CN110075102B

CN110075102B - Application of polycyclic polyketide in preparation of anti-herpes virus drugs

Info

Publication number: CN110075102B
Application number: CN201910468338.2A
Authority: CN
Inventors: 叶文才; 李药兰; 王英; 李满妹; 胡利军; 唐维; 程民井; 苏骏成
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2021-04-09
Anticipated expiration: 2039-05-30
Also published as: CN110075102A

Abstract

The invention discloses application of polycyclic polyketides in preparation of anti-herpes virus drugs. The polyketide compound can inhibit diseases caused by infection of four Herpes viruses, namely Herpes simplex virus I (HSV-1), Herpes simplex virus II (HSV-2), Varicella Zoster Virus (VZV) and Cytomegalovirus (CMV). Compared with the marketed drug acyclovir and the like, the compound shows equivalent activity, but has different action mechanisms, and can overcome the drug resistance of the marketed drug. Therefore, the compounds have good application prospect in treating related diseases caused by HSV-1, HSV-2, VZV and CMV herpes virus infection.

Description

Application of polycyclic polyketide in preparation of anti-herpes virus drugs

Technical Field

The invention relates to a medicament for treating and preventing herpes virus infection. More particularly, the invention relates to application of polycyclic polyketone compounds in preparing medicines for resisting four herpesvirus medicines, namely Herpes simplex virus I (HSV-1), Herpes simplex virus II (HSV-1, HSV-2), Varicella Zoster Virus (VZV) and Cytomegalovirus (CMV).

Background

Herpesviruses (HV) are a class of enveloped, double-stranded DNA genome viruses. The virus of the family can be widely spread in animal and human groups, invisibly infect for a long time and induce the generation of corresponding diseases. The herpesviruses currently found to infect humans can be divided into mainly 3 subfamilies, depending on the genomic sequence, structure and physicochemical properties: the subfamily α -herpesviridae, β -herpesviridae and γ -herpesviridae. The subfamily of the alpha-Herpes virus mainly comprises three viruses of Herpes simplex virus I (HSV-1), Herpes simplex virus II (HSV-2) and Varicella Zoster Virus (VZV), and the virus of the subfamily has the characteristics of short proliferation cycle, main infection of mucosal epithelial cells and the like; the virus of the subfamily beta-herpesviridae mainly comprises Cytomegalovirus (CMV), human herpesvirus VI (HHV-6) and human herpesvirus VII (HHV-7), and has the characteristics of long proliferation period, capability of infecting epithelial cells, lymphocytes and fibroblasts and the like; viruses of the subfamily γ -herpesviridae are tumor-associated viruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpes virus (KSHV). The life cycle of herpesviruses is divided into a typical lytic phase of replication and a latent phase of replication: during the lytic phase of infection, the viral genome replicates, producing large numbers of mature virions and inducing the development of a variety of diseases; in the latent infection process, the genome is in a resting state, only a small amount of virus genes are expressed, but the genome is replicated along with the replication of the cell genome, and under the stimulation of proper conditions, such as ultraviolet irradiation, fever, fatigue, trauma, mental stress, menstruation, certain immunodeficiency diseases and the like, the infection can relapse, and the recurrent diseases are formed. The infection of viruses of the herpesviridae family can cause a variety of diseases, such as herpes, nervous system infections and tumors, among other serious diseases. The disease symptoms that can be produced vary depending on the type of disease source and the site of infection, as shown in Table 1.

TABLE 1 human herpesviruses and related human diseases

At present, the clinical treatment of herpesviridae infections is mainly by the use of antiviral drugs and immunomodulators, such as acyclovir, ganciclovir, gamma-interferon. Antiviral drugs such as acyclovir are a class of virus polymerase inhibitors, and the antiviral mechanism of the antiviral drugs is mainly to inhibit or interfere the synthesis of virus DNA in the splitting phase and block the growth and propagation of the virus. However, the virus has a simple structure, a small amount of protein, and is easy to mutate under the repeated action of antiviral drugs, and the drug resistance is high, so that the application effect of the antiviral drugs used clinically at present is not ideal. Therefore, there is a great need to develop new drugs for treating or preventing herpesvirus infections.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a polycyclic polyketone compound capable of resisting herpes viruses.

In order to achieve the purpose, the invention is realized by the following scheme:

the application of polycyclic polyketides in preparing anti-herpes virus medicaments is disclosed, wherein the polyketides have a structural formula shown in a general formula I:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: r¹、R²、R³And R⁴Each independently is an alkyl group;

R⁵selected from the group consisting of hydrogen atoms, alkyl groups, halogens, haloalkyl groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, wherein said alkyl groups, hydroxyalkyl groups, haloalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are optionally selected from the group consisting of alkyl groups, haloalkyl groups, halogens, amino groups, nitro groups, cyano groups, hydroxy groups, alkoxy groups, haloalkoxy groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, and NR groups¹⁰R¹¹Is substituted with one or more substituents of (1);

R⁶and R⁷Independently selected from the group consisting of hydrogen, alkyl, halogen, haloalkyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C (O) R¹²、-C(O)OR¹²、-S(O)mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S(O)_mNR¹⁰R¹¹Wherein said alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and NR¹⁰R¹¹Substituted with one or more substituents of (a);

R⁸independently selected from the group consisting of a hydrogen atom, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, -C (O) R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Wherein said haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and NR¹⁰R¹¹Substituted with one or more substituents of (a);

R⁹independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, a heteroaryl group, -C (O) R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Wherein said alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and NR¹⁰R¹¹Substituted with one or more substituents of (a);

R¹⁰and R¹¹Each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, halogenAlkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and NR¹⁰R¹¹Is substituted with one or more substituents of (1);

R¹²selected from the group consisting of hydrogen atoms, alkyl groups, haloalkyl groups, hydroxyalkyl groups, amino groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups, wherein said alkyl groups, haloalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are optionally selected from the group consisting of alkyl groups, haloalkyl groups, halogens, amino groups, nitro groups, cyano groups, hydroxy groups, alkoxy groups, haloalkoxy groups, hydroxyalkyl groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, and NR groups¹⁰R¹¹Is substituted with one or more substituents of (1);

m is 0, 1 or 2.

In a preferred embodiment of the invention, the compound of formula I, wherein R is¹＝R²＝R³＝R⁴＝CH₃Which has the formula shown in formula II:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: r⁵、R⁶、R⁷、R⁸And R⁹As previously defined.

In a preferred embodiment of the invention, the compound of formula II, wherein R is⁷Is H, having the formula shown in formula III:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: r⁵、R⁶、R⁸And R⁹As previously defined;

in a preferred embodiment of the present invention, the compounds of formula III include, but are not limited to:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the invention, the compound of formula II, wherein R is⁶Is H, having the formula shown in formula IV:

or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: r⁵、R⁷、R⁸And R⁹As previously defined;

in a preferred embodiment of the present invention, the compounds of formula IV include, but are not limited to:

The invention relates to a pharmaceutical composition, which contains a therapeutically effective dose of polyketide compounds shown in general formulas I, II, III and IV, or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

The invention relates to application of polyketone compounds shown in general formulas I, II, III and IV, or tautomers, racemes, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof containing therapeutically effective dose in preparation of anti-HSV-1, HSV-2, VZV and CMV herpes virus drugs.

The invention relates to application of polyketone compounds shown in general formulas I, II, III and IV, or tautomers, racemates, enantiomers, diastereomers, or mixture forms thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof containing therapeutically effective dose in preparation of drugs for treating diseases such as keratitis, encephalitis, skin herpes, oral herpes, herpes labialis, herpes genitalis, herpes neonatorum, varicella, herpes zoster and the like caused by herpes virus infection of HSV-1, HSV-2, VZV and CMV.

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group of 1 to 20 carbonsLinear or branched groups of atoms, preferably alkyl groups of 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-dimethylpentyl, 2-dimethylhexyl, 3-dimethylpentyl, 2-ethylhexyl, 3-dimethylhexyl, 2, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxyRadicals, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio, -OR¹²、-C(O)R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Is substituted with one or more substituents.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring being selected from 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, most preferably 3 to 6 carbon atoms, for example 3, 4, 5 or 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl and the like, with cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl being preferred; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The cycloalkyl group may be fused to an aryl, heteroaryl or heterocycloalkyl ring, where the ring to which the parent structure is attached is a cycloalkyl group, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like. Cycloalkyl groups may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -OR¹²、-C(O)R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Is substituted with one or more substituents.

The term "heterocyclyl" refers to a saturated and/or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent, mono-or bicyclic hydrocarbon ring selected from a ring of 4 to 10 carbon atoms, heteroatom containing group selected from oxygen, sulfur, nitrogen, preferably wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2)The ring moiety of a molecule, but not comprising-O-O-, -O-S-or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, for example 1,2, 3 or 4 are heteroatoms; more preferably heterocyclyl contains 3 to 10 ring atoms, most preferably heterocyclyl contains 3 to 6 ring atoms, for example 3, 4, 5 or 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydropyranyl, pyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and

etc., preferably azetidinyl, oxetanyl, pyrrolyl and piperidinyl; polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

The heterocyclyl group may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -OR¹²、-C(O)R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Is substituted with one or more substituents.

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group which is a polycyclic (i.e., rings which carry adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

heteroaryl may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -OR¹²、-C(O)R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Is substituted with one or more substituents.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, amino, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -OR¹²、-C(O)R¹²、-C(O)OR¹²、-S(O)_mR¹²、-C(O)NR¹⁰R¹¹、-NR¹⁰R¹¹and-S (O)_mNR¹⁰R¹¹Is substituted with one or more substituents.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

The term "hydroxy" refers to an-OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to the group-NH₂。

The term "cyano" refers to — CN.

The term "nitro" means-NO₂。

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

m and R¹～R¹²As defined in formula I.

Drawings

FIG. 1 shows the inhibitory effect of Compound 39 on different viral titers HSV-1.

FIG. 2 is an inactivation of HSV-1 by compound 39.

FIG. 3 is a graph of the effect of Compound 39 on different time points of the replication cycle of HSV-1;

FIG. 4 is a graph of the effect of Compound 39 on HSV-1 gene transcription.

FIG. 5 is a graph showing the effect of Compound 39 on HSV-1 adsorption.

FIG. 6 is a graph of the effect of Compound 39 on HSV-1 membrane fusion.

FIG. 7 is the effect of Compound 39 on HSV-1 late protein.

FIG. 8 is a graph of the effect of Compound 39 on HFF cell proliferation.

FIG. 9 is a graph of the effect of compound 39 on CMV-GFP viral replication.

FIG. 10 is a graph showing the effect of compound 39 on the replication cycle of CMV-GFP virus.

FIG. 11 is a graph of the therapeutic effect of Compound 39 on HSV-1 infection of the mouse ear: (a) body weight change in mice; (b) herpes score of mouse ear; (c) mouse skin virus titer; (d) thickness of mouse ear.

FIG. 12 is a graph of the therapeutic effect of Compound 39 on the cornea of the eyes of HSV-1 keratitis model mice.

FIG. 13 is a graph of the effect of Compound 39 on ocular keratovirus titer in HSV-1 keratitis model mice.

FIG. 14 is a graph of the therapeutic effect of Compound 39 on vaginal infection of guinea pigs with HSV-2: (a) guinea pig vaginal virus titer; (b) guinea pig vaginal clinical symptoms score.

Figure 15 is the effect of compound 39 on the improvement of CMV growth and development in offspring guinea pigs: (a) the rate of dead tires; (b) infection rate of pregnant guinea pig, placenta and fetus.

Detailed Description

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. delta.) of 10^-6The units in (ppm) are given. NMR was measured using a Bruker AVANCE-300, Bruker AVANCE-400, Bruker AVANCE-500 or Bruker AVANCE-600 nuclear magnetic spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS).

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

Known starting materials of the present invention may be synthesized by or according to methods known in the art, or may be purchased from Acros Organics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), carbofuran, Annage, Darrill Chemicals, and the like.

In the examples, the reaction can be carried out in an argon atmosphere or a nitrogen atmosphere, unless otherwise specified.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

In the examples, the solution means an aqueous solution unless otherwise specified.

In the examples, the reaction temperature is, unless otherwise specified, from 20 ℃ to 30 ℃ at room temperature.

The monitoring of the progress of the reaction in the examples employed Thin Layer Chromatography (TLC), a developing solvent used for the reaction, a system of eluents for column chromatography used for separation and purification of compounds, and a developing solvent system for thin layer chromatography including: a: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: acetone, E: dichloromethane/acetone system, F: ethyl acetate/dichloromethane system, G: ethyl acetate/dichloromethane/n-hexane, H: ethyl acetate/dichloromethane/acetone, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of basic or acidic reagents such as triethylamine, acetic acid and the like can be added for adjustment.

The specific compounds prepared in the following examples include, but are not limited to, those shown in tables 1-53 above.

Example 1

Preparation of 6, 8-dihydroxy-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione intermediate (52)

First step of

Phloroglucinol 52a (2.5g,20mmol) was dissolved in nitromethane solution at room temperature, anhydrous aluminum trichloride (10.7g,80mmol) and isobutyryl chloride (2.3g,22mmol) were added successively, and the temperature was raised to reflux. After 12 hours of reaction, the reaction mixture was slowly poured into ice water, and a saturated sodium potassium tartrate solution (100mL) was added thereto and stirred vigorously. The reaction mixture was extracted 3 times with ethyl acetate (100 mL. times.3). The organic phases were combined and washed with saturated NaCl solution. After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography to give compound 52c (3.6g, 85% yield).

Second step of

Compound 52c (2.1g,10mmol) was dissolved in MeOH (30mL) at room temperature and NaOMe (5mmol/mL,9mL,45mmol) in MeOH was added. After reacting for 10min at room temperature, methyl iodide (3.1mL,50mmol) is added into the reaction system, the temperature is raised to 55 ℃, the reaction is carried out for 15 min, the temperature is reduced to 0 ℃, and 1N HCl is used for acidification treatment. The reaction solution was extracted three times with ethyl acetate (3X 10mL), and the organic phases were combined and washed with saturated NaCl solution (5 mL). After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography to give compound 52d as a yellow solid (2.4g, 91% yield).

The third step

Compound 52d (2.4g,9.1mmol) was dissolved in tetrahydrofuran solution and after cooling to-78 deg.C, diisopropylaluminum hydride was added dropwise. After 2 hours of reaction, the reaction was quenched by addition of saturated aqueous sodium potassium tartrate solution (100 mL). After stirring at room temperature for 3 hours, the reaction mixture was extracted with ethyl acetate 3 times (100 mL. times.3). The organic phases were combined and washed with saturated NaCl solution. After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was isolated and purified by silica gel column chromatography to give compound 52e (2.0g, yield 89%).

The fourth step

Compound 52a (630mg,5mmol) was dissolved in tetrahydrofuran at room temperature and added to compound 52e (1.8g,7.5 mmol). After 4 hours of reaction, p-toluenesulfonic acid monohydrate (2.9g,15mmol) was added and the temperature was raised to reflux. After reacting for 2 hours, the reaction mixture was cooled to room temperature, and then quenched by adding saturated aqueous sodium bicarbonate (100mL), and the reaction mixture was extracted 3 times with ethyl acetate (100 mL. times.3). The organic phases were combined and washed with saturated NaCl solution. After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was isolated and purified by silica gel column chromatography to give compound 52(896mg, yield 50%).

¹H NMR(600MHz,CD₃OD)δ6.17(d,J＝2.3Hz,1H),6.10(d,J＝2.3Hz,1H),4.18(t,J＝5.7Hz,1H),3.33(dt,J＝3.3,1.6Hz,1H),1.54(s,3H),1.49(m,2H),1.45(s,3H),1.39(m,1H),1.36(s,3H),1.34(s,3H),0.85(d,J＝6.5Hz,3H),0.81(d,J＝6.4Hz,3H)；¹³C NMR(150MHz,CD₃OD)δ212.4,198.2,168.5,156.8,155.7,152.4,113.5,104.6,98.8,94.0,55.4,47.0,45.6,25.5,24.7,23.9,23.8,23.6,23.3,22.9,22.3；HR-ESI-MS m/z 359.1847[M+H]⁺。

Example 2

Preparation of 6, 8-dihydroxy-9-cyclobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione intermediate (53)

First step of

Phloroglucinol 52a (2.5g,20mmol) was dissolved in nitromethane solution at room temperature, and anhydrous aluminum trichloride (10.7g,80mmol) and cyclobutyryl chloride (2.6g,22mmol) were added successively, and the temperature was raised to reflux. After 12 hours of reaction, the reaction mixture was slowly poured into ice water, and a saturated sodium potassium tartrate solution (100mL) was added thereto and stirred vigorously. The reaction mixture was extracted 3 times with ethyl acetate (100 mL. times.3). The organic phases were combined and washed with saturated NaCl solution. After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was isolated and purified by silica gel column chromatography to give compound 53c (3.4g, yield 83%).

Second step of

Compound 53c (2.1g,10mmol) was dissolved in MeOH (30mL) at room temperature and NaOMe (5mmol/mL,9mL,45mmol) in MeOH was added. After reacting for 10 minutes at room temperature, methyl iodide (3.1mL,50mmol) was added to the reaction system, the temperature was raised to 55 ℃ for reaction for 15 minutes, the temperature was lowered to 0 ℃ and the reaction system was acidified with 1N HCl. The reaction solution was extracted three times with ethyl acetate (3X 10mL), and the organic phases were combined and washed with saturated NaCl solution (5 mL). After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The crude product was isolated and purified by silica gel column chromatography to give compound 53d as a yellow solid (2.4g, 90% yield).

The third step

Compound 53d (2.4g,9.0mmol) was dissolved in tetrahydrofuran solution and after cooling to-78 deg.C, diisopropylaluminum hydride was added dropwise. After 2 hours of reaction, the reaction was quenched by addition of saturated aqueous sodium potassium tartrate solution (100 mL). After stirring at room temperature for 3 hours, the reaction mixture was extracted with ethyl acetate 3 times (100 mL. times.3). The organic phases were combined and washed with saturated NaCl solution. After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was isolated and purified by silica gel column chromatography to give compound 53e (1.9g, yield 85%).

The fourth step

Compound 52a (630mg,5mmol) was dissolved in tetrahydrofuran at room temperature and added to compound 53e (1.8g,7.5 mmol). After 4 hours of reaction, p-toluenesulfonic acid monohydrate (2.9g,15mmol) was added and the temperature was raised to reflux. After reacting for 2 hours, the reaction mixture was cooled to room temperature, and then quenched by adding saturated aqueous sodium bicarbonate (100mL), and the reaction mixture was extracted 3 times with ethyl acetate (100 mL. times.3). The organic phases were combined and washed with saturated NaCl solution. After drying over anhydrous sodium sulfate and filtration, the organic phase was evaporated to dryness under reduced pressure. The obtained crude product was isolated and purified by silica gel column chromatography to give compound 53(927mg, yield 52%).

¹H NMR(300MHz,CD₃OD)δ6.19(d,J＝2.2Hz,1H),6.12(d,J＝2.2Hz,1H),4.12(d,J＝5.7Hz,1H),2.59(dt,J＝9.0,6.8Hz,1H),1.65(m,5H),1.53(s,3H),1.42(s,3H),1.35(s,3H),1.31(s,3H)；¹³C NMR(75MHz,CD₃OD)δ212.4,198.5,169.2,156.8,155.8,152.8,111.1,102.3,98.9,94.0,55.5,41.9,29.6,24.7,24.2,23.9,23.6,23.5,17.2；HR-ESI-MS m/z 357.1693[M+H]⁺。

Example 3

Preparation of 6, 8-dihydroxy-5-acetyl-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (5)

Compound 52(44.2mg,0.1mmol) was dissolved in glacial acetic acid solution (4mL) at room temperature, acetic anhydride (20.6. mu.L, 0.22mmol) and boron trifluoride etherate (13.6. mu.L, 0.105mmol) were added, respectively, and the temperature was raised to 100 ℃. After 3 hours of reaction, the temperature was lowered to room temperature, and the reaction was quenched by addition of 1N aqueous sodium hydroxide (4mL) and extracted 3 times with ethyl acetate (8 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was recrystallized from a mixture of dichloromethane and n-hexane to give compound 5(16.0mg, 40% yield).

¹H NMR(500MHz,CDCl₃)δ13.54(s,1H),7.09(s,1H),6.28(s,1H),4.33(t,J＝6.1Hz,1H),2.82(s,3H),1.65(s,3H),1.50(s,3H),1.45(s,3H),1.45(m,3H),1.42(s,3H),0.90(d,J＝5.5Hz,3H),0.89(d,J＝6.1Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ211.7,201.6,198.4,167.3,164.6,160.0,153.3,114.5,106.0,105.4,100.1,56.1,47.2,46.9,33.2,25.4,25.0,24.8,24.7,24.5,24.3,23.4,23.1；HR-ESI-MS m/z 401.1957[M+H]⁺。

Example 4

Preparation of 6, 8-dihydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-dimethyl-4, 9-hydro-1H-xanthene-1, 3(2H) -dione (12)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And isobutyryl chloride (0.11mmol) were added to a solution of compound 52(0.1mmol) in dichloromethane, respectively, and stirred at room temperature. After 24 hours of reaction, the reaction was quenched by addition of 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography to give the corresponding product 12(15.5mg, 35% yield).

¹H NMR(500MHz,CDCl₃)δ13.05(s,1H),7.58(s,1H),6.13(s,1H),4.29(t,J＝5.3Hz,1H),3.01(m,2H),2.29(m,1H),1.73(s,3H),1.57(s,3H),1.45(s,3H),1.43(s,3H),1.42(m,3H),1.39(s,3H),1.01(s,3H),0.99(s,3H),0.89(d,J＝5.8Hz,3H),0.85(d,J＝5.6Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.2,206.5,197.9,167.1,162.6,158.5,155.6,114.2,107.6,106.6,94.8,56.1,53.2,47.2,45.9,25.2,25.1,24.7,24.6,24.6,24.2,23.5,23.2,22.8,22.8；HR-ESI-MS m/z 443.2428[M+H]⁺。

Example 5

Preparation of 6, 8-hydroxy-7-carboxaldehyde-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (18)

Phosphorus oxychloride (46.0mg,0.3mmol) was added to a solution of compound 52(44.2mg,0.1mmol) in N, N-dimethylformamide (3mL) at room temperature. After the reaction was stirred overnight, the reaction was quenched by addition of saturated aqueous sodium bicarbonate (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 18(27.0mg, yield 70%).

¹H NMR(400MHz,CDCl₃)δ12.01(s,1H),10.24(s,1H),8.16(s,1H),6.27(s,1H),4.30(t,J＝5.7Hz,1H),1.50(s,3H),1.47(m,3H),1.45(s,3H),1.42(s,3H),1.27(s,3H),0.88(d,J＝6.3Hz,3H),0.87(d,J＝7.5Hz,3H)；¹³C NMR(100MHz,CDCl₃)δ213.0,200.3,191.6,168.9,165.1,164.0,155.2,115.9,107.4,106.3,100.8,57.6,48.9,47.6,31.1,26.5,26.5,26.4,26.2,25.5,24.9,24.6；HR-ESI-MS m/z 387.1801[M+H]⁺。

Example 6

Preparation of 6, 8-dihydroxy-7-acetyl-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (19)

Compound 52(44.2mg,0.1mmol) was dissolved in glacial acetic acid (4mL) at room temperature, acetic anhydride (20.6. mu.L, 0.22mmol) and boron trifluoride etherate (13.6. mu.L, 0.105mmol) were added, respectively, and the temperature was raised to 100 ℃. After 3 hours of reaction, the reaction was quenched by addition of 1N aqueous sodium hydroxide (4mL) and extracted 3 times with ethyl acetate (8 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was recrystallized from a mixture of dichloromethane and n-hexane to give compound 19(16.4mg, yield 41%).

¹H NMR(500MHz,CDCl₃)δ13.12(s,1H),7.73(s,1H),6.17(s,1H),4.30(t,J＝5.5Hz,1H),2.75(s,3H),1.58(s,3H),1.46(m,3H),1.46(s,3H),1.44(s,3H),1.40(s,3H),0.89(d,J＝6.0Hz,3H),0.86(d,J＝5.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.2,204.2,198.1,167.2,162.7,158.8,156.0,114.3,107.6,106.4,94.7,56.1,47.2,45.9,33.2,25.1,24.8,24.6,24.2,23.5,23.2；HR-ESI-MS m/z 401.1957[M+H]⁺。

Example 7

Preparation of 6, 8-dihydroxy-7-propionyl-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (20)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And propionyl chloride (0.11 m)mol) were added to a solution of compound 52(0.1mmol) in dichloromethane, respectively, and stirred at ordinary temperature. After 24 hours of reaction, the reaction was quenched by addition of 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 20(17.8mg, yield 43%).

¹H NMR(500MHz,CD₃OD)δ6.16(s,1H),4.21(t,J＝5.2Hz,1H),3.16(q,J＝7.1Hz,2H),1.55(s,3H),1.46(s,3H),1.38(m,3H),1.37(s,3H),1.35(s,3H),1.17(t,J＝7.1Hz,3H),0.86(d,J＝5.8Hz,3H),0.81(d,J＝5.7Hz,3H)；¹³C NMR(125MHz,CD₃OD)δ211.8,207.6,197.9,167.3,162.6,160.2,155.9,113.9,107.0,104.8,93.6,55.7,46.9,45.2,37.2,25.0,24.9,24.0,23.8,23.5,22.9,22.7,22.3,7.5；HR-ESI-MS m/z 415.2112[M+H]⁺。

Example 8

Preparation of 6, 8-dihydroxy-7-isobutyryl-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (21)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And isobutyryl chloride (0.11mmol) were added to a solution of compound 52(0.1mmol) in dichloromethane, respectively, and stirred at room temperature. After 24 hours of reaction, the reaction was quenched by addition of 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 21(10.7mg, yield 25%).

¹H NMR(500MHz,CDCl₃)δ13.16(s,1H),7.71(s,1H),6.18(s,1H),4.31(s,1H),3.94(m,1H),1.58(s,3H),1.46(s,3H),1.44(s,3H),1.43(m,3H),1.40(s,3H),1.23(t,J＝6.8Hz,6H),0.89(d,J＝4.1Hz,3H),0.87(s,3H)；¹³C NMR(125MHz,CDCl₃)δ212.2,211.2,198.3,167.4,162.9,158.3,155.6,114.3,106.7,106.6,94.9,56.1,47.2,45.9,39.8,25.2,25.1,24.8,24.6,24.6,24.2,23.5,23.2,19.3,19.1；HR-ESI-MS m/z 429.2268[M+H]⁺。

Example 9

Preparation of 6, 8-dihydroxy-7-decanoyl-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (23)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And decanoyl chloride (0.11mmol) were added to a solution of compound 52(0.1mmol) in dichloromethane, respectively, and stirred at room temperature. After 24 hours of reaction, the reaction was quenched by addition of 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 23(20.0mg, 39% yield).

¹H NMR(300MHz,CDCl₃)δ13.56(s,1H),8.33(s,1H),6.26(s,1H),4.33(t,J＝5.1Hz,1H),3.16(t,J＝7.3Hz,2H),1.73(m,2H),1.59(s,3H),1.47(s,3H),1.45(s,3H),1.42(s,3H),1.40(m,15H),0.88(t,J＝4.9Hz,9H)；¹³C NMR(75MHz,CDCl₃)δ212.1,207.4,198.9,168.0,162.9,158.9,155.7,114.3,107.5,106.2,94.74,56.1,47.3,45.9,44.6,31.9,29.6,29.5,29.5,29.3,25.1,24.8,24.7,24.6,24.3,23.6,23.2,22.7,14.1；HR-ESI-MS m/z 513.3210[M+H]⁺。

Example 10

Preparation of 6, 8-dihydroxy-7- (cyclohexylcarbonyl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (27)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And cyclohexyl acid chloride (0.11mmol) were added to a solution of compound 52(0.1mmol) in dichloromethane, respectively, and stirred at room temperature. After 24 hours of reaction, the reaction was quenched by addition of 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 27(17.3mg, 37% yield).

¹H NMR(300MHz,CDCl₃)δ12.66(s,1H),8.65(s,1H),6.21(s,1H),4.35(d,J＝3.1Hz,1H),3.02(tt,J＝15.8,7.9Hz,2H),2.31(m,1H),1.62(m,6H),1.61(s,3H),1.48(s,3H),1.44(s,3H),1.40(s,3H),1.10(m,2H),1.02(d,J＝1.2Hz,3H),0.99(d,J＝1.2Hz,3H),0.99(m,5H)；¹³C NMR(75MHz,CDCl₃)δ212.2,206.7,198.5,168.5,162.0,159.3,156.6,112.1,107.9,104.6,94.8,56.2,53.3,47.4,44.9,31.6,29.8,29.2,26.7,26.5,26.3,25.2,25.1,25.0,24.6,24.1,22.9,22.8；HR-ESI-MS m/z 469.2583[M+H]⁺。

Example 11

Preparation of 6, 8-dihydroxy-7- (3-methylbutyryl) -9-cyclobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (28)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And cyclobutylylchloride (0.11mmol) were added to a solution of compound 53(0.1mmol) in dichloromethane, respectively. After the reaction was stirred for 24 hours, the reaction was quenched by adding 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 28(18.0mg, 41% yield).

¹H NMR(500MHz,CDCl₃)δ11.74(s,1H),9.40(s,1H),6.23(s,1H),4.43(d,J＝5.5Hz,1H),3.05(m,2H),2.70(m,1H),2.32(m,1H),1.62(m,6H),1.59(s,3H),1.47(s,6H),1.42(s,3H),1.01(d,J＝3.5Hz,3H),1.00(d,J＝3.5Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.0,206.6,198.6,168.5,161.2,160.2,156.0,111.5,108.0,103.1,95.5,56.0,53.4,47.3,41.3,29.4,25.4,25.0,24.6,24.6,24.4,22.9,22.8,17.8；HR-ESI-MS m/z441.2268[M+H]⁺。

Example 12

Preparation of 8-hydroxy-6-propargyloxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (30)

Propargyl bromide (17.8mg,0.15mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) at room temperature and the temperature was raised to reflux. After the reaction was allowed to stand overnight, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography to give compound 30(31.7mg, 66% yield).

¹H NMR(500MHz,CDCl₃)δ14.17(s,1H),6.20(s,1H),4.80(s,2H),4.31(t,J＝5.2Hz,1H),2.96(m,2H),2.64(s,1H),2.25(m,1H),1.59(s,3H),1.47(s,3H),1.43(m,3H),1.42(s,3H),1.38(s,3H),1.01(s,3H),0.99(s,3H),0.89(d,J＝5.6Hz,3H),0.85(d,J＝5.5Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.2,206.4,197.4,166.6,163.9,158.6,155.8,114.3,108.5,108.3,91.2,56.5,56.2,53.6,47.1,46.0,25.4,25.2,24.8,24.6,24.6,24.1,23.4,23.1,22.8,22.7；HR-ESI-MS m/z 481.2582[M+H]⁺。

Example 13

Preparation of 8-hydroxy-6- (3-bromopropoxy) -9-isobutyl-7- (3-methylbutyryl) -2,2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (31)

1, 3-dibromopropane (30.3mg,0.15mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) at room temperature and the temperature was raised to reflux. After the reaction was allowed to stand overnight, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 31(10.1mg, yield 18%).

¹H NMR(300MHz,CDCl₃)δ14.13(s,1H),6.14(s,1H),4.25(m,3H),3.62(t,J＝6.3Hz,2H),2.92(dd,J＝6.8,5.0Hz,2H),2.43(m,2H),2.26(m,1H),1.56(s,3H),1.44(s,3H),1.42(m,3H),1.39(s,3H),1.36(s,3H),0.98(s,3H),0.96(s,3H),0.86(d,J＝6.0Hz,3H),0.83(d,J＝5.8Hz,3H)；¹³C NMR(75MHz,CDCl₃)δ212.2,205.9,197.4,166.6,163.9,159.8,156.0,114.3,108.4,107.6,90.8,66.7,56.1,53.6,47.1,45.9,31.9,31.4,30.2,29.3,25.2,24.8,24.7,24.7,24.5,24.1,23.4,23.2,22.8,22.7；HR-ESI-MS m/z 563.2001[M+H]⁺。

Example 14

Preparation of 8-hydroxy-6-isobutoxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (32)

Isobutyl iodide (27.6mg,0.15mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) under argon protection and the temperature was raised to reflux. After 6 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography to give compound 32(18.9mg, 38% yield).

¹H NMR(500MHz,CDCl₃)δ14.22(s,1H),6.12(s,1H),4.30(t,J＝5.3Hz,1H),3.84(m,2H),3.03(dd,J＝10.9,6.9Hz,2H),2.30(m,1H),2.23(m,1H),1.61(s,3H),1.58(s,3H),1.47(s,3H),1.47(m,3H),1.42(s,3H),1.38(s,3H),1.13(s,3H),1.12(s,3H),1.00(s,3H),0.99(s,3H),0.88(d,J＝5.7Hz,3H),0.86(d,J＝5.5Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.3,206.2,197.4,166.7,163.8,160.5,156.0,114.4,108.4,107.1,90.7,75.9,56.1,53.7,47.1,45.9,28.3,25.18,24.7,24.7,24.6,24.5,24.1,23.5,23.2,22.8,22.7,19.6；HR-ESI-MS m/z 499.3050[M+H]⁺。

Example 15

Preparation of 8-hydroxy-6-isopropoxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (33)

Isopropyl bromide (25.5mg,0.15mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) at room temperature and warmed to reflux. After 6 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography to give compound 33(19.8mg, 41% yield).

¹H NMR(500MHz,CDCl₃)δ14.18(s,1H),6.10(s,1H),4.70(m,1H),4.29(t,J＝5.4Hz,1H),3.00(dd,J＝15.3,6.8Hz,1H),2.92(dd,J＝15.3,7.0Hz,1H),2.25(m,1H),1.58(s,3H),1.48(s,3H),1.46(s,6H),1.45(m,3H),1.41(s,3H),1.38(s,3H),0.99(s,3H),0.98(s,3H),0.89(d,J＝5.8Hz,3H),0.85(d,J＝5.7Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.3,206.6,197.4,166.7,163.9,159.1,156.0,114.4,108.9,106.8,91.2,71.2,56.1,53.7,47.1,46.0,25.2,25.2,25.2,24.8,24.7,24.5,24.2,23.5,23.2,22.8,22.7,22.0,21.9；HR-ESI-MS m/z 499.3050[M+H]⁺。

Example 16

Preparation of 6-hydroxy-8-methoxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (34)

Palladium on carbon (10% wt; 3.0mg) was added to a solution of compound 37(27.3mg,0.05mmol) in methanol (2mL) at room temperature under hydrogen, and stirred at room temperature. After the reaction was over night, the crude product was obtained by direct filtration and concentration under reduced pressure. The resulting crude product was purified by silica gel chromatography to give compound 34(14.8mg, 65% yield).

¹H NMR(500MHz,CDCl₃)δ12.57(s,1H),6.54(s,1H),4.22(t,J＝5.7Hz,1H),3.82(s,3H),3.24(dd,J＝15.6,6.6Hz,1H),2.76(dd,J＝15.6,7.0Hz,1H),2.26(m,1H),1.59(s,3H),1.47(s,3H),1.43(m,3H),1.43(s,3H),1.40(s,3H),0.99(s,3H),0.98(s,3H),0.90(d,J＝5.9Hz,3H),0.88(d,J＝5.8Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.0,206.9,197.6,167.1,162.7,160.6,156.4,113.9,113.1,112.0,101.4,63.4,56.1,51.6,47.3,47.2,25.8,25.8,25.0,25.0,24.9,24.4,23.8,23.3,23.0,22.8,22.6；HR-ESI-MS m/z 457.2582[M+H]⁺。

Example 17

Preparation of 8-hydroxy-6- (allyloxy) -7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (35)

Allyl bromide (18.15mg,0.15mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) at room temperature and warmed to reflux. After the reaction was allowed to stand overnight, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by silica gel chromatography to give compound 35(33.8mg, yield 70%).

¹H NMR(500MHz,CDCl₃)δ14.18(s,1H),6.13(m,1H),6.13(s,1H),5.49(t,J＝13.2Hz,1H),5.40(d,J＝10.6Hz,1H),4.63(d,J＝5.3Hz,2H),4.29(t,J＝5.1Hz,1H),2.99(m,1H),2.91(m,1H),2.23(m,1H),1.55(s,3H),1.45(s,3H),1.43(m,3H),1.41(s,3H),1.37(s,3H),0.97(s,3H),0.96(s,3H),0.87(d,J＝5.6Hz,3H),0.84(d,J＝5.4Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.2,206.4,197.4,166.6,163.9,159.8,155.9,131.9,119.2,114.3,108.4,107.4,91.0,70.1,56.1,53.6,47.1,45.9,25.2,25.2,25.2,24.7,24.6,24.5,24.1,23.5,23.2,22.8,22.7；HR-ESI-MS m/z 483.2741[M+H]⁺。

Example 18

Preparation of 8-hydroxy-6- (benzyloxy) -7- (3-methylisobutyroyl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (36)

Benzyl bromide (26mg,0.15mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) at room temperature and warmed to reflux. After the reaction was allowed to stand overnight, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The resulting crude product was purified by silica gel chromatography to give compound 36(34.1mg, yield 64%).

¹H NMR(500MHz,CDCl₃)δ14.27(s,1H),7.45(m,5H),6.25(s,1H),5.12(d,J＝1.2Hz,2H),4.32(t,J＝5.5Hz,1H),2.87(dd,J＝15.4,6.8Hz,1H),2.80(dd,J＝15.4,7.0Hz,1H),2.14(m,1H),1.59(s,3H),1.47(s,3H),1.45(m,3H),1.42(s,3H),1.39(s,3H),0.90(d,J＝5.9Hz,3H),0.86(d,J＝5.7Hz,3H),0.77(t,J＝6.2Hz,6H)；¹³C NMR(125MHz,CDCl₃)δ212.3,206.4,197.4,166.7,164.0,160.0,156.0,135.2,128.8,128.8,128.5,114.4,108.4,107.6,100.0,91.0,71.6,56.2,53.6,47.1,45.9,25.2,25.2,25.1,24.8,24.7,24.6,24.1,23.5,23.2,22.5,22.4；HR-ESI-MS m/z 433.2890[M+H]⁺。

Example 19

Preparation of 6- (benzyloxy) -8-methoxy-7-isobutyryl-9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (37)

Methyl iodide (71.0mg,0.5mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 36(26.6mg,0.05mmol) in acetone (3mL) at room temperature and the temperature was raised to reflux. After 8 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 37(24.0mg, yield 88%).

¹H NMR(500MHz,CDCl₃)δ7.37(m,5H),6.55(s,1H),5.07(d,J＝3.1Hz,2H),4.20(t,J＝5.8Hz,1H),3.81(s,3H),2.70(t,J＝7.1Hz,2H),2.21(m,1H),1.58(s,3H),1.47(s,3H),1.43(m,3H),1.42(s,3H),1.39(s,3H),0.94(d,J＝6.7Hz,3H),0.91(t,J＝5.9Hz,6H),0.84(d,J＝6.1Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.3,203.8,197.6,167.4,155.4,154.9,152.7,135.9,128.6,128.2,127.4,123.2,113.5,113.0,96.8,70.8,63.4,56.0,53.9,47.3,47.2,25.8,25.0,24.9,24.8,24.6,24.2,24.0,23.3,23.1,22.6,22.5；HR-ESI-MS m/z 533.2893[M+H]⁺。

Example 20

Preparation of 8-hydroxy-6-triisopropylsiloxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (38)

Triisopropylsilyltrifluoromethanesulfonate (46.0mg,0.15mmol) and imidazole (13.6mg,0.2mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in N, N-dimethylformamide (3mL) at room temperature. After reacting for 3 hours, the reaction was quenched by addition of saturated aqueous ammonium chloride (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 38(46.7mg, yield 78%).

¹H NMR(500MHz,CDCl₃)δ13.83(s,1H),6.06(s,1H),4.30(t,J＝5.6Hz,1H),3.59(t,J＝6.2Hz,2H),3.19(t,J＝6.8Hz,2H),1.87(m,5H),1.59(s,3H),1.44(s,3H),1.44(m,3H),1.42(s,3H),1.38(s,3H),1.20(s,3H),1.19(s,3H),1.18(s,3H),1.18(s,3H),0.89(d,J＝6.1Hz,4H),0.84(d,J＝5.9Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.3,205.6,197.4,166.7,163.2,157.7,155.7,114.4,110.2,107.7,97.3,56.1,47.1,45.8,44.8,43.2,32.1,25.2,25.2,24.9,24.7,24.5,24.0,23.5,23.1,21.4,18.0,18.0,13.5；HR-ESI-MS m/z 599.3759[M+H]⁺。

Example 21

Preparation of 6, 8-dihydroxy-7- (cyclobutylcarbonyl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (39)

Titanium tetrachloride (0.4mmol,1.0M in CH) was added at room temperature₂Cl₂) And cyclobutyl chloride (0.11mmol) were added to a solution of compound 52(0.1mmol) in dichloromethane, respectively, and stirred at room temperature. After 24 hours of reaction, the reaction was quenched by addition of 1N aqueous hydrochloric acid solution and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 39(18.9mg, 43% yield).

¹H NMR(300MHz,CDCl₃)δ13.50(s,1H),8.26(s,1H),6.20(s,1H),4.27(m,2H),2.34(m,4H),1.96(m,2H),1.58(s,3H),1.48(m,3H),1.46(s,3H),1.44(s,3H),1.41(s,3H),0.88(d,J＝4.5Hz,3H),0.86(s,3H)；¹³C NMR(75MHz,CDCl₃)δ212.2,207.2,198.6,167.7,163.0,158.8,155.7,114.3,106.4,106.1,94.6,77.5,77.0,76.6,56.1,47.3,46.6,45.9,25.2,25.1,24.9,24.8,24.6,24.2,23.6,23.2,17.6；HR-ESI-MS m/z441.2271[M+H]⁺。

Example 22

Preparation of 6, 8-Diallyloxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (40)

Allyl bromide (60.5mg,0.5mmol) and potassium carbonate (41.4mg,0.3mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in acetone (3mL) at room temperature and warmed to reflux. After the reaction was allowed to stand overnight, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with ethyl acetate (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 40(49.1mg, yield 94%).

¹H NMR(400MHz,CDCl₃)δ6.47(s,1H),6.03(m,1H),5.40(dd,J＝17.2,3.2Hz,1H),5.28(dd,J＝17.2,10.5Hz,2H),4.55(m,1H),4.41(m,2H),4.20(t,J＝5.8Hz,1H),2.71(dd,J＝6.7,3.0Hz,2H),2.22(m,1H),1.58(s,3H),1.47(s,3H),1.43(m,3H),1.42(s,3H),1.39(s,3H),1.00(s,3H),0.98(s,3H),0.90(d,J＝6.1Hz,3H),0.82(d,J＝6.0Hz,3H)；¹³C NMR(100MHz,CDCl₃)δ212.2,203.8,197.5,167.4,154.7,154.0,152.6,133.0,132.2,123.6,118.2,118.0,113.6,113.3,96.9,70.0,56.0,53.9,47.2,26.0,24.9,24.9,24.6,24.3,24.0,23.3,23.2,22.7,22.7；HR-ESI-MS m/z 523.3050[M+H]⁺。

Example 23

Preparation of 6, 8-diacetoxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (42)

Acetic anhydride (30.6mg,0.3mmol) and triethylamine (40.4mg,0.4mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature and heated to reflux. After overnight reaction was cooled to room temperature, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by silica gel chromatography to give compound 42(48.4mg, 92% yield).

¹H NMR(300MHz,CDCl₃)δ6.92(s,1H),3.99(t,J＝6.0Hz,1H),2.60(dd,J＝6.7,4.6Hz,2H),2.29(s,3H),2.26(s,3H),2.19(dd,J＝13.2,6.5Hz,1H),1.52(s,3H),1.42(s,3H),1.35(s,3H),1.34(m,3H),1.32(s,3H),0.94(s,3H),0.92(s,3H),0.89(d,J＝6.1Hz,3H),0.79(d,J＝6.1Hz,3H)；¹³C NMR(75MHz,CDCl₃)δ211.6,199.7,197.2,168.4,168.1,167.4,152.0,146.5,145.4,125.1,118.7,112.9,109.0,56.0,52.6,47.2,47.1,26.3,24.8,24.5,24.4,24.2,24.1,23.2,22.8,22.5,22.5,21.0,20.7；HR-ESI-MS m/z 527.2637[M+H]⁺。

Example 24

Preparation of 6-methanesulfonyloxy-8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (43)

Methanesulfonyl chloride (13.7mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 43(42.1mg, yield 81%).

¹H NMR(300MHz,CDCl₃)δ13.27(s,1H),6.75(s,1H),4.35(d,J＝5.2Hz,1H),3.29(s,3H),2.97(m,2H),2.28(m,1H),1.56(s,3H),1.45(s,6H),1.41(m,3H),1.40(s,3H),1.37(s,3H),0.99(s,3H),0.96(s,3H),0.89(d,J＝5.3Hz,3H),0.84(d,J＝5.2Hz,3H)；¹³C NMR(75MHz,CDCl₃)δ211.7,205.0,197.4,166.6,162.5,154.8,147.8,114.4,113.9,111.4,101.6,56.2,52.7,47.1,46.1,38.7,25.4,25.2,25.0,24.7,24.6,24.5,24.0,23.2,23.1,22.6,22.6；HR-ESI-MS m/z 521.2204[M+H]⁺。

Example 25

Preparation of 6-trimethylacetoxy-8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (44)

tert-Butylacetyl chloride (16.1mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 44(18.4mg, yield 35%).

¹H NMR(500MHz,CDCl₃)δ13.56(s,1H),6.22(s,1H),4.36(t,J＝4.9Hz,1H),2.81(d,J＝6.6Hz,2H),2.35(m,1H),1.56(s,3H),1.46(s,3H),1.44(s,9H),1.44(m,3H),1.42(s,3H),1.38(s,3H),0.99(d,J＝3.5Hz,3H),0.98(d,J＝3.7Hz,3H),0.90(d,J＝4.9Hz,3H),0.86(d,J＝4.9Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.0,204.5,197.4,176.9,166.6,162.5,154.9,151.5,113.9,112.8,111.9,102.2,56.2,52.3,47.1,45.9,39.5,27.2,25.5,25.2,24.7,24.7,24.4,24.2,24.2,23.3,23.2,22.7,22.7；HR-ESI-MS m/z 527.3001[M+H]⁺。

Example 26

Preparation of 6-benzoyloxy-8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (45)

Benzoyl chloride (16.9mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The resulting crude product was purified by silica gel chromatography to give compound 45(23.5mg, yield 43%).

¹H NMR(500MHz,CDCl₃)δ13.54(s,1H),8.14(d,J＝7.8Hz,2H),7.64(d,J＝7.3Hz,1H),7.50(t,J＝7.6Hz,2H),6.42(s,1H),4.31(d,J＝5.4Hz,1H),2.70(d,J＝6.8Hz,2H),2.13(m,1H),1.47(s,3H),1.41(m,3H),1.37(s,3H),1.34(s,3H),1.31(s,3H),0.84(d,J＝5.2Hz,3H),0.80(d,J＝5.0Hz,3H),0.71(t,J＝5.8Hz,6H)；¹³C NMR(125MHz,CDCl₃)δ212.0,204.7,197.5,166.7,164.7,162.8,155.0,150.6,134.5,130.3,129.0,128.6,114.0,113.2,111.5,103.0,56.2,52.7,47.2,46.1,25.5,25.2,24.7,24.6,24.42,24.3,23.3,23.2,22.5,22.4；HR-ESI-MS m/z 549.2687[M+H]⁺。

Example 27

Preparation of 6-Trimethylsulfonyloxy-8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (46)

Trifluoromethanesulfonyl chloride (20.2mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 46(27.0mg, 47% yield).

¹H NMR(500MHz,CDCl₃)δ13.19(d,J＝15.9Hz,1H),6.61(d,J＝8.6Hz,1H),4.38(m,1H),2.93(m,2H),2.29(m,1H),1.59(s,3H),1.47(s,3H),1.45(m,3H),1.43(s,3H),1.39(d,J＝2.5Hz,3H),0.99(d,J＝2.5Hz,3H),0.97(s,3H),0.92(d,J＝5.0Hz,3H),0.85(d,J＝5.0Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ211.5,204.4,197.3,166.5,162.5,154.7,147.2,115.8,113.9,110.9,101.5,56.2,52.6,47.1,46.0,25.5,25.2,25.1,24.7,24.6,24.5,23.9,23.2,23.1,22.5,22.4；HR-ESI-MS m/z 575.1918[M+H]⁺。

Example 28

Preparation of 6-phenylacetyloxy-8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (47)

Phenylacetyl chloride (18.6mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 47(21.8mg, yield 39%).

¹H NMR(500MHz,CDCl₃)δ13.49(s,1H),7.40(m,5H),6.36(s,1H),4.35(t,J＝5.1Hz,1H),3.95(s,2H),2.71(d,J＝6.6Hz,2H),2.27(m,1H),1.56(s,3H),1.45(m,3H),1.44(s,3H),1.41(s,3H),1.38(s,3H),0.94(s,3H),0.93(s,3H),0.90(d,J＝5.1Hz,3H),0.85(d,J＝5.0Hz,3H)；HR-ESI-MS m/z 587.2843[M+H]⁺。

Example 29

Preparation of 6-isobutyroyloxy-8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (48)

Isobutyryl chloride (12.8mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) were added to a solution of compound 12(4.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 48(22.0mg, yield 43%).

¹H NMR(500MHz,CDCl₃)δ13.57(s,1H),6.34(s,1H),4.36(t,J＝5.1Hz,2H),2.87(m,1H),2.81(d,J＝6.7Hz,2H),2.32(m,1H),1.56(s,3H),1.46(m,3H),1.46(s,3H),1.42(s,3H),1.41(s,3H),1.39(s,3H),1.39(s,3H),0.99(s,3H),0.98(s,3H),0.90(d,J＝5.2Hz,3H),0.86(d,J＝5.2Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ212.0,204.6,197.5,175.0,166.7,162.7,154.9,150.8,114.0,112.9,111.4,102.5,56.2,52.5,47.2,46.0,34.7,25.4,25.2,24.7,24.6,24.4,24.4,24.2,23.3,23.1,22.7,22.7,18.8,18.8；HR-ESI-MS m/z 513.2843[M+H]⁺。

Example 30

Preparation of 6, 8-diacetoxy-7- (5-chlorovaleryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (49)

Acetic anhydride (30.6mg,0.3mmol) and triethylamine (40.4mg,0.4mmol) were added to a solution of compound 24(47.6mg,0.1mmol) in dichloromethane (2mL) at room temperature and heated to reflux. After overnight reaction was cooled to room temperature, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 49(47.6mg, yield 85%).

¹H NMR(400MHz,CDCl₃)δ6.95(s,1H),4.06(t,J＝6.1Hz,1H),3.57(t,J＝6.0Hz,2H),2.79(dd,J＝6.1,4.2Hz,2H),2.33(s,3H),2.31(s,3H),1.82(m,4H),1.56(s,3H),1.46(s,3H),1.40(s,3H),1.39(m,3H),1.36(s,3H),0.93(d,J＝6.2Hz,3H),0.84(d,J＝6.3Hz,3H)；¹³C NMR(100MHz,CDCl₃)δ211.7,199.9,197.3,168.6,168.2,167.4,152.3,146.5,145.4,124.8,118.7,113.0,108.9,56.0,47.3,47.2,44.6,42.6,31.9,26.3,24.9,24.8,24.5,24.5,24.2,23.2,22.8,21.1,20.8；HR-ESI-MS m/z 561.2248[M+H]⁺。

Example 31

Preparation of 6- (5-bromovaleryloxy) -8-hydroxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (50)

5-Bromopentanoyl chloride (24.0mg,0.12mmol) and 4-dimethylaminopyridine (24.4mg,0.2mmol) are added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature. After 2 hours of reaction, the reaction was quenched by addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The resulting crude product was purified by silica gel chromatography to give compound 50(24.1mg, 40% yield).

¹H NMR(400MHz,CDCl₃)δ13.56(s,1H),6.40(s,1H),4.35(t,J＝5.4Hz,1H),3.49(t,J＝6.1Hz,2H),2.78(d,J＝6.7Hz,2H),2.70(t,J＝7.0Hz,2H),2.30(m,1H),2.01(m,4H),1.56(s,3H),1.46(m,3H),1.45(s,3H),1.41(s,3H),1.38(s,3H),1.00(s,3H),0.98(s,3H),0.90(d,J＝5.7Hz,3H),0.86(d,J＝5.6Hz,3H)；¹³C NMR(100MHz,CDCl₃)δ212.0,204.4,197.4,170.8,166.6,162.7,154.9,150.2,114.0,113.1,111.1,102.6,56.2,52.5,47.1,46.1,33.9,32.7,31.8,25.4,25.2,24.7,24.6,24.5,24.4,24.2,23.3,23.2,23.1,22.8,22.7；HR-ESI-MS m/z 605.2102[M+H]⁺。

Example 32

Preparation of 6, 8-diisobutyroniyloxy-7- (3-methylbutyryl) -9-isobutyl-2, 2,4, 4-tetramethyl-4, 9-dihydro-1H-xanthene-1, 3(2H) -dione (51)

Isobutyryl chloride (32.0mg,0.3mmol) and triethylamine (40.4mg,0.4mmol) were added to a solution of compound 12(44.2mg,0.1mmol) in dichloromethane (2mL) at room temperature and heated to reflux. After the reaction was allowed to stand overnight, the temperature was reduced to room temperature, the reaction was quenched by the addition of 1N aqueous HCl (3mL) and extracted 3 times with dichloromethane (3 mL. times.3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The resulting crude product was purified by silica gel chromatography to give compound 51(47.1mg, yield 81%).

¹H NMR(500MHz,CDCl₃)δ6.84(s,1H),3.95(t,J＝5.7Hz,1H),2.75(m,1H),2.69(m,1H),2.56(m,2H),2.09(m,1H),1.48(s,3H),1.38(s,3H),1.34(m,3H),1.31(s,3H),1.29(s,3H),1.27(d,J＝7.0Hz,3H),1.24(d,J＝7.2Hz,3H),1.23(s,3H),1.22(s,3H),0.89(s,3H),0.88(s,3H),0.82(d,J＝6.0Hz,3H),0.68(d,J＝6.0Hz,3H)；¹³C NMR(125MHz,CDCl₃)δ211.9,199.7,197.3,174.6,174.4,167.5,151.9,146.6,145.4,125.5,118.4,112.6,108.7,55.9,52.8,47.3,46.2,34.2,34.0,26.6,24.9,24.8,24.6,24.5,24.4,24.1,23.6,23.1,22.7,22.7,18.8,18.8,18.7；HR-ESI-MS m/z 583.3076[M+H]⁺。

Evaluation of antiviral Activity of polyketides

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Example 33

Evaluation of Activity of polyketides against HSV-1, HSV-2, VZV and CMV in vitro

(1) Cells, viruses and test materials

Host cells of herpes simplex virus type 1 (HSV-1, F strain), HSV-1 drug-resistant strain (RV-117strain, TK mutation), herpes simplex virus type 2 (HSV-2, MS strain) and HSV-2 drug-resistant strain (AG-3strain, TK mutation) are African green monkey kidney cell (Vero) and human retinal epithelial cell (ARPE-19), respectively. Host cells of varicella-zoster virus (VZV, p-oka strain) and VZV resistant strain (TK-specific strain, TK mutation) are human retinal epithelial cells (ARPE-19). Host cells of human cytomegalovirus (CMV, AD169strain) and CMV-resistant strains (RC314strain, UL97mutation) are Human Foreskin Fibroblasts (HFFs). Cells were grown in DMEM medium with 10% volume fraction Fetal Bovine Serum (FBS); the maintenance solution (MM) was DMEM medium containing FBS in a volume fraction of 2%.

(2) Experimental methods

Cytotoxicity test of polyketides on Vero cells: vero cells were cultured at 1.2X 10⁴The cells/well were seeded in 96-well plates, cultured overnight to form a monolayer, treated with polyketides at 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, three duplicate wells per compound, and none at allThe drug-added cell control group was incubated at 37 ℃ with 5% CO₂In the incubator, after 24 hours of culture, the medium is discarded, 30 μ L of MTT is added to each well, the incubator is incubated for 4 hours in the dark, then, the MTT is aspirated off, 200 μ L of DMSO is added to each well to dissolve the generated formazan, the formazan is shaken on a plate shaker for 10min, and the absorbance (OD) is detected at 570nm of an microplate reader. Calculating the formula: the survival rate (%) of the cells was defined as the OD value of the drug-added group/OD value of the cell control group × 100%. According to the survival rate, the CC of the compound is calculated by fitting in Graph prism 5.0 software₅₀。

Cytotoxicity experiments of polyketides against ARPE-19 cells: ARPE-19 cells at 1X 10⁴Inoculating each cell/well in 96-well plate, culturing overnight, treating with polyketides of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, and 3.125 μ M, respectively, setting three multiple wells for each polyketide, setting cell control group without drug, placing at 37 deg.C and 5% CO₂The cells were cultured in an incubator for 2 days, and then the cytotoxicity was measured by the MTT method.

Cytotoxicity experiments of polyketides on HFF cells: HFF cells were cultured at 1X 10⁴Inoculating each cell/well in 96-well plate, culturing overnight, treating with polyketides of 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, and 3.125 μ M, respectively, setting three multiple wells for each polyketide, setting cell control group without drug, placing at 37 deg.C and 5% CO₂The cells were cultured in an incubator for 5 days, and then the cytotoxicity was measured by the MTT method.

Evaluation of the Activity of polyketides against HSV-1 and HSV-2 in vitro: HSV-1 and HSV-2 were assessed for activity using Vero and ARPE-19 cells, respectively. Vero and ARPE-19 cells were 2X 10⁴Each cell/well was seeded in a 96-well plate, cultured overnight to form a monolayer of cells, and according to toxicity experiments, compounds were diluted with medium containing 2% serum with the maximum non-toxic concentration of the compound as the starting concentration, each compound was set at 6 double dilutions, 50. mu.L of each compound was added at different concentrations, followed by 100TCID ₅₀50 μ L of virus, and a virus control group and a cell control group were simultaneously set, each group having three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in incubator, detecting after 48 hrDegree of cytopathic effect (CPE), and the IC of the compound was counted according to the CPE results₅₀. "-" indicates acellular lesions; "+" indicates that 0-25% of the cells had CPE; "+ +" indicates that 25-50% of the cells had CPE; "+ + + +" indicates that 50-70% of the cells had CPE; "+ ++" indicates 75-100% of the cells have CPE.

Evaluation of the Activity of polyketides against VZV in vitro: ARPE-19 cells at 2X 10⁴Each cell/well was seeded in a 96-well plate, and after monolayer formation, compounds were diluted with medium containing 2% serum with the maximum non-toxic concentration of the compound as the starting concentration, each compound set at 6-fold dilutions, 50. mu.L of different concentrations of compound were added to each well, followed by 100TCID ₅₀50 μ L of virus, and a virus control group and a cell control group were simultaneously set, each group having three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 3-4 days or taking the complete lesion of the model group as an end point. Observing cytopathic degree of each group under microscope, and counting IC of the compound according to CPE result₅₀。

Evaluation of the Activity of polyketides against CMV in vitro: HFF cells were cultured at 1X 10⁴Each cell/well was seeded in a 96-well plate, and after monolayer formation, compounds were diluted with medium containing 2% serum with the maximum non-toxic concentration of the compound as the starting concentration, each compound set at 6-fold dilutions, 50. mu.L of different concentrations of compound were added to each well, followed by 100TCID ₅₀50 μ L of virus, and a virus control group and a cell control group were simultaneously set, each group having three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 6-7 days or taking the complete lesion of the model group as an end point. Observing cytopathic degree of each group under microscope, and counting IC of the compound according to CPE result₅₀。

Evaluation of the activity of polyketide in vitro anti-HSV-1 (acyclovir) and HSV-2 (acyclovir) resistant strains: vero and ARPE-19 cells were grown at 1.2X 10⁴Inoculating each cell/well into 96-well plate, culturing overnight to form monolayer cells, and after the monolayer cells are formed, using the maximum nontoxic concentration of the compound as the initial concentration, and using the concentration of the compound as 2%Serum media dilution of compounds, each compound set at 6 two-fold dilutions, 50 μ L of different concentrations of compound per well, followed by 100TCID ₅₀50 μ L of virus, and a virus control group and a cell control group were simultaneously set, each group having three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in incubator, detecting cytopathic effect (CPE) degree after 48 hr, and calculating IC of compound according to CPE result₅₀。

Evaluation of the Activity of polyketides against VZV-resistant strains in vitro (resistance to Acyclovir): ARPE-19 cells at 2X 10⁴Each cell/well was seeded in a 96-well plate, and after monolayer formation, compounds were diluted with medium containing 2% serum with the maximum non-toxic concentration of the compound as the starting concentration, each compound set at 6-fold dilutions, 50. mu.L of different concentrations of compound were added to each well, followed by 100TCID ₅₀50 μ L of virus, and a virus control group and a cell control group were simultaneously set, each group having three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 3-4 days or taking the complete lesion of the model group as an end point. Observing cytopathic degree of each group under microscope, and counting IC of the compound according to CPE result₅₀。

Evaluation of the Activity of polyketides against CMV-resistant Strain in vitro (Ganciclovir resistance): HFF cells were cultured at 1X 10⁴Each cell/well was seeded in a 96-well plate, and after monolayer formation, compounds were diluted with medium containing 2% serum with the maximum non-toxic concentration of the compound as the starting concentration, each compound set at 6-fold dilutions, 50. mu.L of different concentrations of compound were added to each well, followed by 100TCID ₅₀50 μ L of virus, and a virus control group and a cell control group were simultaneously set, each group having three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 6-7 days or taking the complete lesion of the model group as an end point. Observing cytopathic degree of each group under microscope, and counting IC of the compound according to CPE result₅₀。

(3) Results of the experiment

The biological activity of the compounds of the present invention was measured by the above experimental methodIC of compounds against HSV-1, HSV-2, VZV and CMV activity in vitro₅₀Value and cytotoxicity CC against Vero, ARPE-19, HFF cells, respectively₅₀The values are shown in Table 2. As shown in table 2, compounds 12, 14, 24, 39, 44, 45, 46 and 49 all showed good antiviral activity against the four herpesviruses mentioned above, with compound 39 having the most significant antiviral activity. In addition, as shown in table 3, the series of compounds also showed good antiviral activity against acyclovir-resistant virus strains (HSV-1, HSV-2 and VZV) and ganciclovir-resistant virus strain (CMV), indicating that the antiviral activity targets and action mechanisms of the series of compounds are different from those of acyclovir and ganciclovir.

TABLE 2 Activity test results of Compounds 1-51 against HSV-1, HSV-2, VZV and CMV in vitro

^aIC₅₀The half maximal inhibitory Concentration (50% Inhibition Concentration) was determined by IC₅₀(μ M) represents:

^bCC₅₀means half the Cytotoxic Concentration (50% cytoxic Concentration) in CC₅₀(μ M) represents;

^cSI refers to the Selectivity Index (Selectivity Index) with the value CC₅₀/IC₅₀；

^dn.A. denotes the concentration at half the cytotoxic concentration CC₅₀No anti-viral activity;

^e"-" indicates no SI value or no screening for the positive drug.

TABLE 3 Activity test results of Compounds 1-51 against resistant strains of HSV-1, HSV-2, VZV and CMV in vitro

^e"-" indicates no SI value or no screening for the positive drug.

Example 34

Research on action mechanism of polyketone compound 39 for resisting HSV-1

(1) Effect of Compound 39 on different Titers of HSV-1 replication

The experimental method comprises the following steps: the antiviral activity of the compound 39 selected for best activity was further evaluated quantitatively using plaque reduction experiments. First, toxicity (CC) of Compound 39 on Vero cells was evaluated by MTT method₅₀) Calculating MNCC; the half Inhibitory Concentration (IC) of Compound 39 on viral proliferation in Vero cells was then determined using MNCC as the starting concentration for the antiviral experiment₅₀) Calculating the selectivity index (SI ═ CC) of the compound for inhibiting HSV-1₅₀/IC₅₀)。

The experimental results are as follows: as shown in fig. 1, compound 39 has good inhibitory effect on different titers of HSV-1 virus (moi ═ 0.5,1, and 5), where IC was 1 when moi was 1₅₀At 0.37 μ M, compound 39 also inhibited well replication of high titer viruses when the virus titer was increased to moi of 5.

(2) Study of mode of action of Compound 39 in inhibition of HSV-1 Virus replication

The experimental method comprises the following steps: by direct inactivationThe mode of action of compound 39 on viral replication was investigated for direct virucidal effect. 50 μ L of each of the compound 39 solutions diluted at the serial sesqui-concentration was mixed with 50 μ L of HSV-1, and 50 μ L of the cell maintenance solution and 50 μ L of HSV-1 were mixed in the virus control group, and incubated at 37 ℃ for 2 hours. Subsequently, the mixture of the virus and the samples or the maintenance solution at each concentration was diluted 1000-fold with the maintenance solution so that the concentration of the virus was 30 PFU/well. The culture medium in the previously plated 24-well plate was discarded, washed 1 time with PBS, 200. mu.L of the diluted solution was added to Vero cells, and incubated at 37 ℃ for 2 hours. Meanwhile, a normal cell control group, a virus control group and an acyclovir control group are set. After 2 hours, the plate was discarded, and 500. mu.L of the agarose mixed medium (medium mixed with 2 XMM and 1.2% agarose in a ratio of 1: 1) was added to each well along the walls of the well, and after the agarose had solidified, 500. mu.L of MM was added. At 37 ℃ with 5% CO₂After culturing in an incubator for 4 days, fixing cells and inactivating viruses by 6% formaldehyde solution, after overnight, adding 200-300 μ L of crystal violet solution with the concentration of 1% by mass into each hole, dyeing at room temperature for 20min, washing off the crystal violet solution, recording the number of plaques in each group, and calculating the plaque inhibition rate.

The experimental results are as follows: as shown in FIG. 2, Compound 39 is much higher than IC₅₀Has no obvious inactivation effect on HSV-1 under the concentration of the compound, which indicates that the mechanism of action of the compound 39 for resisting HSV-1 is not to directly inactivate HSV-1.

(3) Effect of Compound 39 on the replication cycle of HSV-1 Virus

The experimental method comprises the following steps: time-point experiments (Time of addition assay) were used to test at which stage in the HSV-1 replication cycle compound 39 exerted inhibitory effects: vero cells were seeded in 96-well plates at a cell density of 1.2X 10⁴One well, cultured overnight to form a monolayer of cells, medium was discarded, Vero cells were infected with HSV-1(MOI 0.5), drugs were added at different time points (0, 1,2, 6, 10 and 18 hours) after viral infection, both virus and drug were added to the cells at 0 hour, viral supernatant was aspirated off at 2 hours, bottom cells were washed 3 times with PBS, drug was re-added at the same concentration, and supernatant was collected after freeze-thawing 3 times at-80 ℃ at 24 hours after viral infection. Air conditionerViral titers were determined by plaque reduction.

The experimental results are as follows: the results of the time point experiments are shown in FIG. 3, and compound 39 acts mainly 6-18 hours after viral infection, and the stage is mainly viral DNA replication, early gene and late gene transcription and translation.

(4) Effect of Compound 39 on the transcription of HSV-1 Virus genes

The experimental method comprises the following steps: the effect of compounds on HSV-1 viral transcription was studied using RT-PCR experiments (RT-PCR assay). Vero cells were seeded in 12-well plates at a cell density of 3X 10⁵One/well, at 37 ℃ and 5% CO₂Culturing in an incubator. Vero cells were infected with HSV-1(MOI ═ 1) for 1 hour at 4 ℃, washed 1 time with pre-cooled PBS and simultaneously with varying concentrations of compound 39(0.25 μ M,0.5 μ M,1 μ M,2 μ M) and the positive drug acyclovir (2 μ M) added, incubated in a 37 ℃ incubator for 4, 7 and 10 hours, respectively. Total RNAs were extracted with RNA rapid extraction kit for 4, 7 and 10 hours, respectively, quantified and reverse-transcribed into cDNAs, and the expression levels of immediate early genes, early genes and late genes were determined by Light Cycler 480PCR instrument.

The experimental results are as follows: RT-PCR experiments detect the influence of the compound 39 on HSV-1 gene transcription, and the results are shown in figure 4, the compound 39 can inhibit the transcription of early genes and late genes, has more obvious inhibition effect on the late genes, presents concentration dependence and is consistent with the experimental results of time points.

(5) Effect of Compound 39 on the adsorption of HSV-1 Virus to the cell Membrane

The experimental method comprises the following steps: vero cells were digested with non-enzymatic cell separation (versene solution) and the digested cells were transferred to 2mL round bottom EP tubes. After centrifugation at 1500rpm for 3 minutes at 4 ℃, the supernatant was discarded, and compound 39 prepared as a maintenance solution and HSV-1(MOI ═ 1) were mixed in advance, added to the cells, and adsorbed at 4 ℃ for 1 hour. Centrifuging at 4 deg.C and 1500rpm for 3 min, discarding supernatant, washing cell precipitate with 4 deg.C refrigerator precooled PBS, repeatedly washing for 3 times, centrifuging, adding precooled 4% paraformaldehyde into cell precipitate, and fixing cells on ice for 15 min. Centrifuging at 4 deg.C and 1500rpm for 3 min, discarding supernatant, and pre-cooling with 4 deg.C PBSWashing the cell sediment, repeatedly washing for 3 times, adding 5% BSA diluted HSV-1gD protein primary antibody, and incubating for 2 hours at 37 ℃. Centrifuging at 1500rpm for 3 min, discarding the supernatant, washing 3 times with PBS, centrifuging, adding 5% BSA diluted Alexa

488 fluorescent secondary antibody, incubated at 37 ℃ for 1 hour. After 3 times of PBS wash, 500. mu.L of PBS was added to resuspend the cells, and the mean fluorescence intensity of the cells was measured at 488nm excitation wavelength of the flow cytometer.

The experimental results are as follows: the flow cytometer detects the effect of the compound 39 on HSV-1 adsorption to Vero cells, and the result is shown in figure 5, heparin (4 mu M) is a virus adsorption inhibitor, and can obviously inhibit HSV-1 adsorption. Acyclovir (4. mu.M) is a DNA replication inhibitor and has no inhibitory effect on the adsorption process. The research result shows that the compound 39 has no inhibition effect on the virus adsorption process, which indicates that the action stage of the compound 39 is not the virus adsorption process.

(6) Effect of Compound 39 on HSV-1 Virus fusion

The experimental method comprises the following steps: 1mL of Vero cell suspension was seeded into a confocal dish at a cell density of 1X 10⁵each/mL, at 37 ℃ with 5% CO₂A cell culture box. After 24 hours, 200. mu. L R18/DiOC18 dye-labeled HSV-1 suspension was added, adsorbed for 1 hour at 4 ℃, the medium was aspirated, washed 1 time with 4 ℃ pre-cooled PBS, and unadsorbed virus was washed away. Adding the compound 39 diluted by the maintenance solution or the maintenance solution with the same volume, and putting the mixture into a cell culture box for culture. After 2 hours of virus infection, the supernatant was aspirated off, fixed with 4% paraformaldehyde, washed with PBS 3 times, and the fluorescence intensity of the two dyes at the excitation wavelengths was measured under a laser confocal microscope.

The experimental results are as follows: a laser confocal experiment detects the influence of a compound 39 on the fusion of HSV-1 and a cell membrane, virus particles are marked by R18 and DiOC18, when the virus membrane and a host cell membrane are not fused, the distances between fluorescent groups of DiOC18 and R18 are very close, a self-quenching effect and an energy resonance transfer phenomenon can occur, fluorescence released by DiOC18 can be transferred to R18 molecules, and a confocal probe is more sensitive to red fluorescence, so that the fluorescence of DiOC18 cannot be detected. When the viral membrane is fused with the cell membrane, the dye is diluted and diffuses to the cell membrane, and the fluorescence released by DiOC18 is no longer covered by R18, so that the fluorescence of DiOC18 can be detected. The results are shown in FIG. 6, which indicates that Compound 39 has no inhibitory effect on the membrane fusion process of the virus.

(7) Effect of Compound 39 on HSV-1 Virus gD protein expression

The experimental method comprises the following steps: HSV-1(MOI ═ 0.5) and various concentrations of compound 39 were allowed to act simultaneously on Vero cells, and after 24 hours, 4% paraformaldehyde was added to fix the cells. After 3 washes with PBS, 100 μ L of 5% BSA formulated in PBS was added for blocking for 30 min. The blocking solution was discarded and 5% BSA was added to make up HSV-1gD protein primary antibody. After incubation for 1 hour at 37 ℃, primary antibody was discarded, washed 3 times with PBS, and secondary antibody formulated with 5% BSA was added: alexa

488 fluorescent secondary antibody, incubated at 37 ℃ for 1 hour. Adding a saturated DAPI solution to stain cell nuclei for 1-2 minutes, and washing with PBS for 3 times. Observing and photographing under a fluorescence microscope with the magnification of 20 times of that of a high content screening instrument, recording, randomly taking 20 pictures of each multiple hole, analyzing the pictures by using software of the instrument, and calculating the average fluorescence intensity of each hole.

The experimental results are as follows: the effect of the compound on the expression of the late protein was detected by immunofluorescence, and the results are shown in fig. 7, and compound 39 significantly reduced the expression level of the late protein gD, suggesting that it may exert inhibitory effects after the virus enters the cells, before assembly and release. The results of the above experiments indicate that compound 39 is effective intracellularly, acting at the middle and late stages of viral replication.

Example 35

Action mechanism research of anti-CMV (cytomegalovirus) of polyketide 39

(1) Effect of Compound 39 on CMV viral replication

Experimental materials: human cytomegalovirus (CMV, AD169strain), recombinant human cytomegalovirus (CMV-GFP, AD169-GFP), and Human Foreskin Fibroblast (HFF) as host cell. The cells were grown in DMEM medium with Fetal Bovine Serum (FBS) at a concentration of 10% by mass; the maintenance solution is DMEM medium containing FBS at a concentration of 2% by mass.

The experimental method comprises the following steps: firstly, the MTT method is used for testing the influence of the compound on the growth of HFF cells, and the specific method is as follows: HFF cells were cultured overnight in 96-well plates at 1X 104 cells/well, treated with 12. mu.M, 9. mu.M, 6. mu.M, 3. mu.M, 1. mu.M, 0.5. mu.M, 0.25. mu.M, 0.125. mu.M, 0.0625. mu.M, 0.0313. mu.M of compound 39, three duplicate wells per compound, and drug-free cell controls placed at 37 ℃ in 5% CO₂The cells were cultured in an incubator for 5 days, and then the cytotoxicity was measured by the MTT method. The virus replication adopts a CMV-GFP fluorescent virus strain for experiment, and the specific method comprises the following steps: HFF cells were cultured at 1X 10⁴Each cell/well was seeded in a 96-well plate and cultured overnight, with the maximum non-toxic concentration of the compound as the starting concentration, the compound was diluted with a medium containing 2% serum, compound 39 was set at 6 double dilutions, i.e. 2 μ M,1 μ M,0.5 μ M, 0.25 μ M, 0.125 μ M, 0.0625 μ M, 50 μ L of the compound at different concentrations was added per well, followed by 50 μ L of CMV-GFP with MOI of 0.1, while a virus control and a cell control were set, each set of three duplicate wells. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 7 days. Subsequently, the medium was discarded, and 100. mu.L of PBS was added thereto, and the fluorescence intensity of GFP was measured under a microplate reader.

The experimental results are as follows: FIG. 8 shows that compound 39 has some effect on the proliferation of HFF cells with increasing compound concentration, its CC₅₀10.10. mu.M. Further examination of the effect of this compound on viral replication revealed that compound 39 was found to inhibit CMV-GFP expression in a concentration-dependent manner (FIG. 9), indicating that compound 39 is capable of inhibiting CMV replication in vitro and that compound 39 inhibits CMV-GFP expression at half the Inhibitory Concentration (IC)₅₀) At 0.16. mu.M, the Selectivity Index (SI) was 63.13.

(2) Effect of Compound 39 on the replication cycle of CMV Virus

The experimental method comprises the following steps: HFF cells were seeded into 96-well plates at a cell density of 1X 10 using a time point addition experiment⁴One/well, cultured overnight to formMonolayer cells were removed, medium was discarded, cells were infected with CMV (MOI ═ 0.1), compound 39(1 μ M) or ganciclovir (GCV,2 μ M) was added at various time points (6, 18, 30, 42, 48, 54, 66, 70 hours) after viral infection, and supernatants were collected after freeze-thawing 3 times at-80 ℃ 72 hours after viral infection. Viral titers were determined by plaque reduction.

The experimental results are as follows: the results of the time point experiments are shown in fig. 10. The DNA polymerase inhibitor ganciclovir acts primarily early in the CMV replication cycle, while the addition of compound 39 late in CMV replication still significantly inhibits CMV replication, suggesting that the compound exerts an inhibitory effect after CMV entry into the cell, but before the late phase of viral replication.

Example 36

Preparation of Compound 39 cream

(1) Preparation of the aqueous phase

Weighing: polyoxyethylene stearate: 210mg of

Glycerol: 1000mg

Distilled water: 4mL (4000mg)

Hydroxyphenyl ethyl ester: 10mg of

Total weight: 5220mg

Mixing the weighed polyoxyethylene stearate, glycerol and distilled water, heating to 80 ℃, adding ethylparaben, uniformly mixing, reducing the temperature to 65 ℃, and keeping the temperature;

high dose group: weighing 26.5% of the total weight, namely 1383mg, from the water phase, transferring to a new EP tube, and keeping the temperature at 65 ℃;

low dose group: 26.7% of the total weight, i.e. 1397mg, was weighed out from the above aqueous phase and transferred to a new EP tube and incubated at 65 ℃.

(2) Preparation of the oil phase

Weighing: vaseline: 3500mg

Oleic acid: 200mg of

Cetyl alcohol: 100mg of

Octadecanol: 300mg

Total weight: 4100mg

Mixing the weighed vaseline, oleic acid, propylene glycol, hexadecanol and octadecanol, heating to 80 ℃ to melt the vaseline, oleic acid, propylene glycol, hexadecanol and octadecanol, uniformly mixing, cooling to 60 ℃, and keeping the temperature;

high dose group: 26.5% of the total weight, 1086mg, was weighed out from the above oil phase, transferred to a new EP tube, incubated at 60 ℃, and then 50.4mg of compound 39 was added to the mixture, stirred until dissolved;

low dose group: 26.7% of the total weight, i.e. 1095mg, was weighed out from the above oil phase, transferred to a new EP tube, incubated at 60 ℃, and then 25.2mg of compound 39 was added to the mixture, stirred until dissolved.

(3) Emulsification

Slowly adding the water phase (65 deg.C) of the high-dose group, the low-dose group and the blank control group into the oil phase (60 deg.C), stirring continuously in the same direction, and continuously stirring until the mixture is condensed to obtain compound 39 cream.

Example 37

Preparation of compound 39 ophthalmic gel

(1) Preparation of high dose group gels

Weighing: compound 39: 400mg of

Carbomer 940 NF: 160mg of

Glycerol: 400mg of

Sodium chloride: 340mg of

2% sodium hydroxide solution: 0.2mL

Hydroxyphenyl ethyl ester: 12mg of

Water for injection: 40mL

Preparation: taking carbomer 940NF, adding glycerol, grinding, fully wetting, adding 8mL of water for injection, and soaking overnight to obtain a gel matrix; sequentially adding 2% sodium hydroxide solution, sodium chloride and ethylparaben into another compound 39, then adding 8mL of water for injection, and stirring until the mixture is dissolved; adding the mixture containing the compound 39 into the gel matrix, adjusting the pH to 8 with 0.5mol/L NaOH under stirring to form gel, adding injection water to 40mL, and stirring to obtain the high-dose (20mg/kg) ophthalmic gel of the compound 39.

(2) Preparation of low-dose group gel

Weighing: compound 39: 200mg of

Carbomer 940 NF: 160mg of

Glycerol: 400mg of

Sodium chloride: 340mg of

2% sodium hydroxide solution: 0.2mL

Hydroxyphenyl ethyl ester: 12mg of

Water for injection: 40mL

Preparation: taking carbomer 940NF, adding glycerol, grinding, fully wetting, adding 8mL of water for injection, and soaking overnight to obtain a gel matrix; sequentially adding 2% sodium hydroxide solution, sodium chloride and ethylparaben into another compound 39, then adding 8mL of water for injection, and stirring until the mixture is dissolved; adding the mixture containing the compound 39 into the gel matrix, adjusting the pH to 8 with 0.5mol/L NaOH under stirring to form gel, adding injection water to 40mL, and stirring to obtain the low-dose (10mg/kg) ophthalmic gel of the compound 39.

Example 38

Research on treatment effect of polyketide 39 on mouse dermatitis caused by HSV-1 infection

The experimental method comprises the following steps: compound 39 was evaluated for in vivo activity using a BALB/c mouse (3 weeks old) infection model. The 3-week-old BALB/c female mice were divided into 5 groups of 10 mice each, namely a normal control group, a virus control group, an acyclovir group (20mg/kg/d), a low dose administration group (10mg/kg/d), and a high dose administration group (20 mg/kg/d). On day 0, the skin of the right auricle was pricked with a 1mL syringe, 10 wells per mouse ear, HSV-1 virus was dropped on the wound surface of the auricle, and the cream described above was applied 30 minutes after viral infection for 6 consecutive days, weighing daily, scoring the wound surface and measuring the thickness of the right auricle with a vernier caliper. On day 10, the skin of the right auricle of the mouse is weighed and placed into a marked EP tube, DMEM is added according to the proportion of 10 mu L/mg, and the mixture is homogenized and subpackaged for measuring each index.

The experimental results are as follows: FIG. 11 shows that compound 39 can reduce weight loss of mice caused by HSV-1 infection at both concentrations, and reduce skin wound score and pinna thickness, as well as reduce viral titer in the ear, indicating that compound 39 can inhibit replication of HSV-1 virus in vivo, and has good in vivo antiviral effect.

Example 39

Research on treatment effect of polyketide 39 on mouse viral keratitis caused by HSV-1 infection

The molding and administration method comprises the following steps: BALB/c mice are adopted as infection models, females are 20g in weight, the mice without eye diseases are checked under a slit lamp, and 0.25% chloramphenicol eye drops are added one drop per eye twice a day before molding. Newly purchased mice were subjected to adaptation feeding in a P2-grade animal laboratory for 3 days and then molded (corneal epithelium scratching method). After the anesthesia of the abdominal cavity injection with the chloral hydrate solution, the mice are placed under a slit lamp microscope, and the corneal epithelial layer of the right eye is scratched by a No. 4.5 sterile needle head in a crossing way for a plurality of times, and the thickness of the corneal epithelial layer is 1mm²The conjunctival sac is scratched in a shape of a small well and then 5. mu.L of the composition is dropped into the conjunctival sac to obtain a titer of 1X 10⁷PFU/mL HSV-1 virus solution and massaged for 30 seconds to fully inoculate it. Among them, 10 mice were randomly drawn and no virus was given as a normal control group. After molding for 12 hours, mouse cornea is stained by fluorescein sodium, corneal pathological changes are observed under a slit lamp microscope, the cornea is stained in a punctiform, map-shaped and dendritic mode, mouse corneal fluorescein sodium is positively stained, and the success of HSK molding is confirmed. Mice successfully modelled were randomly and equally divided into 4 groups, namely: model group, acyclovir group (20mg/kg/d), low dose group (10mg/kg/d), and high dose group (20 mg/kg/d). After the molding was successful, the gel was administered, and mice in the low dose group, the high dose group and the acyclovir group were dosed once a day. The normal control group and the model group were given a relative amount of gel matrix. The administration was continued for 2 weeks. Observation method and detection method: firstly, the general condition of the mouse is observed, and secondly, the mental state, the fur condition, the activity and the like of the mouse are observed during the experiment.

(1) Effect of Compound 39 on growth status in mice

The experimental results are as follows: the normal group of mice grows relatively fast, the weight is increased, the hair color is white and glossy, and the activity is sensitive; after 12 hours, BALB/c mice successfully inoculated with HSV-1 virus and modeled show symptoms such as photophobia and lacrimation, the mental state and the activity become worse, the symptoms of the mice are correspondingly relieved after 1 week of treatment, and the symptoms such as activity, weight, photophobia and lacrimation are obviously improved in each treatment group compared with a model group.

TABLE 4 appearance and growth of the groups of mice

(2) Effect of Compound 39 on the pathological conditions of keratitis in mice

The experimental method comprises the following steps: and observing by a slit lamp microscope. After modeling, each group was stained with fluorescein on

days

3, 7, and 14 after drug administration, and corneal epithelial layer lesions were observed under a slit lamp microscope and scored for Trousdale: 0min, no corneal lesion; 1 minute, the corneal lesion is less than 25 percent of the total corneal area; 2 minutes, the keratopathy becomes total corneal area 25% -50%; dividing by 3, and changing the corneal disease into 50-75% of total corneal area; 4 min, the corneal lesion is larger than 75% of the total corneal area.

The experimental results are as follows: the statistical result is shown in fig. 12, the degree of keratopathy of the mice in the high dose group (20mg/kg/d) on day 3 of administration is obviously relieved compared with that in the model group (P <0.05), and the statistical significance is achieved; compared with the model group, the degree of the corneal lesion of the mice in the low-dose group (10mg/kg/d) and the acyclovir group has no statistical significance, which indicates that the degree of the corneal lesion is not obviously improved; compared with the model group in the corneal lesion degree of each treatment group on the 7 th and 14 th days (P is less than 0.05), the difference has statistical significance, and the corneal inflammation symptom is obviously improved; compared with the polyketide group, the acyclovir group has no statistical significance (P >0.05), which indicates that the compound 39 has equivalent curative effect on treating viral keratitis to the acyclovir serving as a positive control drug.

(3) Effect of Compound 39 on viral keratitis mouse corneal Virus replication

The experimental method comprises the following steps: after 12 hours from the last administration, the mice were anesthetized by intraperitoneal injection of chloral hydrate, and the eyeballs of the mice were extracted. Wherein, the eyeballs of three mice are randomly drawn from each group and fixed in 4% formaldehyde solution for subsequent cornea separation for HE staining. And respectively picking eyeballs of the remaining mice of each group, grinding the mice by using a culture medium, and taking the supernatant for a plaque reduction experiment to detect the HSV-1 titer in the eyeballs.

The experimental results are as follows: as shown in figure 13, the compound 39 administration group and the positive drug acyclovir group can reduce the cornea HSV-1 titer of infected mice, and have significant difference. The experimental results show that the compound 39 has the function of treating HSV-1 viral keratitis.

Example 40

Studies on therapeutic Effect of polyketide 39 on genital herpes of Guinea pig caused by HSV-2 infection

The experimental method comprises the following steps: the best active compound 39 selected in vitro was evaluated for in vivo activity using a Hartley female guinea pig (5 weeks old) infection model. 5-week-old Hartley female guinea pigs were divided into 5 groups of 10 animals, each of which was a normal control group, a virus control group, acyclovir (20mg/kg/d), a low dose group (10mg/kg/d), and a high dose group (20 mg/kg/d). Keeping the constant temperature, humidity and sufficient feed in the room in an isolated breeding mode. On the beginning day of the experiment, the suppositories prepared from different groups of drugs are inserted into the vagina of the guinea pig in sequence according to the above groups, and are completely dissolved after 50 min. In each of the other groups of animals except the normal control group, 50. mu.L of each virus was intravaginally inoculated at one time with a micro-injector, and the administration was continued for 10 days, and the development of lesions in external genitalia was observed for 10 consecutive days after virus inoculation, and the score was estimated based on the degree of lesions. And 3, 5, 7, 9 and 10 days after virus inoculation, cleaning the vulva by using physiological saline, then sampling in a guinea pig vagina by using a self-made sterile fine cotton swab, placing in virus transfer liquid, centrifuging, taking supernatant, inoculating the supernatant on newly formed monolayer cells of a 96-hole micro-culture plate, observing the formation condition of virus plaques by adopting a cell culture technology, and calculating the PFU value according to the number of the plaques.

The experimental results are as follows: as shown in fig. 14, compound 39 at both concentrations reduced the clinical symptom score and also reduced the vaginal viral titer, indicating that compound 39 was able to inhibit HSV-2 virus replication in vivo, with good antiviral effect.

EXAMPLE 41

Research on influence of CMV virus infection on growth and development of offspring guinea pigs and therapeutic effect of polyketide 39 on offspring guinea pigs

The experimental method comprises the following steps: greater than 6 months old Hartley guinea pigs weighing 675 + -25 g. In the experiment, Nested-polymerase chain reaction (N-PCR) is used for screening male and female guinea pigs with CMV DNA negative, the male and female guinea pigs are arranged in the same cage according to the proportion of 4: 1, vaginal secretion of the male and female guinea pigs is taken day by day for smear examination, and the day of sperm is determined as the 0 th day of pregnancy. Then, guinea pigs at the early stage of pregnancy (gestational age 1-20 days) were randomly selected to be divided into 5 groups of 25: blank control group was not given special treatment, and virus control group was inoculated with 1mL (10) of virus suspension per peritoneal cavity⁷TCID₅₀) Ganciclovir (20mg/kg/d), low dose (10mg/kg/d) and high dose (20mg/kg/d) groups were administered by gavage with virus inoculation of ganciclovir and different doses of compound 39(3 times per day for 14 consecutive days, the dose was converted to a human and guinea pig weight dose conversion factor and the dose was adjusted daily according to the change in body weight). Observing the change of weight, fur, food intake, sleep, etc. of each group of pregnant guinea pigs, killing female guinea pigs in the middle stage of pregnancy, counting the number of live and dead fetus, collecting the organ tissues of the maternal blood, placenta and fetus such as salivary gland, thymus, kidney, lung, etc., and detecting the presence or absence of virus infection.

The experimental results are as follows: as shown in table 5 and fig. 15, the blank control group of 25 female guinea pigs had no signs of abnormality. In the virus control group, 25 female guinea pigs, placentas and fetuses are all infected by CMV, the death rate of offspring is obviously increased, and the phenomena of obvious weight reduction, hair reversion, less eating and more sleeping and the like are caused. The compound 39 with two concentrations can reduce the virus titer of guinea pigs, placentas and fetuses, reduce the death rate of offspring, and improve the symptoms of the weight of the guinea pigs such as obvious weight reduction, hair loss, poor appetite and sleep. Indicating that compound 39 can inhibit the replication of CMV virus in vivo, thereby reducing the effect of CMV virus infection on growth and development of offspring guinea pigs.

TABLE 5 appearance and growth of the groups of mice

It should be noted that the above detailed description is only for exemplary purposes, and the present invention is not limited to the above described embodiments. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. The application of polycyclic polyketides in preparing anti-herpes virus medicaments is disclosed, wherein the polyketides have a structural formula shown in a general formula I:

or a pharmaceutically acceptable salt thereof, wherein: r⁵Selected from isobutyl, isopropyl, phenyl; r⁶Selected from hydrogen atom, isovaleryl, 2-methylbutyryl; r⁷Selected from hydrogen atom, isovaleryl group, 2-methylbutyryl group, isobutyryl group, acetyl group, propionyl group, n-butyryl group, decanoyl group; r⁸Selected from hydrogen atom, acetyl, tert-butyl formyl, benzoyl, trifluoromethanesulfonyl and phenylacetyl; r⁹Selected from hydrogen atom and acetyl.

2. Use according to claim 1, wherein R is⁷H, the polyketide has a structural formula shown in a general formula III:

or a pharmaceutically acceptable salt thereof, wherein: r⁵、R⁶、R⁸And R⁹As defined in claim 1.

3. Use according to claim 2, said polyketides being selected from:

or a pharmaceutically acceptable salt thereof.

4. Use according to claim 1, wherein R is⁶H, the polyketide has a structural formula shown in a general formula IV:

or a pharmaceutically acceptable salt thereof, wherein: r⁵、R⁷、R⁸And R⁹As defined in claim 1.

5. Use according to claim 4, said polyketides being selected from:

or a pharmaceutically acceptable salt thereof.

6. Use of polyketides of general formulae I, II, III and IV according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of HSV-1, HSV-2, VZV and CMV herpes virus.

7. Use of a polyketide of the general formulae I, II, III and IV according to any of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of keratitis, skin herpes caused by HSV-1 herpes virus infection; genital herpes caused by HSV-2 herpes virus infection; herpes zoster caused by VZV herpes virus infection; the application of the medicine for treating retinitis diseases caused by CMV herpes virus infection.