyl, 3-trifluorophenyl Fluoromethylphenyl, 2-chloro-4-fluorophenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 3,4,5-trifluorophenyl, benzyl, 2- Fluorobenzyl, 2-chlorobenzyl, 2-trifluoromethylbenzyl, 3-fluorobenzyl, 3-chlorobenzyl, 3-trifluoromethylbenzyl,
  • the R4 is preferably selected from any one of the following groups: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl.
  • the preparation method of the compound shown in the formula I of the present invention includes but not limited to the preparation method of the compound shown in the formula II and the compound shown in the formula III, the preparation method of the compound shown in the formula II: the compound shown in the formula IV is The raw material, under reflux conditions, reacts with copper bromide using ethyl acetate as a solvent to obtain the brominated product shown in formula V. Then, the brominated product shown in formula V and triphenylphosphine were dissolved in ethyl acetate, and reacted at room temperature to obtain a white solid; sodium hydroxide solution (2N) was added into the dichloromethane solution in which the white solid was dissolved, and adjusted pH to 12.
  • the compound represented by formula VI can be obtained.
  • the compound represented by formula VI and aldehyde were dissolved in dichloromethane, and reacted under reflux for 30 h.
  • the compound represented by formula VII was purified by silica gel chromatography.
  • the compound shown in formula VII is reacted with sodium acetate in ethanol solution to obtain the compound shown in formula VIII.
  • the compound represented by formula VIII and primary amine or hydroxylamine hydrochloride were dissolved in toluene, then titanium tetrachloride was added dropwise, and reacted at room temperature for 24 hours to obtain an oily product.
  • the preparation method of the compound shown in the formula III of the present invention use the compound shown in the formula VIII as a raw material, react with sodium borohydride in methanol to obtain the compound shown in the formula IX; in the presence of sodium hydride, the compound shown in the formula IX The compound shown is reacted with the halogenated hydrocarbon compound to obtain the compound shown in the formula III, and the synthetic reaction steps adopted are as follows:
  • the chroman compounds described in the present invention include but are not limited to
  • the “pharmaceutically acceptable salt” in the present invention refers to the salt formed by the basic functional group (such as -NH2, -NHR, etc.) present in the compound and an appropriate inorganic or organic anion (acid), wherein the acid includes but Not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, lactic acid, acetic acid, tartaric acid, p-toluenesulfonic acid, maleic acid, salicylic acid, citric acid, succinic acid, etc.
  • the basic functional group such as -NH2, -NHR, etc.
  • an appropriate inorganic or organic anion (acid) wherein the acid includes but Not limited to: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, lactic acid, acetic acid, tartaric acid, p-toluenesulfonic acid, maleic acid, salicylic acid, citric acid, succinic acid, etc.
  • the salts formed include, but are not limited to: hydrochloride, hydrobromide, sulfate, phosphate, lactate, acetate, tartrate, p-toluenesulfonate, maleate, salicylate , citrate, succinate, etc.
  • compositions described in the present invention include but are not limited to:
  • the present invention adopts the HepG2.2.15 cell model recognized at home and abroad for HBV DNA transfection to resist the chroman compounds represented by the formula I of the present invention, its isomers or pharmaceutically acceptable salts.
  • the activity of hepatitis B virus was tested, and the half maximal effective concentration of the compound to inhibit the HepG2.2.15 cells transfected with HBV DNA was calculated.
  • tested compound shows better anti-hepatitis B virus effect, and wherein the anti-hepatitis B virus effect of compound 50a and 50b is the strongest, and its IC50 and clinical drug lamivudine (0.043 ⁇ mol/L) are replicated to hepatitis B DNA virus similar, however for HBsAg and HBeAg secreted
  • the inhibitory effect is stronger than that of lamivudine.
  • the present invention adopts the animal model of duck hepatitis B recognized at home and abroad at present to test the therapeutic effect of the compound 50a in the formula I compound in vivo on duck hepatitis B in vivo.
  • the test results show that compound 50a has a good inhibitory effect on the replication of DHBV DNA at low, medium and high doses (10mg/kg, 20mg/kg, 40mg/kg).
  • the DHBV DNA content in serum of T14, T21 and T28 ducks in the compound 50a low-dose group decreased significantly (P ⁇ 0.01); the DHBV DNA content in the serum of T7 duck in the compound 50a middle-dose group decreased significantly ( P ⁇ 0.05), T14, T21 and T28 duck serum DHBV DNA content decreased significantly (P ⁇ 0.01), compound 50a high dose group T7, T14, T21 and T28 duck serum DHBV DNA content decreased significantly (P ⁇ 0.01).
  • the DHBV DNA content in the duck serum of the low, medium and high dose groups P7 and P14 of compound 50a still showed a very significant decrease (P ⁇ 0.01), and there was a trend of continuous inhibition of virus replication. After discontinuation of the drug, the internal organs of the duck were not damaged.
  • the present invention adopts the currently recognized SARS-CoV-2-induced Vero-E6 cell model to treat the chroman compound shown in formula I of the present invention, its isomer or pharmaceutically acceptable Salt was tested for anti-SARS-CoV-2 activity, and the half-inhibitory concentration of the compound against SARS-CoV-2 was calculated.
  • the anti-hepatitis B virus and SARS-CoV-2 medicine or pharmaceutical composition of the present invention can be administered in the form of a unit dose, and the route of administration can be enteral or parenteral, such as oral, intramuscular, nasal cavity, oral mucosa, Skin, transdermal, subcutaneous, intradermal, peritoneal, rectal, intravenous, intramuscular, epidural, intraocular, intracranial, vaginal administration, etc.;
  • the administration route of the anti-hepatitis B virus and SARS-CoV-2 medicine or pharmaceutical composition of the present invention can be injection administration.
  • Injections include intravenous, intramuscular, subcutaneous, Intradermal injection, point injection, intrathecal injection and peritoneal injection etc.
  • the dosage forms for administration may be liquid dosage forms or solid dosage forms.
  • the nature of the solution of the liquid dosage form can be a true solution, a colloid, a microparticle dosage form, an emulsion dosage form, or a suspension dosage form.
  • Liquid dosage form can be syrup, injection solution, non-aqueous solution, suspension or emulsion; solid dosage form such as tablet, lozenge, capsule, drop pill, pill, granule, powder, cream, solution, suppository, dispersible Powders such as freeze-dried powder injections, aerosols, etc.
  • the anti-hepatitis B virus and SARS-CoV-2 medicine or pharmaceutical composition of the present invention can be made into common preparations, and can also be sustained-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.
  • the pharmaceutically acceptable carrier or adjuvant in the present invention includes adjuvant for oral preparation, adjuvant for parenteral administration or external administration.
  • the excipients used include excipients such as lactose, calcium carbonate, calcium phosphate, sodium phosphate; diluents and absorbents such as starch, cyclodextrin, lactose, sucrose, mannitol, microcrystalline cellulose sodium, calcium sulfate, etc.; wetting agents With binders such as water, ethanol, propanol, glycerin, propylene glycol, isopropanol, syrup, honey, glucose, gelatin paste, sodium carboxymethylcellulose, potassium phosphate, etc.; disintegrants such as dry starch, agar powder, carbonic acid Calcium, sodium bicarbonate, sodium lauryl sulfonate, methylcellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.;
  • Tablets can also be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets to delay their disintegration and absorption in the gastrointestinal tract , and thus provide sustained action over a longer period of time.
  • coated tablets such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets to delay their disintegration and absorption in the gastrointestinal tract , and thus provide sustained action over a longer period of time.
  • halogen in the present invention refers to being substituted by a "halogen atom”
  • halogen atom refers to a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
  • the "isomer” in the present invention includes structural isomers and stereoisomers.
  • Structural isomers are divided into (carbon) chain isomers, positional isomers and functional group isomers.
  • Stereoisomers are divided into conformational isomers and configurational isomers, and configurational isomers are further divided into cis-trans isomers and optical isomers (enantiomers).
  • Enantiomers, diastereoisomers, racemates, mesoforms, cis-trans isomers, tautomers, geometric isomers of the compounds of the present invention Isomers, epimers and mixtures thereof are all included in the scope of the present invention.
  • Embodiment 1 the preparation of intermediate I-1
  • Embodiment 2 the preparation of intermediate 1-2
  • Embodiment 3 the preparation of intermediate 1-3
  • Embodiment 4 the preparation of intermediate I-4
  • Triphenylphosphine (10 g, 40.8 mmol) was dissolved in ethyl acetate (40.8 mL), intermediate I-2 (10.7 g, 40.8 mmol) was slowly added at room temperature, and stirred overnight at room temperature. A white solid was obtained by filtration, and dissolved in dichloromethane solvent (20.4 mL). At room temperature, the hydrogen Sodium chloride solution (2N) was slowly added to dichloromethane solution to adjust the pH value to 12. After stirring for 30 minutes, it was separated by extraction and washed with saturated aqueous sodium bicarbonate solution. The organic phases were combined, dried, filtered and concentrated to give the crude product. The crude product was recrystallized to obtain intermediate I-4 with a yield of 74%.
  • Embodiment 5 the preparation of intermediate 1-5
  • Triphenylphosphine (10 g, 40.8 mmol) was dissolved in ethyl acetate (40.8 mL), and intermediate I-3 (8.73 g, 40.8 mmol) was slowly added at room temperature, and stirred overnight at room temperature.
  • the preparation method was the same as in Example 4 to obtain intermediate I-5 with a yield of 98%.
  • Embodiment 6 the preparation of intermediate I-6
  • Embodiment 7 the preparation of intermediate I-7
  • Embodiment 8 the preparation of intermediate I-8
  • Embodiment 9 the preparation of intermediate I-9
  • Embodiment 10 the preparation of intermediate I-10
  • Embodiment 11 the preparation of intermediate I-11
  • Embodiment 12 the preparation of intermediate I-12
  • Embodiment 13 the preparation of intermediate I-13
  • Embodiment 14 the preparation of intermediate I-14
  • Embodiment 15 the preparation of intermediate I-15
  • intermediates I-4 (8.52 g, 20 mmol) were reacted with hexanal and heptanal in sequence to obtain intermediates I-16 and I-17.
  • Embodiment 17 the preparation of intermediate I-18
  • intermediate I-4 (8.52 g, 20 mmol) was reacted with nonanal to obtain intermediate I-19.
  • Dissolve intermediate I-40 (5.26g, 20mmol) in methanol (100mL), add palladium carbon (0.53g), put the glass test tube containing the solution into the autoclave, and feed hydrogen to make the pressure reach 0.5 Mp, react at room temperature for 24h. After the reaction, the crude product was obtained by filtration and concentration under reduced pressure. The obtained crude product was purified by silica gel chromatography to obtain compound 36 with a yield of 96%.
  • HepG2.2.15 cells transfected with hepatitis B virus (HBV) DNA were purchased from Shanghai Fuheng Biotechnology Co., Ltd., and subcultured by themselves after purchase.
  • Cells were cultured in MEM medium (i.e. complete medium: containing 10% fetal bovine serum, 400 ⁇ g/mL G418, 100 ⁇ g/mL penicillin, 100 ⁇ g/mL streptomycin), cultured in a 37°C, 5% CO2 incubator . Replace every other day The culture medium was used once, and the cells were overgrown in about 3 days.
  • MEM medium i.e. complete medium: containing 10% fetal bovine serum, 400 ⁇ g/mL G418, 100 ⁇ g/mL penicillin, 100 ⁇ g/mL streptomycin
  • HepG2.2.15 cells were made into cell suspension, and the cells were adjusted The density is 5 ⁇ 10 5 cells/mL, inoculated into 96-well cell culture plate at 100 ⁇ L/well, and cultivated in an incubator at 37°C and 5% CO 2 for 24 hours. Add different concentrations of drug-containing culture solution (administration group), drug-free culture solution containing 1 ⁇ DMSO (cell control group), and complete culture solution (blank control group, no cells), and each group has 3 replicate wells.
  • Cell survival rate (%) (A administration group -A blank group )/(A cell control group -A blank group ) ⁇ 100% (1)
  • a administration group is the absorbance of the drug experimental group with different mass concentrations
  • a blank group is the absorbance of the blank control group
  • a cell control group is the absorbance of the cell control group containing 1 ⁇ DMSO culture solution.
  • HepG2.2.15 cells into a cell suspension, adjust the cell density to 5 ⁇ 105 cells/mL, inoculate 100 ⁇ L/well into a 96-well cell culture plate, and culture in an incubator at 37 °C and 5% CO2 for 24 Hour.
  • 200 ⁇ L of complete culture medium with different concentrations of drugs was added to each well to treat the cells and the cell control group (drug-free medium containing 1 ⁇ DMSO), and each group had 3 replicate wells.
  • Fresh media containing different concentrations of compounds were replaced every 3 days. After culturing for 12 days in an incubator at 37°C with 5% CO 2 , the supernatant was collected.
  • HBsAg hepatitis B surface s antigen
  • HBeAg e antigen
  • a drug administration group is the absorbance of the drug experimental group with different mass concentrations
  • a blank group is the absorbance of the blank control group operated in parallel without adding enzyme binding substrate when the kit is measured
  • Absorbance of the treated cell control group is the absorbance of the drug experimental group with different mass concentrations
  • HepG2.2.15 cells into cell suspension, adjust the cell density to 5 ⁇ 10 5 cells/mL, inoculate 300 ⁇ L/well into 24-well cell culture plates, and culture in an incubator at 37°C and 5% CO 2 for 24 Hour.
  • 500 ⁇ L of complete culture solution of different concentrations of drugs was added to each well to treat the cells and the cell control group (drug-free medium containing 1 ⁇ DMSO), with lamivudine (3-TC) as the positive control, each group Set up 3 duplicate holes. Fresh media containing different concentrations of compounds were replaced every 3 days.
  • HBV DNA inhibition rate (%) (1-C administration group /C cell control group ) ⁇ 100% (3)
  • C administration group is the HBV DNA copy number of the drug experimental group with different mass concentrations
  • C cell control group is the HBV DNA copy number of the cell control group without drug treatment.
  • n 3.
  • n 3.
  • the shelduck ducks screened out were randomly divided into 5 groups, model group (normal saline group), lamivudine group (20mg/kg), drug low dose group (10mg/kg), middle dose group (20mg/kg), In the high-dose group (40mg/kg), 6 rats in each group were intragastrically administered once a day for 28 consecutive days. then stop Medicine for 14 days. During the dosing period, blood was collected from duck foot veins once a week, and the serum was separated by centrifugation, stored in a -80°C refrigerator for inspection, and body weight was weighed once a week.
  • the experimental results are express.
  • the GraphPad prism 6 software was used for statistical analysis, and the t test was used to analyze the changes in serum DHBV DNA levels of animals at various time points before and after drug treatment. Compared with the model group, there was a significant difference at p ⁇ 0.05, which was statistically significant.
  • test results of the inhibitory effect of compound 50a on congenital hepatitis B virus shelduck serum DHBV DNA are shown in Figure 1 and Table 3.
  • the experimental results showed that, compared with the blank group, the low, medium and high dose groups of compound 50a had no effect on the growth of DHBV duckling body weight (see Figure 1).
  • the test results show that compound 50a has a good inhibitory effect on the replication of DHBV DNA at low, medium and high doses (10mg/kg, 20mg/kg, 40mg/kg).
  • the DHBV DNA content in serum of T14, T21 and T28 ducks in the compound 50a low-dose group decreased significantly (P ⁇ 0.01); the DHBV DNA content in the serum of T7 duck in the compound 50a middle-dose group decreased significantly ( P ⁇ 0.05), T14, T21 and T28 duck serum DHBV DNA content decreased significantly (P ⁇ 0.01), compound 50a high dose group T7, T14, T21 and T28 duck serum DHBV DNA content decreased significantly (P ⁇ 0.01).
  • Vero-E6 cell model induced by SARS-CoV-2 Wang ML, Cao RY, Zhang LK, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res.
  • Vero-E6 cells were purchased from the American Type Culture Collection (ATCC), SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019 isolate, GISAID accession number EPI_ISL_402124) was isolated and subcultured by Wuhan Institute of Virology, Chinese Academy of Sciences. Cells were cultured in MEM medium (each 100 mL containing 10% fetal bovine serum, 100 U/mL each of penicillin, streptomycin and kanamycin, NaHCO 3 5%), cultured in a 37°C, 5% CO 2 incubator 3d or so covered with cells.
  • MEM medium each 100 mL containing 10% fetal bovine serum, 100 U/mL each of penicillin, streptomycin and kanamycin, NaHCO 3 5%
  • the detection of drug toxicity to cells was determined by CCK8 kit (Beiyuntian). The specific steps are as follows: Inoculate 1 ⁇ 104 Vero-E6 cells in a 96-well cell culture plate and culture at 37°C for 16 hours; dilute the drug with DMSO to the appropriate concentration of the mother solution, and then dilute it with MEM medium containing 2% FBS When the desired concentration is reached, the original culture medium in the 96-well plate is discarded, and 100 ⁇ L of drug-containing MEM medium is added to the corresponding wells, and three replicate wells are made for each concentration.
  • Negative control in the cell well, add PBS and MEM medium containing 2% FBS, no drug
  • blank control no cells, add DMSO and MEM medium containing 2% FBS.
  • the cells were placed in a 37°C, 5% CO2 incubator for 24 hours; then 10 ⁇ L of CCK8 solution was added to the wells to be tested, placed in a 37°C incubator for 2 hours, and the cells in each well were measured on a microplate reader.
  • A is the absorbance value measured by the microplate reader.
  • Inoculate Vero E6 cells into a 48-well cell culture plate, about 1 ⁇ 10 5 cells per well, and wait for the experiment the next day. Firstly, 100 ⁇ L of the medium containing the drug at the corresponding concentration was added to the cell culture plate, and the cells were pretreated for 1 hour, then 20 ⁇ L of diluted virus (MOI 0.05) was added, and incubated in an incubator for 1 hour. Then discard the virus culture medium, wash away the uninfected residual virus with PBS, then add the drug medium containing the corresponding concentration, and add the medium containing 0.1% DMSO in the control group, and then put it in a 37°C, 5% CO2 incubator to continue Cultivate for 48h.
  • MOI diluted virus
  • RNA extraction using the kit produced by TaKaRa Company (TaKaRa MiniBEST Viral RNA/DNA Extraction Kit), the steps are as follows: (1) Take 100 ⁇ L of the supernatant from the culture plate to be tested and add it to a nuclease-free EP tube, then add 321 ⁇ L of lysate (100 ⁇ L PBS, 200 ⁇ L buffer VGB, 2 ⁇ L proteinase K, 1 ⁇ L carrier RNA), mix well and put it at 56°C for 15min to digest; (2) Add 200 ⁇ L absolute ethanol to the mixture obtained in (1), and mix well; (3) Transfer the mixture obtained in (2) above to In the RNase-free spin column, centrifuge at 12000rpm for 15s, discard the waste solution; (4) add 500 ⁇ L Buffer RW1, centrifuge at 12000rpm for 15s, discard the waste solution; (5) add 650 ⁇ L Buffer RW2, centrifuge at 12000rpm for 15s, discard the waste solution; (6) ) Add 650 ⁇
  • RNA reverse transcription the steps are as follows: In the experiment, the reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser) produced by TaKaRa Company was used for RNA reverse transcription.
  • the reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser) produced by TaKaRa Company was used for RNA reverse transcription.
  • Removal of gDNA Collect RNA samples from each experimental group, and take 3 ⁇ L of RNA for reverse transcription.
  • Real-time PCR detection Fluorescent quantitative PCR uses TB Green Premix (Takara, Cat#RR820A) to mix the reaction system, and performs amplification reaction and reading on the StepOne Plus Real-time PCR instrument. Calculate the copy number per milliliter of the original virus solution. The steps are as follows: (1) Establish standard: Dilute plasmid pMT-RBD to 5 ⁇ 10 8 copies/ ⁇ L, 5 ⁇ 10 7 copies/ ⁇ L, 5 ⁇ 10 6 copies/ ⁇ L, 5 ⁇ 10 5 copies/ ⁇ L, 5 ⁇ 10 4 copies/ ⁇ L, 5 ⁇ 10 3 copies/ ⁇ L, 5 ⁇ 10 2 copies/ ⁇ L.

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Abstract

本发明涉及具有抗乙肝病毒和冠状病毒特别是SARS-CoV-2作用的苯并二氢吡喃类化合物及其制备方法和应用。具体而言，本发明提供了一种式I所示的化合物的制备方法及其用途，其用途用于预防、治疗或减轻乙肝病毒感染性疾病和冠状病毒感染性疾病。本发明的化合物具有较强的抗乙肝病毒和冠状病毒特别是SARS-CoV-2活性，用其作为活性成分与药学上可接受的载体或辅料制备成抗乙肝病毒或抗冠状病毒药物或药物组合物。

Description

具有抗乙肝病毒和冠状病毒作用的苯并二氢吡喃类化合物 技术领域
本发明涉及具有抗乙肝病毒和冠状病毒的苯并二氢吡喃类化合物，尤其涉及苯并二氢吡喃类化合物在制备抗乙肝病毒或抗SARS-CoV-2药物中的用途。
背景技术
病毒是一种个体微小、结构简单，以DNA或RNA为遗传物质，必须在活细胞内寄生并以复制方式增殖的非细胞型生物(Woolhouse M,Scott F,Hudson Z,et al.Human viruses:discovery and emergence[J].Philos Trans R Soc Lond B Biol Sci,2012,367:2864-2871)。病毒感染按照病毒在机体内滞留的时间，可分为流感病毒、新型冠状肺炎病毒(SARS-CoV-2)等引发的急性病毒感染和人类免疫缺陷病毒(HIV)、乙肝病毒(HBV)等引发的持续性病毒感染。病毒的生命周期大致可分为入侵、生物合成、装配和释放等阶段，一旦有足够数量的病毒被复制，子代病毒就会溶解或破坏宿主细胞，进而感染其他细胞。病毒的高度变异性以及各种新型病毒的出现，使病毒感染性疾病的治愈始终面临着严峻的挑战，一直是严重威胁着人类健康的重大公共安全卫生问题。药物是抗击病毒感染疾病的有力武器，目前已针对9个病毒家族研发出百余种抗病毒药物(De Clercq E,Li G.Approved antiviral drugs over the past 50years[J].Clin Microbiol Rev,2016,29:695-747；Chaudhuri S,Symons JA,Deval J.Innovation and trends in the development and approval of antiviral medicines:1987-2017and beyond[J].Antiviral Res,2018,155:76-88)。但慢性难治性病毒感染患者长期服用药物易产生耐药性导致原有抗病毒药物失效。更为严重的是，耐药性的进一步发展使全球公共卫生体系面临严峻的挑战。因此，研发新型广谱抗耐药性 的抗病毒药物是临床长期迫切需求。
HBV是一种能导致机体产生急性或慢性乙型肝炎的嗜肝性DNA病毒，其能够在人体内连续不断的复制进而引起肝脏病变。目前全球约有3.5％的人口感染慢性乙肝病毒，慢性乙肝如未得到及时治疗，极易发展为肝硬化、肝衰竭，甚至肝癌，严重威胁人类的健康，每年约有100万患者死于慢性乙型肝炎相关性疾病(Yuen MF,Chen DS,Dusheiko GM,et al.Hepatitis B virus infection[J].Nat Rev Dis Primers,2018,4:18035)。我国受其影响最为严重，国内乙肝病毒感染者约9000万，占全球1/4。目前口服抗乙肝病毒药物虽然可以抑制病毒载量，减少肝脏相关并发症，但终生治疗、成本高及潜在毒性仍然是治疗过程中需要面对的主要问题。常用抗乙肝病毒药物主要有两大类，分别是干扰素和核苷类似物。作为宿主细胞对抗病毒病原体等外部刺激的天然防御，干扰素(IFN)是一组从宿主细胞中释放出来的细胞因子，可有效地促进cccDNA清除或沉默。但是IFN治疗对于不同基因型病毒感染存在显著差异，并且只有约10％的病人可以实现持续的病毒应答。核苷类似物通过抑制病毒的逆转录过程，直接作用于病毒复制；长期核苷类似物治疗可有效的改善肝脏疾病、降低肝癌发病率及死亡率，但是该类药物却表现出了非常低的乙肝表面抗原(HBsAg)清除率，且只能功能性抑制慢性乙型肝炎，难以实现对乙型肝炎病毒的功能性治愈。
冠状病毒(CoV)在分类上属于尼多病毒目、冠状病毒科、冠状病毒属，其基因组长度约为25000nt～30000nt。目前用于COVID-19治疗的抗病毒药物主要包括小分子药物(如蛋白酶抑制剂、聚合酶抑制剂)、生物大分子药物(如高效价免疫球蛋白、恢复期血浆、单克隆抗体)等。其中小分子药物具有易吸收、分子体积小、能够穿透细胞膜、易于大规模工业化生产等优点，是药物研发的重点。在具有抗SARS-CoV-2作用的小分子药物中，以病毒蛋白剪切步骤为作用靶点的蛋白酶抑制剂(如帕克洛维)与依赖核糖核酸(RNA)的RNA聚合酶抑制剂(如莫那匹韦)取得突破性进展(Owen DR,Allerton CMN,Anderson  AS,et al.An oral SARS-CoV-2Mpro inhibitor clinical candidate for the treatment of COVID-19[J].Science,2021(10):eabl4784；Gordon CJ,Tchesnokov EP,Schinazi RF,et al.Molnupiravir promotes SARS-CoV-2mutagenesis via the RNA template[J].J Biol Chem,2021,297(1):100770)。目前，可用于对抗SARS-CoV-2感染的抗病毒药物有限，在当前COVID-19全球大流行以及SARS-CoV-2持续变异的背景下，研发出有效、安全的抗病毒药物并广泛推广应用是降低疾病危害的重要举措。
发明内容
(一)要解决的技术问题
为了解决现有技术中存在的上述问题，本发明提供一种苯并二氢吡喃类化合物、苯并二氢吡喃类化合物的制备方法、在预防、治疗或减轻病毒感染性疾病中的用途、用于抗乙肝病毒或抗冠状病毒特别是SARS-CoV-2的药物或药物组合物及其制备方法以及用于预防、治疗或减轻乙肝病毒感染性疾病和冠状病毒感染性疾病。
(二)技术方案
为了解决上述技术问题，本发明提供式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，式I结构式如下：
其中，R1、R2分别独立的选自H、OH、OMe、卤素；R3选自H、C1-C11烷基、C3-C6环烷基、苯基、苄基、取代的苄基、取代的苯基；R4选自C1-C11烷基；X独立的选自O、S和NH；
所述取代的苄基选自下述任意一种：含有一个或多个卤原子取代的苄基，含有一个三氟甲基取代的苄基，含有一个三氟甲基和一个卤原子取代的苄基，含有一个氰基取代的苄基；
所述取代的苯基选自下述任意一种：含有一个或多个卤原子取代的苯基，含有一个三氟甲基取代的苯基，含有一个三氟甲基和一个卤原子取代的苯基，含有一个氰基取代的苯基。
本发明还提供式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，所述化合物选自：2-丙基-4-羟基苯并二氢吡喃，2-丁基-4-羟基苯并二氢吡喃，2-戊基-4-羟基苯并二氢吡喃，2-己基-4-羟基苯并二氢吡喃，2-庚基-4-羟基苯并二氢吡喃，2-辛基-4-羟基苯并二氢吡喃，2-壬基-4-羟基苯并二氢吡喃，2-十一烷基-4-羟基苯并二氢吡喃，2-丙基-4-羟基-7-甲氧基苯并二氢吡喃，2-丁基-4-羟基-7-甲氧基苯并二氢吡喃，2-戊基-4-羟基-7-甲氧基苯并二氢吡喃，2-己基-4-羟基-7-甲氧基苯并二氢吡喃，2-庚基-4-羟基-7-甲氧基苯并二氢吡喃，2-辛基-4-羟基-7-甲氧基苯并二氢吡喃，2-壬基-4-羟基-7-甲氧基苯并二氢吡喃，2-十一烷基-4-羟基-7-甲氧基苯并二氢吡喃，2-丙基-4,7-二羟基苯并二氢吡喃，2-戊基-4,7-二羟基苯并二氢吡喃，2-己基-4,7-二羟基苯并二氢吡喃，2-庚基-4,7-二羟基苯并二氢吡喃，2-庚基-4-异丁氨基-7-甲氧基苯并二氢吡喃，2-庚基-4-(4-氯苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氯苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(4-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-苄胺基-7-甲氧基苯并二氢吡喃，2-庚基-4-(4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3,4,5-三氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃，2-戊基-4-氨基-7-甲氧基苯并二氢吡喃，2-己基-4-氨基-7-甲 氧基苯并二氢吡喃，2-庚基-4-氨基-7-甲氧基苯并二氢吡喃。
本发明还提供式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，所述化合物与酸形成加成盐，所述酸分别独立的选自盐酸、氢溴酸、硫酸、磷酸、乳酸、乙酸、酒石酸、对甲苯磺酸、马来酸、水杨酸、柠檬酸、琥珀酸。
本发明还提供式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，所述药学上可接受的盐选自：2-庚基-4-异丁氨基-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(4-氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-苄胺基-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3,4,5-三氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐。
本发明还提供式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐的制备方法，包括式II所示化合物或式III所示化合物的制备方法，所述式II所示化合物的制备方法：以式IV所示的化合物为原料，经过溴代反应，得到式V所示的溴代产物，然后先与三苯基磷反应，再与氢氧化钠反应得到式VI所示的化合物；式VI所示的化合物与醛反应，得到式VII所示的化合物；式VII所示的化合物在碱性条件下关环可得式VIII所示的化合物；式VIII所示的化合物与伯胺或盐 酸羟胺进行还原胺化反应得到式II所示化合物，采用的合成反应步骤如下：
所述式III所示化合物的制备方法：以式VIII所示的化合物为原料，与氢化试剂反应得式IX所示的化合物，再与卤代烃类化合物反应得到式III所示化合物，采用的合成反应步骤如下：
本发明还提供一种药物组合物，包括式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐和药学上可接受的载体或辅料。
本发明的药物组合物，其特征在于，所述药物组合物选自片剂、胶囊、丸剂、注射剂；所述药物组合物选自缓释制剂或控释制剂。
本发明的药物组合物为人用药物组合物或动物用药物组合物，其中所述动物包括猪科动物、牛科动物、犬科动物、马科动物、羊科动物、猫科动物、啮齿类动物、灵长类动物，例如人、牛、猪或马。
本发明还提供式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐、包括式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐和药学上可接受的载体或辅料的药物组合物在制备用于预防、治疗或减轻乙肝病毒感染性疾病和冠状病毒感染性疾病药物的用途。
本发明的化合物或药物组合物的用途，其特征在于，用于预防、治疗或减轻新型冠状病毒(SARS-CoV-2)感染性疾病。
(三)有益效果
本发明的上述技术方案具有如下优点：
1.本发明的式I化合物对乙肝病毒和冠状病毒特别是SARS-CoV-2展现出较好的抑制作用，提示其具有良好的抗病毒活性，可以作为抗病毒活性成分或先导化合物，有着很好的应用前景。
2.本发明的式I化合物的合成方法步骤少，原料容易获得、成本低，反应条件温和，随后的工业化生产很容易实现。
3.本发明的式I化合物为小分子化合物，制备过程中可通过结晶或采用常规柱层析制备方法获得，化合物制备操作流程简单，所获得的化合物纯度高。
4.本发明首次发现式I化合物的盐对乙肝病毒和冠状病毒特别是SARS-CoV-2具有较好的抑制作用，提示其具有良好的抗病毒活性。
5.本发明的式I化合物的盐的制备方法中可采用常规的酸碱中和制备方法，化合物制备操作流程简单，所获得的化合物纯度高。
附图说明
图1为化合物50a用药前后对麻鸭体重的影响图；
图2为2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(43)，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(48)，顺-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(50a)及2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(51)抑制SARS-CoV-2的量效曲线图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例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 有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明所述式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，式I结构式如下：
其中，R1、R2分别独立的选自H、OH、OMe、卤素；R3选自H、C1-C11烷基、C3-C6环烷基、苯基、苄基、取代的苄基、取代的苯基；R4选自C1-C11烷基；X独立的选自O、S和NH；
所述取代的苄基选自下述任意一种：含有一个或多个卤原子取代的苄基，含有一个三氟甲基取代的苄基，含有一个三氟甲基和一个卤原子取代的苄基，含有一个氰基取代的苄基；
所述取代的苯基选自下述任意一种：含有一个或多个卤原子取代的苯基，含有一个三氟甲基取代的苯基，含有一个三氟甲基和一个卤原子取代的苯基，含有一个氰基取代的苯基。
所述的C1-C11烷基包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、1-甲基丁基、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、正庚基、异庚基、正辛基、6-甲基庚基、正壬基、7-甲基辛基、正癸基、8-甲基壬基、正十一烷基、9-甲基癸基。
所述的C3-C6环烷基包括但不限于：环丙基、环丁基、环戊基、环己基及其异构体，其中环烷基上可以包含杂原子(O、S、N等)。
所述R3优选自下述任意一种基团：氢，丙基，正丁基，异丁基，正戊基，异戊基，正庚基，环戊基，环己基，苯基，4-氟苯基，4-氯苯基，4-溴苯基，3,4-二氟苯基，3-氯-4-氟苯基，4-三氟甲基苯基，3-三 氟甲基苯基，2-氯-4-氟苯基，2,4-二氯苯基，3,4-二氯苯基，3,4,5-三氟苯基，苄基，2-氟苄基，2-氯苄基，2-三氟甲基苄基，3-氟苄基，3-氯苄基，3-三氟甲基苄基，4-氟苄基，4-氯苄基，4-三氟甲基苄基，2,4-二氟苄基，3,4-二氟苄基，2,4-二氯苄基，3,4-二氯苄基，2-氯-4-氟苄基，3-氯-4-氟苄基，2-氟-4-三氟甲基苄基，3-氟-4-三氟甲基苄基，3,4,5-三氟苄基，2-氰基苄基，3-氰基苄基，4-氰基苄基。
所述R4优选自下述任意一种基团：甲基、乙基、正丙基、正丁基、正戊基、正己基、正庚基、正辛基、正壬基、正癸基、正十一烷基。
本发明所述式I所示化合物的制备方法包括但不限于式II所示化合物和式III所示化合物的制备方法，所述式II所示化合物的制备方法：以式IV所示的化合物为原料，在回流条件下，以乙酸乙酯为溶剂与溴化铜反应，得到式V所示的溴代产物。然后，将式V所示的溴代产物与三苯基磷溶于乙酸乙酯中，室温反应得白色固体；将氢氧化钠溶液(2N)加入溶有白色固体的二氯甲烷溶液中，调节pH值至12。室温下搅拌30min后可得式VI所示的化合物。式VI所示的化合物与醛溶于二氯甲烷，回流反应30h，反应后经硅胶色谱法纯化得到式VII所示的化合物。式VII所示的化合物在乙醇溶液中与醋酸钠反应得到式VIII所示的化合物。式VIII所示的化合物与伯胺或盐酸羟胺溶于甲苯，然后滴加四氯化钛，室温反应24h得油状产物。将油状产物溶于甲醇中，加入相应的还原剂(硼氢化钠或Pd/C和H2)，室温反应后产物经硅胶色谱法纯化得到式II所示的化合物，采用的合成反应步骤如下：
本发明所述式III所示化合物的制备方法：以式VIII所示的化合物为原料，与硼氢化钠在甲醇中反应，可得到式IX所示的化合物；在氢化钠存在下，式IX所示的化合物与卤代烃类化合物反应得到式III所示化合物，采用的合成反应步骤如下：
本发明所述的苯并二氢吡喃类化合物包括但不限于



本发明所述的“药学上可接受的盐”是指化合物中存在的碱性官能团(例如―NH2、―NHR等)与适当的无机或者有机阴离子(酸)形成的盐，其中的酸包括但不限于：盐酸、氢溴酸、硫酸、磷酸、乳酸、乙酸、酒石酸、对甲苯磺酸、马来酸、水杨酸、柠檬酸、琥珀酸等。其形成的盐包括但不限于：盐酸盐、氢溴酸盐、硫酸盐、磷酸盐、乳酸盐、乙酸盐、酒石酸盐、对甲苯磺酸盐、马来酸盐、水杨酸盐、柠檬酸盐、琥珀酸盐等。
本发明所述的药学上可接受的盐包括但不限于：


本发明采用目前国内外公认的HBV DNA转染的HepG2.2.15细胞模型，对本发明所述式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐进行抗乙肝病毒活性测试，并计算了所述化合物对HBV DNA转染的HepG2.2.15细胞抑制的半数有效浓度。计算结果表明化合物2，3，5，8，11-13，13a，13b，14-25，27-29，30a，30b，31-49，50a，50b及51-55对HBeAg具有抑制作用，其抑制作用的IC50在28.76～498.64μM之间，其中化合物2和36的活性较强，IC50分别为41.89±3.72和28.76±3.55μM，选择性指数(SI)分别为>19.10，4.56；化合物3-5，8，11-13，13a，13b，14-29，30a，30b，31-49，50a，50b及51-55对HBsAg具有抑制作用，其抑制作用的IC50在0.48～136.20μM之间，其中化合物5，18，31及40的活性较强，IC50分别为1.13±0.49，1.17±0.58，2.08±0.98及0.48±0.15μM，SI分别为143.74，104.68，>1923.08及7032.58。化合物1-13，13a，13b，14-29，30a，30b，31-49，50a，50b及51-55对HBV DNA均具有抑制作用，其抑制作用的IC50在0.029～47.99μM之间，其中化合物12，13，13a，13b，18，20，30a，30b，31，34，39，40，43-45，48，50a，50b，51，52，54及55的活性较强，IC50均小于1.00μM，分别为0.26±0.13,0.083±0.027,0.093±0.061,0.10±0.03,0.78±0.25,0.92±0.24,0.36±0.14,0.51±0.19,0.59±0.27,0.36±0.11,0.70±0.24,0.44±0.18,0.10±0.05,0.15±0.06,0.47±0.11,0.12±0.03,0.029±0.011,0.047±0.028,0.12±0.02,0.24±0.13,0.72±0.28,0.59±0.21μM，SI在500.60～36282.80之间。上述结果表明所测试化合物显示出较好的抗乙肝病毒作用，其中化合物50a和50b的抗乙肝病毒作用最强，其对乙肝DNA病毒复制的IC50与临床用药拉米夫定(0.043μmol/L)相近，然而对HBsAg和HBeAg分泌的 抑制作用比拉米夫定强。
本发明采用目前国内外公认的鸭乙型肝炎动物模型，对本发明所述式I化合物中的化合物50a进行体内鸭乙肝治疗作用测试。测试结果表明化合物50a在低、中、高剂量(10mg/kg，20mg/kg，40mg/kg)下对DHBV DNA的复制均具有较好的抑制作用。与同组T0比较，在灌胃给药期间，化合物50a低剂量组T14、T21及T28鸭血清DHBV DNA含量显著下降(P<0.01)；化合物50a中剂量组T7鸭血清DHBV DNA含量下降明显(P<0.05)，T14、T21及T28鸭血清DHBV DNA含量下降显著(P<0.01)，化合物50a高剂量组T7、T14、T21及T28鸭血清DHBV DNA含量均下降效果显著(P<0.01)。与同组T0比较，在停药期间，化合物50a的低、中、高剂量组P7、P14鸭血清DHBV DNA含量均仍呈极显著下降(P<0.01)，有持续抑制病毒复制的趋势。停药后解剖，鸭内脏无损伤。
本发明采用目前国内外公认的SARS-CoV-2诱导的Vero-E6细胞模型，对本发明所述的式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐进行抗SARS-CoV-2活性测试，并计算了所述化合物对SARS-CoV-2的半数抑制浓度。计算结果表明化合物43，48，50a及51对SARS-CoV-2分离株nCoV-2019BetaCoV/Wuhan/WIV04/2019有明显的抑制作用，在病毒感染的MOI为0.05时，其半数抑制浓度(EC50)分别为3.28，2.27，2.98及5.93μmol/L，相应的选择指数(SI)分别大于122.0，176.2，134.2及67.5。上述结果表明所测试化合物显示出较好的抗SARS-CoV-2作用。
本发明所述的抗乙肝病毒和SARS-CoV-2的药物或药物组合物可以单位剂量形式给药，给药途径可以为肠道、或非肠道，如口服、肌肉、鼻腔、口腔黏膜、皮肤、透皮、皮下、皮内、腹膜、直肠、静脉内、肌内、硬膜外、眼内、颅内、阴道给药等；
本发明所述的抗乙肝病毒和SARS-CoV-2的药物或药物组合物的给药途径可以为注射给药。注射包括静脉注射、肌肉注射、皮下注射、 皮内注射、穴位注射、鞘内注射和腹膜注射等。
给药剂型可以是液体剂型、固体剂型。如液体剂型的溶液性质可以是真溶液类、胶体类、微粒剂型、乳剂剂型、混悬剂型。液体剂型形式可以是糖浆剂、注射溶液、非水溶液、悬浮液或乳剂；固体剂型例如片剂、锭剂、胶囊、滴丸、丸剂、粒剂、粉剂、霜剂、溶液剂、栓剂、可分散粉剂如冻干粉针剂、气雾剂等。
本发明所述的抗乙肝病毒和SARS-CoV-2的药物或药物组合物可以制成普通制剂，也可以是缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
本发明所述的药学上可接受的载体或辅料包括口服制剂辅料、胃肠外途径给药或外用给药的辅料。所用辅料包括，赋形剂如乳糖、碳酸钙、磷酸钙、磷酸钠；稀释剂与吸收剂如淀粉、环糊精、乳糖、蔗糖、甘露醇、微晶纤维素钠、硫酸钙等；湿润剂与粘合剂如水、乙醇、丙醇、甘油、丙二醇、异丙醇、糖浆、蜂蜜、葡萄糖、明胶浆、羧甲基纤维素钠、磷酸钾等；崩解剂如干燥淀粉、琼脂粉、碳酸钙、碳酸氢钠、十二烷基磺酸钠、甲基纤维素等；崩解抑制剂如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂如季铵盐、十二烷基硫酸钠等；润滑剂如滑石粉、三乙胺硬脂酸镁、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡等。还可以将片剂进一步制备成包衣片，如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片，以延迟其在胃肠道中的崩解和吸收，并由此提供在较长时间内的持续作用。
本发明所述“卤素”是指被“卤素原子”取代，“卤素原子”是指氟原子、氯原子、溴原子或碘原子。
本发明所述的“异构体”包含构造异构体和立体异构体。构造异构体又分为(碳)链异构体、位置异构体和官能团异构体。立体异构体分为构象异构体和构型异构体，而构型异构体还分为顺反异构体和旋光异构体(对映异构体)。本发明所述化合物的对映异构体、非对映异构体、外消旋体、内消旋体、顺反异构体、互变异构体、几何异构 体、差向异构体及其混合物等，均包含在本发明范围内。
本发明所述式I所示的苯并二氢吡喃类化合物的异构体，其中的旋光异构体(对映异构体)为式X-式XIII所示化合物，
为了更好的理解本发明，以下通过具体实施例进一步解释或说明本发明内容，但这些例子不应被理解为对本发明保护范围的限制。
下述实施例中所述方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
实施例1：中间体I-1的制备
将2，4-二羟基苯乙酮(15.21g,0.1mol)溶于1L的丙酮中，加入碳酸钾(55.2g,0.4mol)。在冰浴下加入硫酸二甲酯(0.11mol)，滴加完毕，恢复至室温，油浴加热回流5h。反应结束后，加入500mL的冰水，用乙酸乙酯萃取三次。合并有机相并用饱和的NaCl洗涤三次，然后用无水硫酸镁干燥。减压蒸馏浓缩后，经硅胶色谱法纯化后得中间体I-1，产率为90％。
1H-NMR(400MHz,CDCl3),δ12.75(s,1H),7.63(d,J＝8.7Hz,1H),6.44(dd,J＝8.7,2.5Hz,1H),6.42(d,J＝2.4Hz,1H),3.84(s,3H),2.56(s,3H).13C-NMR(100MHz,CDCl3)δ202.5,166.0,165.2,132.3,113.8,107.5,100.8,55.5,26.1.
实施例2：中间体I-2的制备
将中间体I-1(16.62g,0.1mol)溶解在乙酸乙酯(1500mL)中，室温下加入溴化铜，加热回流12h。待冷却至室温后，将混合物进行过滤，再用饱和的碳酸氢钠水溶液进行中和。经乙酸乙酯萃取三次并合并有机相后，用饱和的NaCl洗涤三次，无水硫酸镁干燥，减压蒸馏浓缩得粗品。然后经硅胶色谱法和重结晶纯化后，得中间体I-2，产率为90％。
1H NMR(400MHz,CDCl3)δ12.22(s,1H),7.65(d,J＝8.9Hz,1H),6.48(dd,J＝8.9,2.5Hz,1H),6.45(d,J＝2.5Hz,1H),4.36(s,2H),3.86(s,3H).13C-NMR(100MHz,CDCl3)δ195.3,167.1,166.4,132.1,111.3,108.5,101.3,55.8,29.8.
实施例3：中间体I-3的制备
将2-羟基苯乙酮(13.6g,0.1mol)溶解在乙酸乙酯(1500mL)中，制备方法同实施例2，得中间体I-3，产率为90％。
1H NMR(400MHz,CDCl3)δ11.75(s,1H),7.75(dd,J＝8.1,1.6Hz,1H),7.53(ddd,J＝8.6,7.2,1.6Hz,1H),7.02(dd,J＝8.5,0.9Hz,1H),6.94(ddd,J＝8.2,7.3,1.1Hz,1H),4.46(s,2H).13C-NMR(100MHz,CDCl3)δ197.1,163.3,137.6,130.5,119.4,119.0,117.1,30.2.
实施例4：中间体I-4的制备
将三苯基磷(10g,40.8mmol)溶于乙酸乙酯(40.8mL)中，在室温下将中间体I-2(10.7g,40.8mmol)缓慢的加入，并在室温下搅拌过夜。经过滤得白色固体，再溶入二氯甲烷溶剂(20.4mL)。在室温下，将氢氧 化钠溶液(2N)缓慢的加入二氯甲烷溶液，调节pH值至12。搅拌30分钟后，萃取分离，并用饱和的碳酸氢钠水溶液洗涤。合并有机相，经干燥、过滤和浓缩得粗品。粗品经重结晶可得中间体I-4，产率为74％。
1H NMR(400MHz,CDCl3)δ7.75–7.66(m,7H),7.63–7.55(m,4H),7.51–7.47(m,6H),6.39(d,J＝2.5Hz,1H),6.32(dd,J＝8.7,2.6Hz,1H),3.78(s,3H).13C-NMR(100MHz,CDCl3)δ187.1,164.2,162.9,133.3(3×CH),133.2(3×CH),132.5(4×CH),129.2(4×CH),129.1(3×CH),127.1,126.2,114.4,114.2,105.5,101.3,55.4.
实施例5：中间体I-5的制备
将三苯基磷(10g,40.8mmol)溶于乙酸乙酯(40.8mL)中，在室温下将中间体I-3(8.73g,40.8mmol)缓慢的加入，并在室温下搅拌过夜。制备方法同实施例4，得中间体I-5，产率为98％。
1H NMR(400MHz,CDCl3)δ7.74–7.69(m,7H),7.62–7.57(m,3H),7.52–7.48(m,7H),7.24(ddd,J＝8.5,7.2,1.6Hz,1H),6.88(dd,J＝8.3,1.2Hz,1H),6.75(ddd,J＝8.2,7.2,1.3Hz,1H).13C NMR(100MHz,CDCl3)δ187.6,162.0,133.2(3×CH),133.1(CH×3),132.6(4×CH),132.2,129.2(3×CH),129.1(3×CH),128.0,126.4,125.5,120.9,120.8,118.0,117.6.
实施例6：中间体I-6的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中， 再加入丁醛(1.44g,0.02mol)，添加完毕加热回流30h。反应完毕，经过滤、减压蒸馏得到粗品，经硅胶色谱法纯化后，得中间体I-6，产率为60％。
1H NMR(400MHz,CDCl3)δ12.71(s,1H),7.73(dd,J＝8.0,1.2Hz,1H),7.39(ddd,J＝8.4,7.2,1.3Hz,1H),7.13(dt,J＝15.2,6.9Hz,1H),6.97–6.90(m,2H),6.82(ddd,J＝8.0,7.2,1.0Hz,1H),2.24(qd,J＝7.0,1.1,2H),1.55–1.44(m,2H),0.90(t,J＝7.4Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.7,150.9,136.4,130.0,124.1,119.7,118.9,118.6,35.1,21.5,13.9.
实施例7：中间体I-7的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入戊醛(1.72g,0.02mol)，制备方法同实施例6，得中间体I-7，产率为65％。
1H NMR(400MHz,CDCl3)δ12.71(s,1H),7.74(d,J＝7.8Hz,1H),7.40(t,J＝7.4Hz,1H),7.14(m,1H),6.98–6.91(m,2H),6.83(t,J＝7.6Hz,1H),2.28(q,J＝7.0Hz,2H),1.49–1.40(m,2H),1.36–1.27(m,2H),0.87(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.7,151.2,136.4,130.0,123.9,119.7,118.9,118.6,32.8,30.3,22.5,14.0.
实施例8：中间体I-8的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入己醛(2.00g,0.02mol)，制备方法同实施例6，得中间体I-8，产率为75％。
1H NMR(400MHz,CDCl3)δ12.68(s,1H),7.74(dd,J＝8.1,1.6Hz,1H),7.40(ddd,J＝8.6,7.3,1.6Hz,1H),7.14(dt,J＝15.1,6.9Hz,1H),6.98–6.91(m,2H),6.83(ddd,J＝8.2,7.2,1.2Hz,1H),2.28(qd,J＝7.2,1.4,2H),1.51–1.44(m,2H),1.30–1.5(m,4H),0.84(t,J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.7,151.2,136.4,130.0,124.0,119.7,118.9,118.6,33.1,31.6,28.0,22.6,14.1.
实施例9：中间体I-9的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入庚醛(2.28g,0.02mol)，制备方法同实施例6，得中间体I-9，产率为70％。
1H NMR(400MHz,CDCl3)δ12.79(s,1H),7.81(dd,J＝8.1,1.6Hz,1H),7.47(ddd,J＝8.4,7.2,1.5Hz,1H),7.21(dt,J＝15.2,6.9Hz,1H),7.06–6.99(m,2H),6.90(ddd,J＝8.2,7.2,1.1Hz,1H),2.35(q,J＝7.2Hz,2H),1.57–1.50(m,2H),1.39–1.28(m,6H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.6,151.3,136.4,130.0,123.8,119.7,118.9,118.6,33.1,31.7,29.1,28.2,22.7,14.2.
实施例10：中间体I-10的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入辛醛(2.56g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-10，产率为70％。
1H NMR(400MHz,CDCl3)δ12.79(s,1H),7.81(d,J＝8.1Hz,1H),7.47(t,J＝8.2Hz,1H),7.20(dt,J＝15.1,6.9Hz,1H),7.05–6.98(m,2H), 6.90(t,J＝7.6Hz,1H),2.34(q,J＝7.2Hz,2H),1.57–1.49(m,2H),1.34–1.28(m,8H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.6,151.3,136.4,130.0,123.8,119.6,118.9,118.6,33.1,31.9,29.4,29.2,28.2,22.8,14.2.
实施例11：中间体I-11的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入壬醛(2.84g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-11，产率为60％。
1H NMR(400MHz,CDCl3)δ12.72(s,1H),7.74(d,J＝8.0Hz,1H),7.40(t,J＝7.7Hz,1H),7.14(dt,J＝15.1,6.9Hz,1H),6.98–6.91(m,2H),6.83(t,J＝7.6Hz,1H),2.27(q,J＝7.2Hz,2H),1.49–1.42(m,2H),1.31–1.11(m,10H),0.81(t,J＝6.9Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.6,151.3,136.4,130.0,123.8,119.6,118.9,118.6,33.1,32.0,29.5,29.4,29.3,28.2,22.8,14.3.
实施例12：中间体I-12的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入癸醛(3.12g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-12，产率为55％。
1H NMR(400MHz,CDCl3)δ12.78(s,1H),7.81(dd,J＝8.1,1.2Hz,1H),7.47(ddd,J＝8.5,7.3,1.5Hz,1H),7.21(dt,J＝15.1,7.0Hz,1H),7.05–6.98(m,2H),6.90(ddd,J＝8.1,7.3,1.1Hz,1H),2.34(q,J＝7.2Hz,2H),1.55–1.49(m,2H),1.41–1.20(m,12H),0.88(t,J＝7.0Hz, 3H).13C NMR(100MHz,CDCl3)δ194.3,163.6,151.3,136.4,130.0,123.9,119.7,118.9,118.6,33.1,32.0,29.6,29.6,29.4,29.4,28.3,22.8,14.3.
实施例13：中间体I-13的制备
将中间体I-5(7.92g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入壬醛(3.68g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-13，产率为62％。
1H NMR(400MHz,CDCl3)δ12.77(s,1H),7.82(dd,J＝8.1,1.6Hz,1H),7.48(ddd,J＝8.6,7.3,1.6Hz,1H),7.21(dt,J＝15.0,6.9Hz,1H),7.05–6.99(m,2H),6.91(ddd,J＝8.2,7.3,1.1Hz,1H),2.35(qd,J＝7.2,1.3Hz,2H),1.55–1.50(m,2H),1.31–1.25(m,16H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ194.3,163.7,151.3,136.4,130.0,123.9,119.7,118.9,118.6,33.1,32.1,29.8,29.8,29.7,29.6,29.5,29.4,28.3,22.8,14.3.
实施例14：中间体I-14的制备
将中间体I-4(8.52g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入丁醛(1.44g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-14，产率为65％。
1H NMR(400MHz,CDCl3)δ13.39(s,1H),7.72(d,J＝9.6Hz,1H),7.15(m,1H),6.95(d,J＝15.2Hz,1H),6.46–6.44(m,2H),3.84(s,3H),2.31(q,J＝7.2Hz,2H),1.61–1.52(m,2H),0.97(t,J＝7.2Hz,3H).13C  NMR(100MHz,CDCl3)δ192.5,166.7,166.2,149.7,131.5,124.1,113.7,107.7,101.1,55.7,35.1,21.6,13.9.
实施例15：中间体I-15的制备
将中间体I-4(8.52g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入戊醛(1.72g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-15，产率为68％。
1H NMR(400MHz,CDCl3)δ13.38(s,1H),7.71(d,J＝9.6Hz,1H),7.16(m,1H),6.94(d,J＝15.1Hz,1H),6.46–6.43(m,2H),3.84(s,3H),2.33(q,J＝7.2Hz,2H),1.55–1.47(m,2H),1.42–1.33(m,2H),0.93(t,J＝7.3Hz,3H).13C NMR(100MHz,CDCl3)δ192.5,166.7,166.1,150.0,131.5,123.9,113.7,107.7,101.1,55.7,32.8,30.4,22.5,14.0.
实施例16：中间体I-16和I-17的制备
根据实施例15的方法，将中间体I-4(8.52g,20mmol)依次与己醛和庚醛反应，可以获得中间体I-16和I-17。
实施例17：中间体I-18的制备
将中间体I-4(8.52g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入辛醛(2.56g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-18，产率为70％。
1H NMR(400MHz,CDCl3)δ13.38(s,1H),7.80(d,J＝9.6Hz,1H), 7.15(m,1H),6.93(d,J＝15.1Hz,1H),6.45–6.40(m,2H),3.83(s,3H),2.25(q,J＝7.3Hz,2H),1.53–1.48(m,2H),1.34–1.27(m,8H),0.88(t,J＝6.6Hz,3H).13C NMR(100MHz,CDCl3)δ192.4,166.6,166.0,149.9,131.4,123.8,113.6,107.6,101.0,55.6,33.0,31.8,29.3,29.1,28.2,22.7,14.1.
实施例18：中间体I-19的制备
根据实施例17的方法，将中间体I-4(8.52g,20mmol)与壬醛反应，可以获得中间体I-19。
实施例19：中间体I-20的制备
将中间体I-4(8.52g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入癸醛(3.12g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-20，产率为66％。
1H NMR(400MHz,CDCl3)δ13.38(s,1H),7.71(d,J＝9.3Hz,1H),7.15(m,1H),6.94(d,J＝15.1Hz,1H),6.44–6.41(m,2H),3.83(s,3H),2.31(q,J＝7.3Hz,2H),1.55–1.48(m,2H),1.35–1.26(m,12H),0.87(t,J＝6.6Hz,3H).13C NMR(100MHz,CDCl3)δ192.4,166.7,166.1,150.0,131.5,123.9,113.7,107.7,101.1,55.7,33.1,32.0,29.6,29.6,29.4,29.4,28.3,22.8,14.3.
实施例20：中间体I-21的制备
将中间体I-4(8.52g,20mmol)溶解在干燥的二氯甲烷(60mL)中，再加入十二醛(3.68g,0.02mol)，添加完毕加热回流30h。制备方法同实施例6，得中间体I-21，产率为72％。
1H NMR(400MHz,CDCl3)δ13.38(s,1H),7.71(d,J＝9.6Hz,1H),7.16(m,1H),6.94(d,J＝15.1Hz,1H),6.45–6.40(m,2H),3.84(s,3H),2.32(q,J＝7.3Hz,2H),1.55–1.48(m,2H),1.36–1.25(m,16H),0.87(t,J＝6.6Hz,3H).13C NMR(100MHz,CDCl3)δ192.5,166.7,166.1,150.0,131.5,123.9,113.7,107.7,101.1,55.7,33.1,32.0,29.8,29.7,29.7,29.6,29.5,29.4,28.3,22.8,14.3.
实施例21：中间体I-22的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-6(3.80g,20mmol)加入该溶液中，添加完毕加热回流7h。反应完毕，冷却至室温，经减压蒸馏除去乙醇溶剂，再加入蒸馏水(200mL)，然后经乙酸乙酯萃取、饱和的NaCl洗涤、无水硫酸镁干燥和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得中间体I-22，产率为95％。
1H NMR(400MHz,CDCl3)δ7.86(dd,J＝7.8,1.8Hz,1H),7.45(t,J＝7.8Hz,1H),7.00–6.94(m,2H),4.44(m,1H),2.67(d,J＝8.2Hz,2H),1.86(m,1H),1.66(m,1H),1.59–1.45(m,2H),0.98(t,J＝7.4Hz,3H).13C NMR(100MHz,CDCl3)δ192.9,161.8,136.1,127.0,121.2,121.1,118.0,77.7,43.0,37.1,18.3,14.0.
实施例22：中间体I-23的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-7(4.08g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-23，产率为90％。
1H NMR(400MHz,CDCl3)δ7.85(d,J＝7.8Hz,1H),7.44(m,1H),7.00–6.94(m,2H),4.41(m,1H),2.68–2.65(m,2H),1.86(m,1H),1.70(m,1H),1.52(m,1H),1.42–1.34(m,3H),0.93(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ192.8,161.7,136.0,127.0,121.2,121.0,118.0,78.0,43.0,34.7,27.1,22.6,14.1.
实施例23：中间体I-24的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-8(4.36g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-24，产率为97％。
1H NMR(400MHz,CDCl3)δ7.85(dt,J＝7.8,1.6Hz,1H),7.44(t,J＝7.8Hz,1H),6.99–6.93(m,2H),4.41(m,1H),2.66(d,J＝7.8Hz,2H),1.88(m,1H),1.70(m,1H),1.55(m,1H),1.46(m,1H),1.38–1.32(m,4H),0.91(t,J＝6.7Hz,3H).13C NMR(100MHz,CDCl3)δ192.7,161.7,136.0,127.0,121.2,121.1,118.0,78.0,43.0,35.0,31.6,24.6,22.6,14.1.
实施例24：中间体I-25的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-9(4.64g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-25，产率为97％。
1H NMR(400MHz,CDCl3)δ7.85(d,J＝7.8Hz,1H),7.45(t,J＝7.7Hz,1H),6.99–6.94(m,2H),4.42(m,1H),2.67(d,J＝8.0Hz,2H),1.86 (m,1H),1.68(m,1H),1.53(m,1H),1.42(m,1H),1.36–1.27(m,6H),0.88(t,J＝6.7Hz,3H).13C NMR(100MHz,CDCl3)δ192.8,161.8,136.0,127.0,121.2,121.0,118.0,78.0,43.0,35.0,31.8,29.1,24.9,22.7,14.2.
实施例25：中间体I-26的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-10(4.92g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-26，产率为90％。
1H NMR(400MHz,CD3OD)δ7.86(dd,J＝7.8,1.8Hz,1H),7.46(t,J＝7.8Hz,1H),7.00–6.95(m,2H),4.43(m,1H),2.67(d,J＝8.1Hz,2H),1.87(m,1H),1.69(m,1H),1.53(m,1H),1.43(m,1H),1.33–1.24(m,8H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CD3OD)δ194.7,163.3,137.3,127.6,122.2,122.1,119.1,79.3,43.8,36.0,33.0,30.6,30.4,26.0,23.7,14.5.
实施例26：中间体I-27的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-11(5.2g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-27，产率为97％。
1H NMR(400MHz,CDCl3)δ7.87(d,J＝7.4Hz,1H),7.47(t,J＝7.0Hz,1H),7.01–6.96(m,2H),4.44(m,1H),2.68(d,J＝7.3Hz,2H),1.87(m,1H),1.66(m,1H),1.51(m,1H),1.43(m,1H),1.35–1.25(m,10H),0.87(t,J＝6.4Hz,3H).13C NMR(100MHz,CDCl3)δ193.0,161.8,136.1,127.0,121.3,121.1,118.0,78.0,43.1,35.1,32.0,29.6,29.5,29.4,25.0, 22.8,14.3.
实施例27：中间体I-28的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-12(5.48g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-28，产率为96％。
1H NMR(400MHz,CD3OD)δ7.86(dd,J＝7.8,1.7Hz,1H),7.46(ddd,J＝8.4,7.2,1.8Hz,1H),7.00–6.95(m,2H),4.42(m,1H),2.68(d,J＝7.7Hz,2H),1.87(m,1H),1.68(m,1H),1.53(m,1H),1.43(m,1H),1.32–1.26(m,12H),0.87(t,J＝6.9Hz,3H).13C NMR(100MHz,CD3OD)δ194.7,163.3,137.4,127.7,122.2,122.1,119.1,79.3,43.8,36.0,33.1,30.7,30.7,30.6,30.5,26.1,23.8,14.5.
实施例28：中间体I-29的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-13(6.04g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-29，产率为89％。
1H NMR(400MHz,CD3OD)δ7.73(dd,J＝7.8,1.6Hz,1H),7.44(ddd,J＝8.6,7.3,1.7Hz,1H),6.95–6.90(m,2H),4.38(m,1H),2.61(m,2H),1.78(m,1H),1.63(m,1H),1.50(m,1H),1.40(m,1H),1.27–1.22(m,16H),0.82(t,J＝6.9Hz,3H).13C NMR(100MHz,CD3OD)δ194.8,163.3,137.4,127.6,122.2,122.1,119.1,79.3,43.8,36.0,33.1,30.8,30.8,30.7,30.7,30.6,30.5,26.0,23.8,14.5.
实施例29：中间体I-30的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-14(4.40g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-30，产率为93％。
1H NMR(400MHz,CDCl3)δ7.80(d,J＝8.8Hz,1H),6.56(dd,J＝8.8,2.4Hz,1H),6.41(d,J＝2.4Hz,1H),4.44(m,1H),3.83(s,3H),2.63–2.60(m,2H),1.85(m,1H),1.66(m,1H),1.59–1.47(m,2H),0.98(t,J＝7.3Hz,3H).13C NMR(100MHz,CDCl3)δ191.5,166.1,163.8,128.8,115.0,109.9,100.8,78.2,55.7,42.8,37.1,18.3,14.0.
实施例30：中间体I-31的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-15(4.68g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-31，产率为94％。
1H NMR(400MHz,CDCl3)δ7.78(d,J＝8.8Hz,1H),6.53(dd,J＝8.8,2.0Hz,1H),6.39(d,J＝2.1Hz,1H),4.39(m,1H),3.80(s,3H),2.61–2.59(m,2H),1.84(m,1H),1.67(m,1H),1.50(m,1H),1.45–1.31(m,3H),0.92(t,J＝7.0Hz,3H).13C NMR(100MHz,CDCl3)δ:191.4,166.1,163.7,128.7,115.0,109.9,100.7,78.4,55.6,42.7,34.7,27.1,22.6,14.1.
实施例31：中间体I-32的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中， 将中间体I-16(4.96g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-32，产率为92％。
1H NMR(400MHz,CDCl3)δ7.79(d,J＝8.7Hz,1H),6.54(dd,J＝8.8,2.4Hz,1H),6.39(d,J＝2.3Hz,1H),4.41(m,1H),3.81(s,3H),2.66–2.53(m,2H),1.84(m,1H),1.67(m,1H),1.53(m,1H),1.44(m,1H),1.37–1.30(m,4H),0.89(t,J＝6.7Hz 3H).13C NMR(100MHz,CDCl3)δ191.4,166.1,163.8,128.7,115.0,109.9,100.7,78.4,55.7,42.7,35.0,31.7,24.7,22.6,14.1.
实施例32：中间体I-33的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-17(5.24g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-33，产率为92％。
1H NMR(400MHz,CDCl3)δ7.78(d,J＝8.8Hz,1H),6.53(dd,J＝8.8,2.2Hz,1H),6.39(d,J＝2.2Hz,1H),4.40(m,1H),3.81(s,3H),2.61–2.59(m,2H),1.84(m,1H),1.67(m,1H),1.51(m,1H),1.43(m,1H),1.39–1.23(m,6H),0.87(t,J＝6.7Hz,3H).13C NMR(100MHz,CDCl3)δ191.4,166.1,163.8,128.7,115.0,109.9,100.7,78.4,55.7,42.7,35.1,31.8,29.2,25.0,22.7,14.2.
实施例33：中间体I-34的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-18(5.52g,20mmol)加入该溶液中，添加完毕加热回流7h。 后处理方法同实施例21，得中间体I-34，产率为95％。
1H NMR(400MHz,CDCl3)δ7.79(d,J＝8.8Hz,1H),6.55(dd,J＝8.8,2.4Hz,1H),6.40(d,J＝2.4Hz,1H),4.41(m,1H),3.82(s,3H),2.62–2.60(m,2H),1.85(m,1H),1.68(m,1H),1.53(m,1H),1.43(m,1H),1.36–1.24(m,8H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ191.4,166.1,163.8,128.7,115.0,109.9,100.7,78.4,55.7,42.8,35.1,31.9,29.5,29.3,25.0,22.8,14.2.
实施例34：中间体I-35的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-19(5.80g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-35，产率为90％。
1H NMR(400MHz,CDCl3)δ7.80(d,J＝8.8Hz,1H),6.55(dd,J＝8.8,2.4Hz,1H),6.40(d,J＝2.4Hz,1H),4.41(m,1H),3.82(s,3H),2.63–2.60(m,2H),1.85(m,1H),1.67(m,1H),1.53(m,1H),1.44(m,1H),1.34–1.24(m,10H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ191.5 166.1,163.8,128.7,115.0,109.9,100.8,78.4,55.7,42.8,35.1,32.0,29.6,29.5,29.3,25.0,22.8,14.2.
实施例35：中间体I-36的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-20(6.08g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-36，产率为94％。
1H NMR(400MHz,CDCl3)δ7.79(d,J＝8.8Hz,1H),6.59(dd,J＝8.4,2.4Hz,1H),6.40(d,J＝2.4Hz,1H),4.41(m,1H),3.81(s,3H),2.63–2.58(m,2H),1.85(m,1H),1.67(m,1H),1.51(m,1H),1.43(m,1H),1.34–1.26(m,12H),0.87(t,J＝6.6Hz,3H).13C NMR(100MHz,CDCl3)δ191.7,160.6,134.9,125.9,120.1,119.9,116.9,76.9,41.9,33.9,30.9,28.5,28.5,28.4,28.3,23.9,21.7,13.1.
实施例36：中间体I-37的制备
将无水乙酸钠(16.40g,200mmol)溶解在干燥的乙醇(700mL)中，将中间体I-21(6.64g,20mmol)加入该溶液中，添加完毕加热回流7h。后处理方法同实施例21，得中间体I-37，产率为29％。
1H NMR(400MHz,CDCl3)δ7.79(d,J＝8.8Hz,1H),6.54(dd,J＝8.8,2.4Hz,1H),6.40(d,J＝2.4Hz,1H),4.41(m,1H),3.82(s,3H),2.62–2.60(m,2H),1.85(m,1H),1.67(m,1H),1.52(m,1H),1.43(m,1H),1.38–1.25(m,16H),0.87(t,J＝6.7Hz,3H).13C NMR(100MHz,CDCl3)δ191.4,166.1,163.8,128.7,115.0,109.9,100.7,78.4,55.7,42.7,35.1,32.0,29.7(3×CH2),29.6,29.5,29.5,25.0,22.8,14.2.
实施例37：中间体I-38的制备
将中间体I-33(5.24g,20mmol)溶于200mL无水二氯甲烷中，再加入4mmol的三溴化硼，添加完毕后，室温反应18h。反应完毕，加入一定量的饱和碳酸氢钠溶液，调节pH至8-9。然后经乙酸乙酯萃取、饱和的NaCl洗涤、无水硫酸镁干燥和减压浓缩得粗品。所得粗品再经 硅胶色谱法纯化后，得中间体I-38，产率65％。
1H NMR(400MHz,CDCl3)δ7.66(d,J＝8.8Hz,1H),6.44(dd,J＝8.8,2.4Hz,1H),6.29(d,J＝2.4Hz,1H),4.41(m,1H),2.63–2.50(m,2H),1.84–1.28(m,10H),0.92(t,J＝6.4Hz,3H).13C NMR(100MHz,CDCl3)δ193.8,166.6,165.5,129.8,115.0,111.5,103.8,79.4,43.4,36.0,32.9,30.3,26.0,23.7,14.5.
实施例38：中间体I-39的制备
将中间体I-34(5.52g,20mmol)溶于200mL无水二氯甲烷中，再加入4mmol的三溴化硼，添加完毕后，室温反应18h。后处理方法同实施例37，得中间体I-39，产率63％。
1H NMR(400MHz,CDCl3)δ7.67(d,J＝8.8Hz,1H),6.44(dd,J＝8.8,2.0Hz,1H),6.30(d,J＝2.0Hz,1H),4.42(m,1H),2.65–2.53(m,2H),1.86–1.31(m,12H),0.91(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ193.6,166.6,165.5,129.8,115.0,111.5,103.6,79.4,43.4,36.0,33.0,30.6,30.4,26.1,23.8,14.5.
实施例39：中间体I-40的制备
将中间体I-32(4.96g,20mmol)溶于200mL无水乙醇中，再加入28mmol盐酸羟胺和30mmol的醋酸钠，添加完毕后，加热回流6h。反应完毕，经减压蒸馏除去溶剂，再加入蒸馏水(200mL)，然后经乙酸乙酯萃取、饱和的NaCl洗涤、无水硫酸镁干燥和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得中间体I-40，产率95％。
1H NMR(400MHz,CDCl3)δ7.69(d,J＝8.8Hz,1H),6.53(d,J＝ 8.8Hz,1H),6.41(s,1H),4.07(br.s,1H),3.79(s,3H),3.28(m,1H),2.41(m,1H),1.85–1.21(m,8H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ162.2,158.0,150.4,125.0,110.9,109.4,101.4,75.6,55.4,35.1,31.7,28.3,24.8,22.6,14.0.
实施例40：中间体I-41的制备
将中间体I-33(5.24g,20mmol)溶于200mL无水乙醇中，再加入28mmol盐酸羟胺和30mmol的醋酸钠，添加完毕后，加热回流6h。后处理方法同实施例39，得中间体I-41，产率95％。
1H NMR(400MHz,CDCl3)δ7.69(d,J＝8.8Hz,1H),6.52(d,J＝8.8Hz,1H),6.41(s,1H),4.10(br.s,1H),3.79(s,3H),3.27(m,1H),2.39(m,1H),1.81(m,1H),1.66(m,1H),1.57–1.45(m,2H),1.82–1.24(m,6H),0.88(t,J＝6.0Hz,3H).13C NMR(100MHz,CDCl3)δ162.2,158.0,150.5,125.0,111.0,109.4,101.4,75.6,55.4,35.1,31.7,29.2,28.2,25.1,22.6,14.1.
实施例41：中间体I-42的制备
将中间体I-34(5.52g,20mmol)溶于200mL无水乙醇中，再加入28mmol盐酸羟胺和30mmol的醋酸钠，添加完毕后，加热回流6h。后处理方法同实施例39，得中间体I-42，产率95％。
1H NMR(400MHz,CDCl3)δ7.69(d,J＝8.8Hz,1H),6.53(d,J＝8.8Hz,1H),6.41(s,1H),4.07(br.s,1H),3.80(s,3H),3.28(m,1H),2.40(m,1H),1.83–1.20(m,12H),0.89(t,J＝6.8Hz,3H).13C NMR(100 MHz,CDCl3)δ162.2,158.0,150.4,125.0,111.0,109.4,101.4,75.6,55.4,35.1,31.9,29.5,29.3,28.3,25.1,22.7,14.1.
实施例42 2-丙基-4-羟基苯并二氢吡喃(1)的制备
将中间体I-22(3.80g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。反应完毕后，减压蒸馏除去溶剂，再加入蒸馏水(200mL)，然后经乙酸乙酯萃取、饱和的NaCl洗涤、无水硫酸镁干燥和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得化合物1，产率为63％。
1H NMR(400MHz,CD3OD)δ7.34(d,J＝7.7Hz,1H),7.01(ddd,J＝8.3,7.7,1.1Hz,1H),6.79(td,J＝7.6,1.0Hz,1H),6.63(dd,J＝8.2,0.8Hz,1H),4.79(dd,J＝10.7,6.3Hz,1H),4.02(m,1H),2.14(ddd,J＝12.8,6.3,1.5Hz,1H),1.71–1.37(m,5H),0.92(t,J＝7.1Hz,3H).13C NMR(100MHz,CD3OD)δ156.0,129.4,128.2,127.7,121.2,117.1,75.9,66.0,39.0,38.7,19.4,14.4.
实施例43 2-丁基-4-羟基苯并二氢吡喃(2)的制备
将中间体I-23(4.08g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物2，产率为66％。
1H NMR(400MHz,CD3OD)δ7.33(d,J＝7.7Hz,1H),7.00(ddd,J＝8.2,7.6,0.9Hz,1H),6.78(td,J＝7.5,0.9Hz,1H),6.62(dd,J＝8.2,1.0Hz,1H),4.78(m,1H),3.99(m,1H),2.13(m,1H),1.65–1.28(m,7H),0.88(t,J＝7.1Hz,3H).13C NMR(100MHz,CD3OD)δ156.0,129.5,128.2,127.7,121.2,117.2,76.2,66.0,38.7,36.5,28.5,23.8,14.5.
实施例44 2-戊基-4-羟基苯并二氢吡喃(3)的制备
将中间体I-24(4.36g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物3，产率为66％。
1H NMR(400MHz,CD3OD)δ7.34(d,J＝7.7Hz,1H),7.01(ddd,J＝8.3,7.7,1.1Hz,1H),6.79(td,J＝7.6,1.1Hz,1H),6.63(dd,J＝8.2,1.0Hz,1H),4.79(dd,J＝10.7,6.3Hz,1H),4.00(m,1H),2.14(ddd,J＝12.8,6.3,1.6Hz,1H),1.66–1.27(m,9H),0.86(t,J＝6.9Hz,3H).13C NMR(100MHz,CD3OD)δ156.0,129.5,128.2,127.7,121.2,117.1,76.2,66.0,38.7,36.8,33.0,25.9,23.7,14.4.
实施例45 2-己基-4-羟基苯并二氢吡喃(4)的制备
将中间体I-25(4.64g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物4，产率为64％。
1H NMR(400MHz,CDCl3)δ7.45(d,J＝7.7Hz,1H),7.16(ddd,J＝8.3,7.7,1.2Hz,1H),6.93(td,J＝7.6,0.8Hz,1H),6.79(dd,J＝8.2,1.0Hz,1H),4.90(dd,J＝10.6,6.3Hz,1H),4.09(m,1H),2.28(ddt,J＝12.9,6.4,1.6Hz,1H),1.82–1.32(m,11H),0.91(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ154.7,129.1,127.0,126.1,120.6,116.6,75.1,65.8,38.1,35.6,31.9,29.3,25.2,22.7,14.2.
实施例46 2-庚基-4-羟基苯并二氢吡喃(5)的制备
将中间体I-26(4.92g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物5，产率为65％。
1H NMR(400MHz,CDCl3)δ7.45(d,J＝7.6Hz,1H),7.16(ddd,J＝8.2,7.7,1.1Hz,1H),6.92(t,J＝7.4Hz,1H),6.79(d,J＝8.2Hz,1H),4.90(m,1H),4.08(m,1H),2.28(m,1H),1.80–1.3(m,13H),0.91(t,J＝6.9Hz,3H).13C NMR(100MHz,CDCl3)δ154.7,129.1,127.1,126.1,120.6,116.6,75.1,65.8,38.1,35.6,32.0,29.7,29.4,25.2,22.8,14.3.
实施例47 2-辛基-4-羟基苯并二氢吡喃(6)的制备
将中间体I-27(5.20g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物6，产率为68％。
1H NMR(400MHz,CDCl3)δ7.46(d,J＝7.6Hz,1H),7.16(m,1H),6.93(t,J＝7.4Hz,1H),6.79(d,J＝8.2Hz,1H),4.90(m,1H),4.09(m,1H),2.30(m,1H),1.82–1.72(m,2H),1.67–1.9(m,1H),1.55–1.42(m,2H),1.38–1.29(m,10H),0.90(t,J＝6.7Hz,3H).13C NMR(100MHz,CDCl3)δ154.7,129.1,127.0,126.1,120.6,116.6,75.1,65.8,38.1,35.7,32.0,29.7,29.7,29.4,25.2,22.8,14.3.
实施例48 2-壬基-4-羟基苯并二氢吡喃(7)的制备
将中间体I-28(5.48g,20mmol)溶解在甲醇(400mL)中，然后加入 0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物7，产率为67％。
1H NMR(400MHz,CDCl3)δ7.45(d,J＝7.6Hz,1H),7.16(m,1H),6.92(td,J＝7.6,1.2Hz,1H),6.79(dd,J＝8.4,1.2Hz,1H),4.91(dd,J＝10.4,6.4Hz,1H),2.29(ddd,J＝12.9,6.3,0.9Hz,1H),1.85(m,1H),1.82–1.71(m,2H),1.63(m,1H),1.55–1.41(m,2H),1.37–1.26(m,12H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ154.7,129.1,127.0,126.1,120.6,116.6,75.1,65.8,38.1,35.7,32.0,29.7,29.7,29.5,29.4,25.2,22.8,14.3.
实施例49 2-十一烷基-4-羟基苯并二氢吡喃(8)的制备
将中间体I-29(6.04g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物8，产率为65％。
1H NMR(400MHz,CD3OD)δ7.34(d,J＝7.7Hz,1H),7.01(ddd,J＝8.3,7.8,1.1Hz,1H),6.79(td,J＝7.6,1.1Hz,1H),6.63(dd,J＝8.2,1.0Hz,1H),4.79(dd,J＝10.7,6.3Hz,1H),4.00(m,1H),2.14(ddd,J＝12.8,6.3,1.6Hz,1H),1.66–1.23(m,21H),0.83(t,J＝6.8Hz,3H).13C NMR(100MHz,CD3OD)δ156.0,129.4,128.2,127.7,121.2,117.2,76.2,66.0,38.7,36.8,33.1,30.8(5×CH2),30.5,26.2,23.8,14.5.
实施例50 2-丙基-4-羟基-7-甲氧基苯并二氢吡喃(9)的制备
将中间体I-30(4.40g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物9，产率为63％。
1H NMR(400MHz,CDCl3)δ7.33(dd,J＝8.4,0.8Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.33(d,J＝2.4Hz,1H),4.86(dd,J＝10.4,6.4Hz,1H),4.08(m,1H),3.76(s,3H),2.26(m,1H),1.76(m,1H),1.77-1.30(m,4H),0.97(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.8,128.0,118.6,107.8,101.0,75.1,65.5,55.4,38.3,37.7,18.5,14.1.
实施例51 2-丁基-4-羟基-7-甲氧基苯并二氢吡喃(10)的制备
将中间体I-31(4.68g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物10，产率为68％。
1H NMR(400MHz,CDCl3)δ7.32(d,J＝8.4Hz,1H),6.50(dd,J＝8.4,2.4Hz,1H),6.33(d,J＝2.4Hz,1H),4.84(m,1H),4.06(m,1H),3.75(s,3H),2.25(m,1H),1.77-1.33(m,7H),0.94(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.7,127.9,118.6,107.7,100.9,75.4,65.4,55.4,38.2,35.3,27.4,22.8,14.2.
实施例52 2-戊基-4-羟基-7-甲氧基苯并二氢吡喃(11)的制备
将中间体I-32(4.96g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物11，产率为69％。
1H NMR(400MHz,CDCl3)δ7.33(dd,J＝8.4,0.8Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.34(d,J＝2.4Hz,1H),4.86(dd,J＝10.4,6.4Hz,1H),4.07(m,1H),3.76(s,3H),2.27(m,1H),1.77–1.32(m,9H),0.91(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ:160.4,155.8,127.9,118.6,107.8,101.0,75.4,65.5,55.4,38.3,35.6,31.9,24.9,22.7,14.2.
实施例53 2-己基-4-羟基-7-甲氧基苯并二氢吡喃(12)的制备
将中间体I-33(5.24g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物12，产率为67％。
1H NMR(400MHz,CDCl3)δ7.33(dd,J＝8.4,0.8Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.34(d,J＝2.4Hz,1H),4.86(dd,J＝10.4,6.4Hz,1H),4.07(m,1H),3.76(s,3H),2.27(m,1H),1.80–1.29(m,11H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.8,127.9,118.6,107.8,101.0,75.4,65.5,55.4,38.3,35.6,31.9,29.3,25.2,22.8,14.2.
实施例54 2-庚基-4-羟基-7-甲氧基苯并二氢吡喃(13)的制备
将中间体I-34(5.52g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物13，产率为66％。
1H NMR(400MHz,CDCl3)δ7.33(d,J＝8.4Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.34(d,J＝2.4Hz,1H),4.86(dd,J＝10.4,6.4Hz,1H),4.07(m,1H),3.76(s,3H),2.26(m,1H),1.77–1.26(m,13H),0.89(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.8,127.9,118.6,107.8,101.0,75.4,65.5,55.4,38.3,35.6,32.0,29.6,29.4,25.2,22.8,14.2.
化合物13的手性拆分可通过HPLC手性拆分实现，分离条件如下：手性柱：Chiralpak AD-RH(4.6*150mm)；分离条件：流速：0.3mL/min；流动相：乙腈/水(90：10)；λ＝254nm；t13a＝24.80min,t13b＝48.41min.
(2S,4R)-2-庚基-4-羟基-7-甲氧基苯并二氢吡喃(13a): (2R,4S)-2-庚基-4-羟基-7-甲氧基苯并二氢吡喃(13b):根据已知文献报道(Ma Y,Li J,Ye J,et al.Synthesis of chiral chromanols via a RuPHOX–Ru catalyzed asymmetric hydrogenation of chromones.Chem.Commun.2018,54:13571-13574；Kuninobu K,Yoriko K,Shozo Y,et al.The synthesis and the stereochemistry of 4-chromanones and 4-chromanols with bulky substituents.Bull.Chem.Soc.Jpn.1973,46:1839-1844)，化合物的绝对构型通过对比化合物的比旋光度，以及化合物2位和4位上的氢与3位上的氢的耦合常数对照来确定。
实施例55 2-辛基-4-羟基-7-甲氧基苯并二氢吡喃(14)的制备
将中间体I-35(5.80g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物14，产率为65％。
1H NMR(400MHz,CDCl3)δ7.33(t,J＝8.8,Hz,1H),6.51(m,1H),6.34(t,J＝2.8,Hz,1H),4.86(m,1H),4.07(m,1H),3.75(s,3H),2.26(m,1H),1.77–1.26(m,15H),0.89(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.8,128.0,118.6,107.8,101.0,75.4,65.6,55.5,38.3,35.6,32.0,29.8,29.7,29.4,25.2,22.8,14.3.
实施例56 2-壬基-4-羟基-7-甲氧基苯并二氢吡喃(15)的制备
将中间体I-36(6.08g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物15，产率为66％。
1H NMR(400MHz,CDCl3)δ7.34(d,J＝8.4Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.35(d,J＝2.4Hz,1H),4.86(dd,J＝10.4,2.4Hz,1H),4.08(m,1H),3.76(s,3H),2.28(m,1H),1.79–1.27(m,17H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.8,127.9,118.6,107.8,101.0,75.4,65.5,55.4,38.3,35.6,32.0,29.8,29.8,29.7,29.5,25.3,22.8,14.3.
实施例57 2-十一烷基-4-羟基-7-甲氧基苯并二氢吡喃(16)的制备
将中间体I-37(6.64g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物16，产率为66％。
1H NMR(400MHz,CDCl3)δ7.33(dd,J＝8.4,1.2Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.34(br.s,1H),4.86(t,J＝7.6Hz,1H),4.08(m,1H),3.76(s,3H),2.27(m,1H),1.76–1.27(m,21H),0.89(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.4,155.8,127.9,118.6,107.8,101.0,75.4,65.5,55.4,38.4,35.6,32.0,5×29.7,29.5,25.3,22.8,14.3.
实施例58 2-己基-4,7-二羟基苯并二氢吡喃(17)的制备
将中间体I-38(4.96g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物17，产率为45％。
1H NMR(400MHz,CDCl3)δ7.28(d,J＝8.4Hz,1H),6.41(dd,J＝ 8.4,2.4Hz,1H),6.27(d,J＝2.4Hz,1H),4.88(br.s,1H),4.06(m,1H),2.28(m,1H),1.77–1.31(m,11H),0.89(t,J＝6.4Hz,3H).13C NMR(100MHz,CDCl3)δ156.3,155.8,128.1,118.5,108.3,102.9,75.3,65.5,38.1,35.4,31.8,29.2,25.1,22.6,14.1.
实施例59 2-庚基-4,7-二羟基苯并二氢吡喃(18)的制备
将中间体I-41(5.24g,20mmol)溶解在甲醇(400mL)中，然后加入0.02mol的硼氢化钠，在室温下反应2h。后处理方法同实施例42，得化合物18，产率为47％。
1H NMR(400MHz,CDCl3)δ7.21(d,J＝8.4Hz,1H),6.30(m,1H),6.27(d,J＝2.4Hz,1H),4.80(m,1H),4.00(m,1H),2.20(m,1H),1.75–1.20(m,13H),0.81(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ156.5,155.9,128.3,118.6,108.4,103.1,75.4,65.6,38.3,35.6,32.0,29.7,29.4,25.2,22.8,14.3.
实施例60 2-庚基-4-异丁氨基-7-甲氧基苯并二氢吡喃(19)的制备
将中间体I-34(5.52g,20mmol)和异丁胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。反应后经减压蒸馏除去溶剂，再加入蒸馏水(100mL)，然后经乙酸乙酯萃取、饱和的NaCl洗涤、无水硫酸镁干燥和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得化合物19，产率为40％。
1H NMR(400MHz,CDCl3)δ7.41(d,J＝8.4Hz,1H),6.48(dd,J＝8.4,2.8Hz,1H),6.35(d,J＝2.8Hz,1H),4.03(m,1H),3.94(dd,J＝10.8,5.6Hz,1H),3.76(s,3H),2.52(dd,J＝11.2,6.8Hz,1H),2.41(dd,J＝11.2,6.4Hz,1H),2.26(ddd,J＝11.6,5.6,1.2Hz,1H),1.81–1.67(m,2H),1.65–1.42(m,2H),1.38–1.24(m,10H),0.95(t,J＝7.2,6.8Hz,6H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.7,156.5,128.2,118.1,107.4,101.1,76.1,55.4,53.7,52.6,36.0,35.3,32.0,29.7,29.4,29.3,25.3,22.8,21.0,20.8,14.2.HRESIMS:m/z 356.2554[M+Na]+,calcd.for C21H35NO2Na:356.2560.
实施例61 2-庚基-4-(4-氯苄胺基)-7-甲氧基苯并二氢吡喃(20)的制备
将中间体I-34(5.52g,20mmol)和4-氯苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物20，产率为47％。
1H NMR(400MHz,CDCl3)δ7.48(d,J＝8.4Hz,1H),7.36(d,J＝8.4Hz,2H),7.30(d,J＝8.4Hz,2H),6.51(dd,J＝8.8,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.05–3.95(m,2H),3.90(d,J＝13.2Hz,1H),3.78(d,J＝13.2Hz,1H),3.77(s,3H),2.32(m,1H),1.76(m,1H),1.66–1.44(m,4H),1.39–1.26(m,8H),0.89(t,J＝6.4Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,156.5,139.3,132.7,2×129.4,2×128.6,128.2,117.7,107.5,101.1,76.0,55.4,51.8,49.0,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3. HRESIMS:m/z 424.2010[M+Na]+,calcd.for C24H32ClNO2Na:424.2014.
实施例62 2-庚基-4-(3-氟苄胺基)-7-甲氧基苯并二氢吡喃(21)的制备
将中间体I-34(5.52g,20mmol)和3-氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物21，产率为56％。
1H NMR(400MHz,CDCl3)δ7.51(d,J＝8.4Hz,1H),7.29(m,1H),7.18(d,J＝8.4Hz,2H),6.95(m,1H),6.52(dd,J＝8.4,2.4Hz,1H),6.37(d,J＝2.4Hz,1H),4.06–3.98(m,2H),3.94(d,J＝14.0Hz,1H),3.79(d,J＝14.0Hz,1H),3.77(s,3H),2.33(m,1H),1.76(m,1H),1.64(m,1H),1.55(m,1H),1.45(m,2H),1.33(m,8H),0.91(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ163.1(d,J＝243.9Hz),159.8,156.5,143.7(d,J＝7.0Hz),129.9(d,J＝8.3Hz),128.2,123.5(d,J＝2.7Hz),117.7,114.9(d,J＝21.3Hz),113.9(d,J＝21.1Hz),107.5,101.1,76.0,55.4,51.8,49.2(d,J＝1.6Hz),36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ-113.55.HRESIMS:m/z 408.2307[M+Na]+,calcd.for C24H32FNO2Na:408.2309.
实施例63 2-庚基-4-(3-氯苄胺基)-7-甲氧基苯并二氢吡喃(22)的制备
将中间体I-34(5.52g,20mmol)和3-氯苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物22，产率为51％。
1H NMR(400MHz,CDCl3)δ7.49(dd,J＝8.4,1.2Hz,1H),7.43(br.s,1H),7.29(m,1H),7.26(br.s,1H),7.24(m,1H),6.51(dd,J＝8.4,2.8Hz,1H),6.36(d,J＝2.8Hz,1H),4.06–3.96(m,2H),3.91(d,J＝13.6Hz,1H),3.79(d,J＝13.7Hz,1H),3.76(s,3H),2.32(m,1H),1.74(m,1H),1.67–1.43(m,4H),1.37–1.27(m,8H),0.91(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,156.6,143.1,134.4,129.7,128.2,128.2,127.2,126.2,117.7,107.5,101.2,76.0,55.4,51.9,49.2,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.HRESIMS:m/z 424.2016[M+Na]+,calcd.for C24H32ClNO2Na:424.2014.
实施例64 2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃(23)的制备
将中间体I-34(5.52g,20mmol)和3-三氟甲基苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、 减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物23，产率为49％。
1H NMR(400MHz,CDCl3)δ7.70(br.s,1H),7.63(d,J＝7.6Hz,1H),7.53–7.43(m,3H),6.52(dd,J＝8.4,2.4Hz,1H),6.37(d,J＝2.4Hz,1H),4.10–3.96(m,3H),3.87(d,J＝13.6Hz,1H),3.77(s,3H),2.34(m,1H),1.76(m,1H),1.69–1.44(m,4H),1.37–1.25(m,8H),0.91(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.9,156.6,141.9,131.5,130.8(d,J＝31.9Hz),128.9,128.2,124.8(q,J＝3.8Hz),123.9(q,J＝3.8Hz),123.0,117.5,107.5,101.2,76.0,55.4,52.0,49.2,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ3×(-62.54).HRESI MS:m/z434.2308[M-H]-,calcd.for C25H31F3NO2:434.2312.
实施例65 2-庚基-4-(4-氟苄胺基)-7-甲氧基苯并二氢吡喃(24)的制备
将中间体I-34(5.52g,20mmol)和4-氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物24，产率为39％。
1H NMR(400MHz,CDCl3)δ7.47(d,J＝8.8Hz,1H),7.40-7.33(m,2H),7.02(t,J＝8.4Hz,2H),6.50(dd,J＝8.4,2.4Hz,1H),6.35(d,J＝2.4Hz,1H),4.11–3.95(m,2H),3.89(d,J＝13.2Hz,1H),3.78(d,J＝13.2Hz,1H),3.76(s,3H),2.32(m,1H),1.75(m,1H),1.68–1.48(m,4H), 1.39–1.23(m,8H),0.90(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ162.1(d,J＝243.2Hz),159.9,156.6,2×129.6(d,J＝7.8Hz),128.2,128.2,117.8,2×115.3(d,J＝21.2Hz),107.5,101.2,76.1,55.4,51.9,49.1,36.0,35.3,32.0,29.7,29.4,25.3,22.8,14.2.19F NMR(376MHz,CDCl3)δ-116.14.HRESIMS:m/z 408.2305[M+Na]+,calcd.for C24H32FNO2Na:408.2309.
实施例66 2-庚基-4-苄胺基-7-甲氧基苯并二氢吡喃(25)的制备
将中间体I-34(5.52g,20mmol)和苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物25，产率为54％。
1H NMR(400MHz,CDCl3)δ7.50(dd,J＝8.4,1.2Hz,1H),7.44–7.42(m,2H),7.39–7.33(m,2H),7.30–7.25(m,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.37(d,J＝2.4Hz,1H),4.05–3.99(m,2H),3.95(d,J＝13.2Hz,1H),3.83(d,J＝13.2Hz,1H),3.77(s,3H),2.35(m,1H),1.77(m,1H),1.70–1.43(m,4H),1.41–1.28(m,8H),0.91(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,156.5,140.8,128.5,128.2,128.2,128.1,128.1,127.1,117.9,107.5,101.1,76.1,55.4,51.8,49.8,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.HRESIMS:m/z 366.2437[M-H]-,calcd.for C24H32NO2:366.2439.
实施例67 2-庚基-4-(4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃(26)的制备
将中间体I-34(5.52g,20mmol)和4-三氟甲基苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物26，产率为41％。
1H NMR(400MHz,CDCl3)δ7.60(d,J＝8.4Hz,2H),7.55(d,J＝8.0Hz,2H),7.51(d,J＝8.8Hz,1H),6.52(dd,J＝8.8,2.4Hz,1H),6.37(d,J＝2.4Hz,1H),4.06–3.97(m,3H),3.88(d,J＝14.0Hz,1H),3.77(s,3H),2.33(m,1H),1.77(m,1H),1.69–1.53(m,4H),1.46–1.23(m,8H),0.91(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,156.5,145.0,129.3(q,J＝32.0Hz),128.3,2×128.2,2×125.4(d,J＝3.8Hz),124.4(d,J＝270.5Hz),117.5,107.5,101.1,76.0,55.4,51.9,49.2,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ3×(-62.38).HRESIMS:m/z 458.2275[M+Na]+,calcd.for C25H32F3NO2Na:458.2277.
实施例68 2-庚基-4-(2-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃(27)的制备
将中间体I-34(5.52g,20mmol)和2-氯-4-氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减 压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物27，产率为36％。
1H NMR(400MHz,CDCl3)δ7.53(dd,J＝8.4,6.4Hz,1H),7.47(d,J＝8.8Hz,1H),7.12(dd,J＝8.4,2.4Hz,1H),6.99(td,J＝8.4,2.4Hz,1H),6.50(dd,J＝8.4,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.09–3.98(m,2H),3.96(d,J＝13.6Hz,1H),3.86(d,J＝13.6Hz,1H),3.76(s,3H),2.35(m,1H),1.78(m,1H),1.70–1.53(m,4H),1.43–1.20(m,8H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ162.8,160.4,159.8,156.5,134.1(d,J＝10.4Hz),130.9(d,J＝8.6Hz),128.2,117.5,116.8(d,J＝24.5Hz),114.1(d,J＝20.6Hz),107.5,101.1,76.0,55.4,52.2,46.7,36.0,35.3,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ-113.94.HRESIMS:m/z 442.1916[M+Na]+,calcd.for C24H31ClFNO2Na:4442.1920.
实施例69 2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃(28)的制备
将中间体I-34(5.52g,20mmol)和3，4-二氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物28，产率为31％。
1H NMR(400MHz,CDCl3)δ7.49(dd,J＝8.4,1.2Hz,1H),7.30(dd,J＝8.4,2.0Hz,1H),7.15–7.06(m,2H),6.51(dd,J＝8.4,2.4Hz,1H), 6.36(d,J＝2.4Hz,1H),4.09–3.94(m,2H),3.89(d,J＝14.0Hz,1H),3.77(d,J＝13.6Hz,1H),3.76(s,3H),2.31(m,1H),1.76(m,1H),1.68–1.41(m,4H),1.38–1.26(m,8H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,156.5,151.2(dd,J＝102.7,12.6Hz),148.7(dd,J＝101.6,12.5Hz),138.0,128.2,123.5(dd,J＝6.1,3.4Hz),117.5,117.0(d,J＝16.8Hz),116.8(d,J＝17.0Hz),107.5,101.1,76.0,55.4,51.8,48.7,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ-138.17(d,J＝21.1Hz),-140.73(d,J＝21.3Hz).HRESIMS:m/z426.2211[M+Na]+,calcd.for C24H31F2NO2Na:426.2215.
实施例70 2-庚基-4-(3,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃(29)的制备
将中间体I-34(5.52g,20mmol)和3，4-二氯苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物29，产率为41％。
1H NMR(400MHz,CDCl3)δ7.54(d,J＝1.6Hz,1H),7.48(d,J＝8.4Hz,1H),7.39(d,J＝8.4Hz,1H),7.26(dd,J＝8.0,2.0Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.07–3.94(m,2H),3.88(d,J＝14.0Hz,1H),3.77(d,J＝14.0Hz,1H),3.77(s,3H),2.30(m,1H),1.75(m,1H),1.69–1.49(m,4H),1.41–1.23(m,8H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.9,156.5,141.3,132.4,130.8,130.4,129.9,128.2,127.4,117.5,107.5,101.1,76.0,55.4,51.8, 48.5,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.HRESIMS:m/z458.1624[M+Na]+,calcd.for C24H31Cl2NO2Na:458.1624.
实施例71 2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃(30)的制备
将中间体I-34(5.52g,20mmol)和3-氯-4-氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物30a和30b，产率为56％。
顺-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃(30a)：1H NMR(400MHz,CDCl3)δ7.49(d,J＝2.0Hz,1H),7.47(d,J＝3.6Hz,1H),7.28(m,1H),7.09(t,J＝8.4Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.07–3.94(m,2H),3.88(d,J＝13.6Hz,1H),3.77(s,3H),3.76(d,J＝13.6Hz,1H),2.31(m,1H),1.76(m,1H),1.67–1.50(m,4H),1.40–1.24(m,8H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,157.2(d,J＝245.8Hz),156.5,138.0(d,J＝3.6Hz),130.1,128.2,127.6(d,J＝6.9Hz),120.8(d,J＝17.6Hz),117.5,116.4(d,J＝20.9Hz),107.5,101.1,76.0,55.4,51.8,48.5,36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ-118.39.HRESIMS:m/z 442.1917[M+Na]+,calcd.for C24H31ClFNO2Na:442.1920.
反-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃(30b)：1H NMR(400MHz,CDCl3)δ7.44(dd,J＝7.2,2.4Hz,1H),7.23(m,1H),7.09(t,J＝8.8Hz,1H),7.01(d,J＝8.4Hz,1H),6.46(dd,J＝8.4,2.4Hz, 1H),6.39(d,J＝2.4Hz,1H),4.25(m,1H),3.85(br.s,2H),3.76(s,3H),3.70(dd,J＝3.6,2.0Hz,1H),1.95(dt,J＝13.6,2.0Hz,1H),1.76(m,1H),1.66–1.41(m,4H),1.39–1.25(m,8H),0.89(t,J＝7.2Hz,1H).13C NMR(100MHz,CDCl3)δ160.2,157.2(d,J＝246.0Hz),156.2,138.0(d,J＝3.8Hz),130.6,130.2,127.7(d,J＝6.9Hz),120.9(d,J＝17.7Hz),116.5(d,J＝20.8Hz),116.4,107.5,101.2,71.5,55.4,51.0,50.4,35.6,32.6,32.0,29.8,29.4,25.4,22.8,14.3.
实施例72 2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃(31)的制备
将中间体I-34(5.52g,20mmol)和2,4-二氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物31，产率为45％。
1H NMR(400MHz,CDCl3)δ7.49(d,J＝8.4Hz,1H),7.45(d,J＝8.4Hz,1H),7.44(d,J＝8.8Hz,1H),6.87(ddd,J＝8.4,2.0,1.6Hz,1H),6.80(m,1H),6.50(dd,J＝8.4,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.06–3.97(m,2H),3.91(d,J＝13.6Hz,1H),3.82(d,J＝13.6Hz,1H),3.76(s,3H),2.33(m,1H),1.77(m,1H),1.69–1.54(m,4H),1.45–1.23(m,8H),0.90(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ162.8(dd,J＝105.7,12.1Hz),161.3(dd,J＝141.1,12.0Hz),159.8,156.5,130.9(dd,J＝9.5,6.2Hz),128.1,123.7(dd,J＝14.4,3.4Hz),117.5,111.3(dd,J＝20.8,3.7Hz),107.5,103.8(t,J＝25.4Hz),101.1,76.0,55.4,52.0,42.6(d, J＝2.9Hz),36.0,35.2,32.0,29.7,29.4,25.3,22.8,14.3.19F NMR(376MHz,CDCl3)δ-112.34,-115.42.HRESIMS:m/z 426.2211[M+Na]+,calcd.for C24H31F2NO2Na:426.2215.
实施例73 2-庚基-4-(2-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃(32)的制备
将中间体I-34(5.52g,20mmol)和2-氟-4-三氟甲基苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物32，产率为41％。
1H NMR(400MHz,CDCl3)δ7.69(t,J＝7.6Hz,1H),7.46(d,J＝8.4Hz,1H),7.42(d,J＝8.0Hz,1H),7.31(d,J＝10.0Hz,1H),6.50(dd,J＝8.4,2.4Hz,1H),6.37(d,J＝2.4Hz,1H),4.07–3.99(m,3H),3.92(d,J＝14.4Hz,1H),3.77(s,3H),2.34(m,1H),1.78(m,1H),1.68–1.52(m,4H),1.45–1.24(m,8H),0.91(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ160.6(d,J＝246.4Hz),160.0,156.6,132.2(d,J＝14.1Hz),131.0(dd,J＝41.2,8.2Hz),130.6(d,J＝5.0Hz),128.2,123.6(dd,J＝270.6,2.6Hz),121.2(q,J＝3.8Hz),117.4,112.8(m),107.6,101.2,76.0,55.4,52.2,42.7(d,J＝3.2Hz),36.0,35.3,32.0,29.7,29.4,25.3,22.8,14.2.19F NMR(376MHz,CDCl3)δ3×(-62.58),-117.09.HRESIMS:m/z 476.2181[M+Na]+,calcd.for C25H31F4NO2Na:476.2183.
实施例74 2-庚基-4-(3,4,5-三氟苄胺基)-7-甲氧基苯并二氢吡喃(33)的制备
将中间体I-34(5.52g,20mmol)和3,4,5-三氟苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物33，产率为73％。
1H NMR(400MHz,CDCl3)δ7.48(d,J＝8.8Hz,1H),7.14–7.02(m,2H),6.52(dd,J＝8.4,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.09–3.92(m,2H),3.87(d,J＝14.4Hz,1H),3.77(s,3H),3.76(d,J＝14.0Hz,1H),2.29(m,1H),1.77(m,1H),1.82–1.41(m,4H),1.40–1.27(m,8H),0.91(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ160.0,156.6,152.5(dd,J＝9.9,3.9Hz),150.0(dd,J＝9.8,3.9Hz),139.9(t,J＝15.4Hz),137.5(m),128.1,117.4,111.6(d,J＝5.5Hz),111.5(d,J＝5.5Hz),107.5,101.2,76.0,55.4,51.9,48.5,36.0,35.3,32.0,29.7,29.4,25.3,22.8,14.2.19F NMR(376MHz,CDCl3)δ2×(-134.79)(d,J＝20.7Hz),-162.99(t,J＝20.7Hz).HRESIMS:m/z 444.2118[M+Na]+,calcd.for C24H30F3NO2Na:444.2121.
实施例75 2-庚基-4-(3-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃(34)的制备
将中间体I-34(5.52g,20mmol)和3-氟-4-三氟甲基苄胺(120mmol) 溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物34，产率为44％。
1H NMR(400MHz,CDCl3)δ7.55(t,J＝7.6Hz,1H),7.49(d,J＝8.8Hz,1H),7.33(d,J＝11.2Hz,1H),7.28(d,J＝8.0Hz,1H),6.51(dd,J＝8.4,2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.07–3.94(m,3H),3.87(d,J＝14.4Hz,1H),3.77(s,3H),2.31(m,1H),1.76(m,1H),1.66–1.42(m,4H),1.40–1.23(m,8H),0.90(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ160.1(dd,J＝254.4,2.4Hz),160.0,156.6,148.5(d,J＝8.2Hz),128.2,127.1(m),123.3(d,J＝3.4Hz),122.9(d,J＝269.2Hz),117.4,116.2(d,J＝20.7Hz),107.6,101.3,76.0,55.4,52.0,48.8,36.0,35.3,32.0,29.6,29.4,25.3,22.8,14.2.19F NMR(376MHz,CDCl3)δ3×(-61.11)(d,J＝12.6Hz),-114.81(q,J＝12.4Hz).HRESIMS:m/z 476.2180[M+Na]+,calcd.for C25H31F4NO2Na:476.2183.
实施例76 2-庚基-4-(2,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃(35)的制备
将中间体I-34(5.52g,20mmol)和2,4-二氯苄胺(120mmol)溶解在甲苯(110mL)中，冰浴下，将四氯化钛的甲苯溶液逐滴加入到反应液中。滴加完毕恢复到室温，反应24h。反应完毕，经过滤、减压蒸馏得油状产品。再把油状产品溶于甲醇(110mL)中，并加入硼氢化钠(24mmol)，室温下搅拌2h。后处理方法同实施例60，得化合物35，产率为63％。
1H NMR(400MHz,CDCl3)δ7.42(d,J＝8.4Hz,1H),7.38(d,J＝8.4Hz,1H),7.28(d,J＝2.0Hz,1H),7.15(m,1H),6.40(dd,J＝8.8,2.0Hz,1H),6.27(m,1H),3.99–3.88(m,2H),3.86(d,J＝14.0Hz,1H),3.76(d,J＝14.0Hz,1H),3.67(s,3H),2.24(m,1H),1.68(m,1H),1.58–1.34(m,4H),1.36–1.14(m,8H),0.81(d,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,156.5,137.0,134.2,133.2,130.6,129.2,128.2,127.3,117.5,107.5,101.1,76.0,55.4,52.2,46.7,36.0,35.3,32.0,29.7,29.4,25.3,22.8,14.3.HRESIMS:m/z 458.1625[M+Na]+,calcd.for C24H31Cl2NO2Na:458.1624.
实施例77 2-戊基-4-氨基-7-甲氧基苯并二氢吡喃(36)的制备
将中间体I-40(5.26g,20mmol)溶解在甲醇(100mL)中，加入钯碳(0.53g)后，将装有溶液的玻璃试管放入高压釜中，通入氢气，使压力达到0.5Mp，室温反应24h。反应后经过滤和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得化合物36，产率为96％。
1H NMR(400MHz,CDCl3)δ7.31(d,J＝8.4Hz,1H),6.51(d,J＝8.4,1H),6.35(s,1H),4.10–4.01(m,2H),3.76(s,3H),2.18(dd,J＝13.2,5.6Hz,1H),1.75–1.33(m,9H),0.91(t,J＝6.4Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,155.8,127.6,120.2,107.5,101.1,76.1,55.5,46.9,39.8,35.8,31.9,24.9,22.8,14.2.
实施例78 2-己基-4-氨基-7-甲氧基苯并二氢吡喃(37)的制备
将中间体I-41(5.54g,20mmol)溶解在甲醇(100mL)中，加入 钯碳(0.55g)后，将装有溶液的玻璃试管放入高压釜中，通入氢气，使压力达到0.5Mp，室温反应24h。反应后经过滤和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得化合物37，产率为93％。
1H NMR(400MHz,CDCl3)δ7.31(d,J＝8.4Hz,1H),6.51(d,J＝8.4,1H),6.34(s,1H),4.08–4.00(m,2H),3.76(s,3H),2.17(dd,J＝12.8,5.6Hz,1H),1.75–1.31(m,11H),0.88(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.8,155.8,127.6,120.2,107.5,101.1,76.1,55.4,46.9,39.8,35.8,31.9,29.4,25.2,22.8,14.2.
实施例79 2-庚基-4-氨基-7-甲氧基苯并二氢吡喃(38)的制备
将中间体I-42(5.82g,20mmol)溶解在甲醇(100mL)中，加入钯碳(0.58g)后，将装有溶液的玻璃试管放入高压釜中，通入氢气，使压力达到0.5Mp，室温反应24h。反应后经过滤和减压浓缩得粗品。所得粗品再经硅胶色谱法纯化后，得化合物38，产率为90％。
1H NMR(400MHz,CDCl3)δ7.31(d,J＝8.4Hz,1H),6.51(d,J＝8.8,1H),6.34(s,1H),4.08–4.02(m,2H),3.76(s,3H),2.18(dd,J＝12.4,5.6Hz,1H),1.74–1.29(m,13H),0.89(t,J＝6.8Hz,3H).13C NMR(100MHz,CDCl3)δ159.7,155.7,127.4,119.9,107.4,101.0,75.9,55.3,46.7,39.6,35.7,31.8,29.6,29.2,25.0,22.7,14.1.
实施例80式I所示的苯并二氢吡喃类化合物及其异构体盐酸盐的制备
式I所示的苯并二氢吡喃类化合物及其异构体(17.93mmol)溶于乙酸乙酯(54mL)中，然后滴加HCl的乙酸乙酯溶液(46mL,3.9mol/L)，室温下反应1.5h。反应后经减压浓缩和重结晶可得相应化合物的盐酸盐，分别为：2-庚基-4-异丁氨基-7-甲氧基苯并二氢吡喃盐酸盐(39)， 2-庚基-4-(4-氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(40)，2-庚基-4-(3-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(41)，2-庚基-4-(3-氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(42)，2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(43)，2-庚基-4-(4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(44)，2-庚基-4-苄胺基-7-甲氧基苯并二氢吡喃盐酸盐(45)，2-庚基-4-(4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(46)，2-庚基-4-(2-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(47)，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(48)，2-庚基-4-(3,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(49)，顺-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(50a)，反-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(50b)，2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(51)，2-庚基-4-(2-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(52)，2-庚基-4-(3,4,5-三氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(53)，2-庚基-4-(3-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(54)，2-庚基-4-(2,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(55)。
实施例81所述式I所示的苯并二氢吡喃类化合物、其异构体、药学上可接受的盐的抗乙肝病毒活性检测
采用HBV DNA转染的HepG2.2.15细胞模型(Sells MA,Chen ML,Acs G.Production of hepatitis B virus particles in HepG2cells transfected with cloned hepatitis B virus DNA.Proc.Natl.Acad.Sci.U.S.A.1987,84:1005-1009)，测试了实施例42-80(化合物1-55)的抗乙肝病毒活性。实验方法如下：
(1)HepG2.2.15细胞的来源及培养
乙型肝炎病毒(HBV)DNA转染的HepG2.2.15细胞购自上海富衡生物科技有限公司，购进后自行传代培养待用。细胞培养于MEM培养液(即完全培养液：含10％胎牛血清、400μg/mL G418、100μg/mL青霉素、100μg/mL链霉素)中，在37℃、5％CO2培养箱中培养。隔天更换 培养液一次，3d左右长满细胞。
(2)药物对细胞的毒性实验
根据文献(Mosmann T.Rapid colorimetric assay for cellular growth and survival:application to proliferation in cytoxicity assay.J.Immunol.Meth.1983,65,55-65)，将HepG2.2.15细胞制成细胞悬液，调整细胞的密度为5×105个/mL，以100μL/孔接种到96孔细胞培养板中，在37℃、5％CO2的培养箱中培养24小时。加入不同浓度的含药培养液(给药组)，含1‰DMSO的无药物培养液(细胞对照组)，完全培养液(空白对照组，无细胞)，每组设3个复孔。培养72h后，各孔加入20μL MTT溶液(5mg/mL)，继续培养4h。吸弃各孔上清液，分别加入150μL DMSO溶液，将培养板置于震荡器上避光震荡15min，使结晶物充分溶解。在酶标仪上测各孔的OD值，测定波长490nm，记录结果，根据公式(1)计算药物不同浓度下的细胞存活率，计算半数细胞毒性浓度(CC50)。
细胞存活率(％)＝(A给药组-A空白组)/(A细胞对照组-A空白组)×100％(1)
式中：A给药组为不同质量浓度的药物实验组的吸光度；A空白组为空白对照组的吸光度；A细胞对照组为含1‰DMSO的培养液的细胞对照组的吸光度。
(3)HBsAg与HBeAg的检测
将HepG2.2.15细胞制成细胞悬液，调整细胞密度为5×105个/mL，以100μL/孔接种到96孔细胞培养板中，在37℃、5％CO2的培养箱中培养24小时。在给药组中每孔加入200μL不同浓度药物的完全培养液处理细胞和细胞对照组(含1‰DMSO的无药物培养基)，每组设3个复孔。每3天更换含不同浓度化合物的新鲜培养基。在37℃、5％CO 2的培养箱中培养12天后，收集上清液。取细胞上清液，采用酶联免疫法，按用乙肝表面s抗原(HBsAg)及e抗原(HBeAg)检测试剂盒说明书操作，在酶标仪上以450nm为检测波长测定各孔OD值。根据公式(2)计算不同浓度药物对HBsAg和HBeAg的抑制率，计算半数抑制浓度(IC50)。HBsAg(或HBeAg)的抑制率(％)＝1-(A给药组-A空白组)/(A细胞对照组 -A空白组)×100％(2)
式中：A给药组为不同质量浓度的药物实验组的吸光度；A空白组为试剂盒测定时不加酶结合底物而平行操作的空白对照组的吸光度；A细胞 对照组为不加药物处理的细胞对照组的吸光度。
(4)HBV DNA含量的测定
将HepG2.2.15细胞制成细胞悬液，调整细胞密度为5×105个/mL，以300μL/孔接种到24孔细胞培养板中，在37℃、5％CO2的培养箱中培养24小时。在给药组中每孔加入500μL不同浓度药物的完全培养液处理细胞和细胞对照组(含1‰DMSO的无药物培养基)，以拉米夫定(3-TC)为阳性对照，每组设3个复孔。每3天更换含不同浓度化合物的新鲜培养基。在37℃、5％CO2的培养箱中培养12天后，收集上清液及24孔板中的细胞，按照血液/组织/细胞基因组DNA提取试剂盒(天根生化-北京有限公司)说明进行上清液及24孔板中的细胞提取DNA。
采用Real-time PCR(ABI Steap One)进行HBV DNA含量测定，上引物：5′-ACCAATCGCCAGTCAGGAAG-3′，下引物：5′-ACCAGCAGGGAAATACAGGC-3’(购自北京擎科生物科技有限公司)，在反应管中加入10μL UltraSYBR Mixture荧光染料、1μL上引物、1μL下引物、2μL样本DNA或阳性对照DNA、6μL无菌水，循环程序在95℃运行10min，然后在95℃运行15s，60℃运行1min，循环40次，72℃运行10s，然后设置熔解曲线分析：95℃运行15s，60℃运行1min，95℃运行15s，60℃运行15s。以阳性对照的扩增结果为y轴，阳性对照拷贝数的对数为x轴制作线性标准曲线，计算出药物HBV DNA含量。根据公式(3)计算不同浓度药物对HBV DNA的抑制率，计算半数抑制浓度(IC50)。
HBV DNA抑制率(％)＝(1-C给药组/C细胞对照组)×100％  (3)
式中：C给药组为不同质量浓度的药物实验组的HBV DNA拷贝数；C细胞对照组为不加药物处理的细胞对照组的HBV DNA拷贝数。
所测试化合物对HepG2.2.15细胞分泌HBeAg和HBsAg的抑制作用测试结果见表1。实验结果表明，化合物2，3，5，8，11-13，13a，13b，14-25，27-29，30a，30b，31-49，50a，50b及51-55对HBeAg具有抑制作用，其抑制作用的IC50在28.76～498.64μM之间，其中化合物2和36的活性较强，IC50分别为41.89±3.72和28.76±3.55μM，选择性指数(SI)分别为>19.10，4.56；化合物3-5，8，11-13，13a，13b，14-29，30a，30b，31-49，50a，50b及51-55对HBsAg具有抑制作用，其抑制作用的IC50在0.48～136.20μM之间，其中化合物5，18，31及40的活性较强，IC50分别为1.13±0.49，1.17±0.58，2.08±0.98及0.48±0.15μM，SI分别为143.74，104.68，>1923.08及7032.58。
所测试化合物对HepG2.2.15细胞分泌HBV DNA的抑制作用测试结果见表2。实验结果表明，所测试的化合物(1-13，13a，13b，14-29，30a，30b，31-49，50a，50b及51-55)对HBV DNA均具有抑制作用，其抑制作用的IC50在0.029～47.99μM之间，其中化合物12，13，13a，13b，18，20，30a，30b，31，34，39，40，43-45，48，50a，50b，51，52，54及55的活性较强，IC50均小于1.00μM，分别为0.26±0.13,0.083±0.027,0.093±0.061,0.10±0.03,0.78±0.25,0.92±0.24,0.36±0.14,0.51±0.19,0.59±0.27,0.36±0.11,0.70±0.24,0.44±0.18,0.10±0.05,0.15±0.06,0.47±0.11,0.12±0.03,0.029±0.011,0.047±0.028,0.12±0.02,0.24±0.13,0.72±0.28,0.59±0.21μM，SI在500.60～36282.80之间。
表1苯并二氢吡喃类化合物对HepG2.2.15细胞分泌HBeAg、HBsAg的影响


n＝3。
表2苯并二氢吡喃类化合物体外对HBV DNA的影响

n＝3。
实施例82顺-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(50a)体内抗DHBV DNA活性测试
采用先天鸭乙型肝炎动物模型(Niu J,Wang Y,Dixon R,et al.The use of ampligen alone and in combination with ganciclovir and coumermycin A1for the treatment of ducks congenitally-infected with duck hepatitis B virus.Antiviral Res.1993,21(2):155-71)，测试了顺-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(50a)对乙型肝炎病毒麻鸭的治疗作用。实验方法如下：
(1)乙型肝炎病毒麻鸭的筛选
购买1日龄麻鸭(购自昆明市官渡区康源活禽经营部)，适应性饲养一周后，于鸭足静脉取血，分离血清，按照血液/组织/细胞基因组DNA提取试剂盒(天根生化-北京有限公司)说明提取DNA，按照鸭乙型肝炎病毒染料法荧光定量PCR试剂盒(购自上海联迈生物科技有限公司)说明书进行检测，每周检测一次，连续检测2周，筛选出稳定先天感染DHBV DNA的麻鸭备用。
(2)分组及给药
将筛选出的麻鸭随机分为5组，模型组(生理盐水组)、拉米夫定组(20mg/kg)、药物低剂量组(10mg/kg)、中剂量组(20mg/kg)、高剂量组(40mg/kg)，每组6只，每天灌胃给药一次，连续给药28天。然后停 药14天。给药期间自鸭足静脉取血，每周取一次血，离心分离血清，-80℃冰箱贮藏，待检，并每周称量一次体重。
(3)鸭血清DHBV DNA水平检测
取上述待检鸭血清，按照血液/组织/细胞基因组DNA提取试剂盒(天根生化-北京有限公司)说明进行提取血清中DNA，按照鸭乙型肝炎病毒染料法荧光定量PCR试剂盒(购自上海联迈生物科技有限公司)说明进行DHBV DNA含量检测，以阳性对照的扩增结果为y轴，阳性对照拷贝数的对数为x轴制作线性标准曲线，计算出样本DHBV DNA含量。
(4)统计学方法
实验结果均以表示。采用GraphPad prism 6软件进行统计分析，采用t检验分析药物治疗前、后各时间点动物血清DHBV DNA水平变化，与模型组比较，p<0.05存在显著性差异，具有统计学意义。
化合物50a对先天乙型肝炎病毒麻鸭血清DHBV DNA的抑制作用测试结果见图1和表3。实验结果表明，与空白组相比，化合物50a的低、中、高剂量组均对DHBV麻鸭体重的增长无影响(见图1)。测试结果表明化合物50a在低、中、高剂量(10mg/kg，20mg/kg，40mg/kg)下对DHBV DNA的复制均具有较好的抑制作用。与同组T0比较，在灌胃给药期间，化合物50a低剂量组T14、T21及T28鸭血清DHBV DNA含量显著下降(P<0.01)；化合物50a中剂量组T7鸭血清DHBV DNA含量下降明显(P<0.05)，T14、T21及T28鸭血清DHBV DNA含量下降显著(P<0.01)，化合物50a高剂量组T7、T14、T21及T28鸭血清DHBV DNA含量均下降效果显著(P<0.01)。与同组T0比较，在停药期间，化合物50a的低、中、高剂量组P7、P14鸭血清DHBV DNA含量均仍呈极显著下降(P<0.01)，有持续抑制病毒复制的趋势(见表3)。
表3化合物50a用药前后对鸭血清DHBV DNA含量的影响
续表3化合物50a用药前后对鸭血清DHBV DNA含量的影响

注：##与空白组比较，P<0.01；*与模型组比较，P<0.05，**与模型组比较，P<0.01。
实施例83所述式I所示的苯并二氢吡喃类化合物、其异构体、药学上可接受的盐的抗SARS-CoV-2活性检测
采用SARS-CoV-2诱导的Vero-E6细胞模型(Wang ML,Cao RY,Zhang LK,et al.Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus(2019-nCoV)in vitro.Cell Res.2020,30:269-271)，测试了2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(43)，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(48)，顺-2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(50a)，2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐(51)的抗SARS-CoV-2活性。实验方法如下：
1.Vero-E6细胞的来源及培养
Vero-E6细胞购自美国菌种保藏中心(ATCC)，SARS-CoV-2 (nCoV-2019BetaCoV/Wuhan/WIV04/2019分离株，GISAID登录号EPI_ISL_402124)由中国科学院武汉病毒研究所分离传代培养。细胞培养于MEM培养液(每100mL含胎牛血清10％、青霉素、链霉素及卡那霉素各100U/mL，NaHCO3 5％)中，在37℃、5％CO2培养箱中培养3d左右长满细胞。
2.药物对细胞毒性实验
药物对细胞毒性的检测利用CCK8试剂盒(碧云天)测定。具体步骤如下：在96孔细胞培养板中接种1×104个Vero-E6细胞，37℃培养16小时；将药物用DMSO稀释到合适的母液浓度，再用含2％FBS的MEM培养基稀释到所需浓度，弃96孔板中原培养基，取100μL含药物的MEM培养基加入到对应的孔中，每个浓度做三个复孔。同时设置阴性对照(细胞孔中加PBS和含2％FBS的MEM培养基，不加药物)和空白对照(不含细胞，加DMSO和含2％FBS的MEM培养基)。加药完毕，将细胞放置于37℃、5％CO2培养箱中培养24h；然后向待测孔中加入10μL CCK8溶液，置于37℃培养箱中2h后，在酶标仪上测各孔的OD值，测定波长450nm，记录结果，根据公式(4)计算药物不同浓度下的细胞存活率，计算细胞活性：
细胞活性(％)＝(A药物处理组–A空白对照))/(A阴性对照–A空白对照)×100％
(4)
其中A为酶标仪测得的吸光度数值。
3.药物抗新冠病毒实验
将Vero E6细胞接种到48孔细胞培养板中，每孔约1×105个细胞，待第二天实验。先将100μL含有相应浓度药物的培养基加入细胞培养板中，预处理细胞1h，然后加入20μL稀释的病毒(MOI＝0.05)，置于培养箱孵育1h。然后弃病毒培养液，用PBS洗去未感染的残留病毒，再加入含有相应浓度的药物培养基，对照组加入含有0.1％DMSO培养基，然后放入37℃、5％CO2孵箱中继续培养48h。
RNA提取：采用TaKaRa公司生产的试剂盒(TaKaRa MiniBEST  Viral RNA/DNA Extraction Kit)，步骤如下：(1)取受试培养板的上清液100μL，加入无核酸酶EP管中，然后每孔加入321μL裂解液(100μL PBS,200μL buffer VGB,2μL proteinase K,1μL carrier RNA)，混匀后置于56℃消化15min；(2)向(1)所得混合液加200μL无水乙醇，混匀；(3)将上述(2)中所得混合液转入无RNA酶的离心柱中，12000rpm离心15s，弃废液；(4)加入500μL Buffer RW1,12000rpm离心15s，弃废液；(5)加入650μL Buffer RW2,12000rpm离心15s，弃废液；(6)加入650μL Buffer RW2,12000rpm离心2min，弃废液；(7)将(6)中的离心柱整体转移至新的无RNA酶的2ml收集管，12000rpm离心1min；8)将(7)中的离心柱整体转移至新的1.5ml收集管，向每个离心柱中加入30μl不含RNA酶的水，12000rpm离心2min，洗脱液即含有相应的RNA。
RNA反转录，步骤如下：实验采用TaKaRa公司生产的反转录试剂盒(PrimeScriptTMRT reagent Kit with gDNA Eraser)进行RNA反转录。(1)进行gDNA去除：收集各实验组RNA样品，分别取3μL RNA进行反转录。先向各实验组RNA中加入2μL 5×gDNA Eraser Buffer及1μL gDNA Eraser，然后用RNase Free水补足反应体系至10μl，充分混匀，42℃水浴2min去除样品中可能存在的g DNA；(2)进行逆转录：向(1)中所得样品中加入适量的酶和引物Mix及反应缓冲液，用RNase Free水补足体积至20μl，37℃反应15min，然后85℃处理15s，既可转录得到cDNA。
Real-time PCR检测：荧光定量PCR采用TB Green Premix(Takara，Cat#RR820A)混好反应体系，在StepOne Plus Real-time PCR仪进行扩增反应和读数。计算原病毒液每毫升所含拷贝数。步骤如下：(1)建立标准品：将质粒pMT-RBD稀释成5×108copies/μL,5×107copies/μL,5×106copies/μL,5×105copies/μL,5×104copies/μL,5×103copies/μL,5×102copies/μL。取2μL标准品或cDNA模板用于qPCR反应；(2)实验过程中所用引物序列如下(均为5’-3’方向表示)：RBD-qF:CAATGGTTTAACAGGCACAGG；RBD-qR:CTCAAGTGTCTGTGGA TCACG；(3)反应程序如下：预变性：95℃5分钟；循环参数：95℃15秒，54℃15秒，72℃30秒。共40个循环。按照公式(5)计算化合物的半数抑制浓度(EC50)和半数毒性浓度(CC50)。
抑制率(％)＝(1-A药物处理组)/A空白对照)×100％  (5)
实验结果表明，化合物43，48，50a及51对SARS-CoV-2分离株nCoV-2019BetaCoV/Wuhan/WIV04/2019有明显的抑制作用，在病毒感染复数(MOI)为0.05时，其半数抑制浓度分别为3.28，2.27，2.98及5.93μmol/L，相应的选择指数(SI)分别大于122.0，176.2，134.2及67.5。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (10)

  1. 式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，式I结构式如下：
    其中，R1、R2分别独立的选自H、OH、OMe、卤素；R3选自H、C1-C11烷基、C3-C6环烷基、苯基、苄基、取代的苄基、取代的苯基；R4选自C1-C11烷基；X独立的选自O、S和NH；
    所述取代的苄基选自下述任意一种：含有一个或多个卤原子取代的苄基，含有一个三氟甲基取代的苄基，含有一个三氟甲基和一个卤原子取代的苄基，含有一个氰基取代的苄基；
    所述取代的苯基选自下述任意一种：含有一个或多个卤原子取代的苯基，含有一个三氟甲基取代的苯基，含有一个三氟甲基和一个卤原子取代的苯基，含有一个氰基取代的苯基。
  2. 根据权利要求1所述的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，所述化合物选自：2-丙基-4-羟基苯并二氢吡喃，2-丁基-4-羟基苯并二氢吡喃，2-戊基-4-羟基苯并二氢吡喃，2-己基-4-羟基苯并二氢吡喃，2-庚基-4-羟基苯并二氢吡喃，2-辛基-4-羟基苯并二氢吡喃，2-壬基-4-羟基苯并二氢吡喃，2-十一烷基-4-羟基苯并二氢吡喃，2-丙基-4-羟基-7-甲氧基苯并二氢吡喃，2-丁基-4-羟基-7-甲氧基苯并二氢吡喃，2-戊基-4-羟基-7-甲氧基苯并二氢吡喃，2-己基-4-羟基-7-甲氧基苯并二氢吡喃，2-庚基-4-羟基-7-甲氧基苯并二氢吡喃，2-辛基-4-羟基-7-甲氧基苯并二氢吡喃，2-壬基-4-羟基-7-甲氧基苯并二氢吡喃，2-十一烷基-4-羟基-7-甲氧基苯并二氢吡喃，2-丙基-4,7-二羟基苯并二氢吡喃，2-戊基-4,7-二羟基苯并二氢吡喃，2-己基-4,7-二羟基苯并二氢吡喃，2-庚基-4,7-二羟基苯并二氢吡喃，2-庚基-4-异丁氨 基-7-甲氧基苯并二氢吡喃，2-庚基-4-(4-氯苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氯苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(4-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-苄胺基-7-甲氧基苯并二氢吡喃，2-庚基-4-(4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3,4,5-三氟苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(3-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃，2-庚基-4-(2,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃，2-戊基-4-氨基-7-甲氧基苯并二氢吡喃，2-己基-4-氨基-7-甲氧基苯并二氢吡喃，2-庚基-4-氨基-7-甲氧基苯并二氢吡喃。
  3. 根据权利要求1所述的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，药学上可接受的盐是所述化合物与酸形成加成盐，所述酸分别独立的选自盐酸、氢溴酸、硫酸、磷酸、乳酸、乙酸、酒石酸、对甲苯磺酸、马来酸、水杨酸、柠檬酸、琥珀酸。
  4. 根据权利要求1所述的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐，其特征在于，所述药学上可接受的盐选自：2-庚基-4-异丁氨基-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(4-氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-苄胺基-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3,4- 二氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氯-4-氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2,4-二氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3,4,5-三氟苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(3-氟-4-三氟甲基苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐，2-庚基-4-(2,4-二氯苄胺基)-7-甲氧基苯并二氢吡喃盐酸盐。
  5. 权利要求1的式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐的制备方法，其特征在于，包括式II所示化合物或式III所示化合物的制备方法，所述式II所示化合物的制备方法：以式IV所示的化合物为原料，经过溴代反应，得到式V所示的溴代产物，然后先与三苯基磷反应，再与氢氧化钠反应得到式VI所示的化合物；式VI所示的化合物与醛反应，得到式VII所示的化合物；式VII所示的化合物在碱性条件下关环可得式VIII所示的化合物；式VIII所示的化合物与伯胺或盐酸羟胺进行还原胺化反应得到式II所示化合物，采用的合成反应步骤如下：
    所述式III所示化合物的制备方法：以式VIII所示的化合物为原料，与氢化试剂反应得式IX所示的化合物，再与卤代烃类化合物反应得到式III所示化合物，采用的合成反应步骤如下：
  6. 一种药物组合物，包括权利要求1的式I所示的苯并二氢吡喃 类化合物、其异构体或药学上可接受的盐和药学上可接受的载体或辅料。
  7. 根据权利要求6所述的药物组合物，其特征在于，所述药物组合物选自片剂、胶囊、丸剂、注射剂；所述药物组合物选自缓释制剂或控释制剂。
  8. 根据权利要求6所述的药物组合物，其特征在于，所述药物组合物为人用药物组合物或动物用药物组合物，其中所述动物包括猪科动物、牛科动物、犬科动物、马科动物、羊科动物、猫科动物、啮齿类动物、灵长类动物，例如人、牛、猪或马。
  9. 权利要求1的式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐、包括式I所示的苯并二氢吡喃类化合物、其异构体或药学上可接受的盐和药学上可接受的载体或辅料的药物组合物在制备用于预防、治疗或减轻乙肝病毒感染性疾病和冠状病毒感染性疾病药物的用途。
  10. 如权利要求9所述的用途，其特征在于，用于预防、治疗或减轻新型冠状病毒(SARS-CoV-2)感染性疾病。
PCT/CN2023/077694 2022-02-25 2023-02-22 具有抗乙肝病毒和冠状病毒作用的苯并二氢吡喃类化合物 WO2023160586A1 (zh)

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