CN115819391A

CN115819391A - Baicalein derivative, preparation method and application thereof

Info

Publication number: CN115819391A
Application number: CN202210271409.1A
Authority: CN
Inventors: 沈敬山; 许叶春; 肖庚富; 蒋翔锐; 苏海霞; 谢航; 尚卫娟; 张苏晴; 王震; 张磊砢; 蒋华良
Original assignee: Shanghai Institute of Materia Medica of CAS; Wuhan Institute of Virology of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS; Wuhan Institute of Virology of CAS
Priority date: 2021-09-17
Filing date: 2022-03-18
Publication date: 2023-03-21
Also published as: WO2023040733A1

Abstract

The invention relates to a baicalein derivative, a preparation method and application thereof. The structure of the baicalein derivative is shown in the following general formula I. Pharmacological experiments prove that the baicalein derivative shows inhibitory activity on the novel coronavirus, and can be used as a novel coronavirus resistant medicament or a potential medicament for treating related diseases caused by the novel coronavirus.

Description

Baicalein derivatives, preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and relates to a baicalein derivative, a preparation method thereof and an application thereof in the aspect of antivirus.

Background

Baikal skullcap root, also known as camellia root and native gold tea root, is bitter in taste and cold in nature, has the efficacies of clearing heat, drying dampness, purging intense heat, detoxifying and the like, and is mainly used for treating warm diseases, upper respiratory tract infection, lung heat cough, pneumonia, dysentery, hemoptysis and other symptoms. The scutellaria baicalensis is an effective component in a plurality of heat-clearing and detoxifying Chinese patent medicines such as lung-clearing and detoxifying soup, golden flower cold-clearing granules, shuanghuanglian oral liquid and Lanqin oral liquid.

Baicalein is one of the main active ingredients of radix Scutellariae, and has antiviral effect. Literature reports that baicalein inhibits the EC of SARS virus replication ₅₀ Is 12.5 to 25 mu g/mL (Journa)l of Clinical Virology,2004,31,69-75); EC that inhibit replication of influenza A virus (H1N 1) ₅₀ 0.018. Mu.M (Evidence-Based comparative and Alternative Medicine,2013,750803); at the same time, baicalein also showed inhibitory activity against HIV and dengue virus (Molecules and Cells,2001,12,127-130, scientific Reports, 2014,4,5452). Baicalein has SARS-CoV-2 3CLpro inhibiting effect, and can be used for resisting SARS-CoV-2 replication EC on Vero E6 cell model infected with SARS-Cov-2 ₅₀ At 2.94. Mu.M (Acta Pharmacologa Sinica,2020,41,1167-1177).

Baicalein is well absorbed by gastrointestinal tract and mainly distributed in tissues and organs such as liver, kidney, lung, etc. The metabolite of baicalein in rat body is mainly the conjugate of baicalein 7-hydroxy glucuronic acid, which can be transported to intestinal cavity through bile and intestinal mucosa to form reabsorption. After oral administration of baicalein to rats, the original form of baicalein was hardly detectable in the blood, the elimination half-life was 0.1 hour, the plasma prototype drug concentration was extremely low, and the oral bioavailability was low (Journal of Chinese Mass Spectrometry Society,2004,25,129-133. The finding of baicalein derivatives with antiviral effect and better in vivo pharmacokinetic properties is of great significance.

In view of this, the invention is particularly proposed.

Disclosure of Invention

An object of the present invention is to provide a novel class of baicalein derivatives.

The invention further aims to provide application of the baicalein derivative in preparing medicaments for resisting coronavirus, particularly novel coronavirus.

The invention also aims to provide application of the baicalein derivative in preparing medicaments for resisting enterovirus EV71, coxsackievirus and norovirus.

In order to accomplish the above objects, the present invention provides a baicalein derivative represented by the following general formula I, a stereoisomer or a pharmaceutically acceptable salt thereof:

in the above general formula I, R ₁ And R ₂ Each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aralkyl, C7-C12 aralkyl,

Wherein the substituent of the C1-C6 alkyl is selected from cyano,

- O(CH ₂ ) _m OR ₁₂ 、-OC(＝O)OR ₁₃ And

R ₁₁ selected from C1-C6 alkyl, C3-C10 cycloalkyl; in particular, from C1-C4 alkyl, C3-C6 cycloalkyl;

R ₁₂ selected from C1-C6 alkyl, C3-C10 cycloalkyl; in particular, it is selected from C1-C4 alkyl, C3-6 cycloalkyl;

m represents 1, 2 or 3;

R ₁₃ selected from C1-C6 alkyl; in particular from C1-C4 alkyl;

wherein R is ₉ And R ₁₀ Each independently selected from H, C-C6 alkyl, by R ₁₄ OC (= O) -substituted C1-C10 alkyl (e.g. with R) ₁₄ OC (= O) -substituted C1-C6 alkyl); r ₁₄ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, in particular selected from C1-C4 alkyl, C5-C6 cycloalkyl;

in particular, R ₉ And R ₁₀ Each independently selected from H, C-C4 alkyl, C1-C4 alkoxy formyl substituted C1-C4 alkyl, C5-C6 cycloalkyl oxy formyl substituted C1-C4 alkyl;

in particular, it is possible to use, for example,

can be selected from

In particular, it is possible to use, for example,

can be selected from

In particular, it is possible to use, for example,

can be selected from

In particular, R ₁ And R ₂ May all be H;

R ₃ a substituent selected from H, cyano, halogen, nitro, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C2-C6 fatty acyl, C6-C12 arylalkyl, or C7-C12 aryloyl, wherein C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, or C2-C6 fatty acyl is selected from halogen and hydroxy; in particular, R ₃ A substituent selected from H, cyano, halogen, unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted C2-C4 alkenyl, unsubstituted or substituted C3-C4 cycloalkyl, or unsubstituted or substituted C2-C4 fatty acyl, wherein C1-C4 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl, or C2-C4 fatty acyl is selected from halogen and hydroxy; more particularly, R ₃ Selected from H, cyano, F, cl, br, I, unsubstituted or substituted C1-C3 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl, or C2-C4 fatty acyl, wherein the substituents of the C1-C3 alkyl group are selected from F, cl and hydroxyl, and the number of substituents can be one or more, for example 1, 2 or 3; more particularly, R ₃ Selected from the group consisting of H, cyano, F, cl, br, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, acetyl, trifluoromethyl, cyclopropyl, hydroxymethyl, vinyl, 1-propenyl, 2-propenyl, allyl, nitro;

R ₄ -R ₈ each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ Cyano, C6-C10 aryl which is unsubstituted or substituted by C1-C4 alkyl, C2-C6 fatty acyl, wherein the substituent of C1-C6 alkyl is halogen, R ₁₅ And R ₁₆ Each independently selected from H and C2-C6 fatty acyl; in particular, R ₄ -R ₈ Each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ Cyano, C6-C10 aryl which is unsubstituted or substituted by methyl or ethyl, wherein the substituent of C1-C6 alkyl is halogen, R ₁₅ And R ₁₆ Each independently selected from H and C2-C4 fatty acyl; more particularly, R ₄ -R ₈ Each independently selected from H, halogen, C1-C3 alkyl substituted with one or more (e.g. 1, 2 or 3) F, nitro, amino, acetamido, cyano, phenyl, tolyl, ethylphenyl; more particularly, R ₄ -R ₈ Each independently selected from H, cyano, halogen, trifluoromethyl, methyl, ethyl, nitro, amino, acetamido, phenyl, tolyl;

represents a single bond or a double bond;

provided that R is ₁ -R ₈ Not simultaneously H, and compounds of formula I are excluded

In a specific embodiment, the baicalein derivative of formula I is represented by the following formulae II-IV:

in the above general formulae II-III, R ₄ -R ₈ Are as defined above, respectively; in the above formula IV, except R ₃ Not being other than H, R ₃ And R ₄ -R ₈ Are as defined above.

For example,

in a particular embodiment of the method of the present invention,

R ₁ and R ₂ Each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aralkyl, C7-C12 aralkyl,

- SO ₂ OH、

Wherein the substituent of the C1-C6 alkyl is selected from cyano,

- O(CH ₂ ) _m OR ₁₂ 、-OC(＝O)OR ₁₃ And

R ₁₁ selected from C1-C6 alkyl;

R ₁₂ selected from C1-C6 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ selected from C1-C6 alkyl;

wherein R is ₉ And R ₁₀ Each independently selected from H, C-C6 alkyl, by R ₁₄ OC (= O) -substituted C1-C10 alkyl; r is ₁₄ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, in particular R ₁₄ Selected from C1-C4 alkyl, C5-C6 cycloalkyl;

R ₃ a substituent selected from H, cyano, halogen, nitro, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C2-C6 fatty acyl, C6-C12 arylalkyl, or C7-C12 aryloyl, wherein C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, or C2-C6 fatty acyl is selected from halogen and hydroxy;

R ₄ -R ₈ each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ Cyano, C6-C10 aryl which is unsubstituted or substituted by C1-C4 alkyl, C2-C6 fatty acyl, wherein the substituent of C1-C6 alkyl is halogen, R ₁₅ And R ₁₆ Each independently selected from H and C2-C6 fatty acyl;

represents a single bond or a double bond.

In one embodiment of the present invention, the substrate is,

- SO ₂ OH、

Wherein the substituent of the C1-C6 alkyl is selected from cyano,

- O(CH ₂ ) _m OR ₁₂ 、-OC(＝O)OR ₁₃ And

R ₁₁ selected from C1-C6 alkyl;

R ₁₂ selected from C1-C6 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ selected from C1-C6 alkyl;

wherein R is ₉ And R ₁₀ Each independently selected from H, C-C6 alkyl, by R ₁₄ OC (= O) -substituted C1-C10 alkyl; r ₁₄ Selected from C1-C6 alkyl, C3-C8 cycloalkyl, in particular selected from C1-C4 alkyl, C5-C6 cycloalkyl;

R ₃ selected from H, cyano, F, cl, br, I, unsubstituted or substituted C1-C3 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl, or C2-C4 fatty acyl, wherein the substituents of C1-C3 alkyl are selected from F, cl and hydroxyl, and the number of substituents can be one or more, for example, 1, 2 or 3;

R ₄ -R ₈ each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ Cyano, C6-C10 aryl which is unsubstituted or substituted by methyl or ethyl, wherein the substituent for C1-C6 alkyl is halogen, R ₁₅ And R ₁₆ Each independently selected from H and C2-C4 fatty acyl;

represents a single bond or a double bond.

In a particular embodiment of the method of the present invention,

R ₁ and R ₂ Are all H;

represents a single bond or a double bond.

In a particular embodiment of the method of the present invention,

R ₁ and R ₂ Are all H;

R ₃ selected from H, cyano, F, cl, br, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, acetyl, trifluoromethyl, cyclopropyl, hydroxymethyl, vinyl, 1-propenyl, 2-propenyl, allyl, nitro;

R ₄ -R ₈ each independently selected from H, cyano, halogen, trifluoromethyl, methyl, ethyl, nitro, amino, acetamido, phenyl, tolyl;

represents a single bond or a double bond.

In a specific embodiment, the baicalein derivative represented by the general formula I of the present invention is selected from one of the following compounds:

in another aspect, the present invention also provides a method for preparing the baicalein derivative represented by the above general formula I, which is carried out by one of the routes comprising the steps of:

route one:

a) The compound I '-1 reacts with iodine in dimethyl sulfoxide under inert atmosphere such as nitrogen atmosphere at 0-150 ℃ for 1-36h to generate a compound I' -1,

b) Compound I' -1 and BBr ₃ Reacting in dichloromethane to generate a compound I-1,

wherein R is ₁ 、R ₂ Is C1-C6 alkyl, R ₃ -R ₈ Each as defined hereinbefore;

or

And a second route:

c) Reacting the intermediate I '-1 with anhydrous sodium acetate in anhydrous ethanol under inert atmosphere such as nitrogen atmosphere at 0-100 deg.C for 1-24h to obtain compound I' -2;

d) Compound I' -2 and BBr ₃ In dichloromethane to produce compound I-2, wherein R ₁ 、R ₂ Is C1-C6 alkyl, R ₃ -R ₈ Each as defined hereinbefore;

or

When R in the formula I ₃ Selected from CN, CF ₃ Cyclopropyl and halogen, alternatively, by the following route:

and a third route:

e) Reacting the compound of formula I '-2 with a halogenating agent (such as N-chlorosuccinimide (NCS) or N-iodosuccinimide (NIS)) in acetonitrile or trifluoroacetic acid at 0-100 deg.C for 1-24h to obtain a compound of formula I' -3;

f) Reacting the compound of the general formula I '-3 in DMF in the presence of methyl fluorosulfonyl difluoroacetate and cuprous iodide at 0-130 ℃ for 1-15h to obtain the compound of the general formula I' -4 (R) ₃ = trifluoromethyl); or in 1,4-dioxane, in PdCl ₂ (DPPF)-CH ₂ Cl ₂ Reacting in the presence of potassium carbonate and cyclopropyl borate at 0-120 ℃ for 1-48h in an inert atmosphere such as nitrogen atmosphere to obtain the compound (R) with the general formula I' -4 ₃ = cyclopropyl group),

g) A compound of formula I' -4 with BBr ₃ Mixing and reacting at-78 ℃ in dichloromethane under an inert atmosphere, such as nitrogen, at 0-50 ℃ to form the compound of formula I-3,

wherein R is ₁ 、R ₂ Is C1-C6 alkyl, R ₄ -R ₈ Each as defined hereinbefore, R ₃ Selected from trifluoromethyl, cyclopropyl;

or

f1 A compound of a general formula I '-3 reacts for 1 to 28 hours at 0 to 150 ℃ in the presence of dichlorodicyanoquinone (DDQ), copper acetate, silver carbonate and NMP to obtain a compound of a general formula I' -5,

g1 A compound of the formula I' -5 with BBr ₃ Mixing and reacting at-78 ℃ in dichloromethane under an inert atmosphere, such as nitrogen, at 0-50 ℃ to form the compound of formula I-4,

wherein R is ₁ 、R ₂ And R ₄ -R ₈ Each as defined hereinbefore, R ₃ Is cyano;

or

f2 A compound of the formula I' -3 with BBr ₃ Mixing and reacting at-78 ℃ in dichloromethane under an inert atmosphere, such as nitrogen, at 0-50 ℃ to form the compound of formula I-5,

wherein R is ₁ 、R ₂ Is C1-C6 alkyl, R ₄ -R ₈ Each as defined hereinbefore, R ₃ Is a halogen, and the halogen is a halogen,

in particular, formula I' -1 is obtained by the following reaction:

reacting the compound of the general formula I-A with the compound of the general formula I-B in methanol under alkaline conditions, such as 10-50% sodium hydroxide or potassium hydroxide aqueous solution, at-20-50 ℃ for 0.1-24h to obtain the compound of the general formula I' -1.

In a particular embodiment, when R ₃ When methyl, the compounds of formula I-A are obtained using the following reaction scheme:

h) Reacting Compounds I-E with CHCl ₂ OMe in TiCl in dichloromethane ₄ Reacting in the presence of ice-water bath to obtain a compound I-D,

i) Reacting the compound I-D with trifluoroacetic acid at 0-50 ℃ in the presence of triethylsilane to obtain a compound I-C,

j) Reacting the compound I-C with boron trifluoride diethyl etherate and acetic acid at 0-100 ℃ to obtain a compound I-A.

In a particular embodiment, when R ₃ In the case of acetyl, the compounds of formula I-A are obtained using the following reaction scheme:

mixing the compound I-F, acetic anhydride and nitromethane with aluminum trichloride at the temperature of-20-20 ℃, and reacting for 1-24h at the temperature of 0-50 ℃ to obtain the compound I-A.

Preferably, the present invention also provides a method for preparing the baicalein derivatives of the above general formulae II-IV,

as shown in the above reaction formula, the method comprises:

1) The initial reactant I1 reacts with benzaldehyde compounds to generate an intermediate I1',

2) The intermediate I1 'reacts with iodine simple substance in dimethyl sulfoxide to generate a compound with a general formula II',

3) A compound of the formula II' with BBr ₃ Reacting in dichloromethane to generate a compound of a general formula II;

or alternatively

2) Reacting the intermediate I1 'with anhydrous sodium acetate in anhydrous ethanol to generate a compound with a general formula III';

3) Compounds of the formula III' with BBr ₃ Reacting in dichloromethane to generate a compound shown in a general formula III;

further, the compound of formula II or II' is reacted with a reactive agent, such as a halogenating agent (e.g., N-chlorosuccinimide (NCS) or N-iodosuccinimide (NIS)), dichlorodicyanoquinone (DDQ), cyclopropylboronic acid ester, or methyl Fluorosulfonyldifluoroacetate (FSO) ₂ CF ₂ COOCH ₃ ) Formed at R) by reaction ₃ A compound of formula IV 'or formula IV substituted in position, a compound of formula IV' and BBr ₃ Reaction in dichloromethane produces the compound of formula IV.

In a particular embodiment, when R is prepared ₃ In the case of the compound of formula IV, which is a methyl group, the product prepared according to the above reaction scheme is used in place of I1 in the above reaction scheme and further the same procedure as in the synthesis of the compound of formula II is carried out.

Specifically, as shown in the above reaction scheme:

1) Mixing 3,4,5-trimethoxyphenol with CHCl ₂ OMe in TiCl in dichloromethane ₄ Reacting in the presence of ice-water bath;

2) Reacting the reaction product of the step 1) with trifluoroacetic acid at room temperature in the presence of triethylsilane;

3) Reaction of the reaction product of step 2), boron trifluoride etherate and acetic acid at, for example, 95 ℃ and subsequent preparation of R ₃ A compound of formula IV which is methyl;

in a particular embodiment, when R is prepared ₃ In the case of compounds of formula IV, which are acetyl groups, the product prepared according to the following reaction scheme is used instead of I1 in the above reaction scheme and further the same steps as the synthesis of compounds of formula II are carried out:

specifically, as shown in the above reaction scheme:

mixing 3,4,5-trimethoxyphenol, acetic anhydride and nitromethane with aluminum trichloride at 0 ℃ for reaction for 1-24h at 0-50 ℃, and using the obtained reaction product for subsequent preparation of R ₃ A compound of formula IV which is acetyl.

In a particular embodiment, when R is prepared ₃ In the case of compounds of the general formula IV, which are ethyl or isobutyl, for example compounds Ic10 and Ic11, the synthetic route is as follows:

the compound 3,4,5-trimethoxyphenol and acetic acid or isobutyryl chloride are subjected to acylation reaction in the presence of boron trifluoride ethyl ether; then, carrying out reduction reaction under the action of trifluoroacetic acid and triethylsilane; the obtained reaction product is subjected to acetylation reaction in the presence of boron trifluoride ethyl ether and acetic acid, and then reacts with o-chlorobenzoyl chloride to generate corresponding ester; the generated ester is subjected to rearrangement reaction under the action of alkali, the obtained rearrangement crude product is further subjected to ring closure reaction under the action of strong acid (such as concentrated sulfuric acid), and the ring-closed product is demethylated under the action of boron tribromide to obtain a final product Ic10 or Ic11, wherein R is methyl or isopropyl.

In a specific embodiment, the synthesis of a specific compound Ic18 is as follows:

the methylation product of the compound Ic15 of the present application undergoes a reduction reaction under the action of stannous chloride dihydrate to generate a corresponding amino compound, acetic anhydride is added to the reaction product to undergo an acetylation reaction to generate a corresponding amide compound, and the amide compound undergoes a demethylation reaction under the action of boron tribromide to generate the compound Ic18.

In a particular embodiment, when R is prepared ₃ In the case of the isopropenyl compound of formula IV, for example compound Ic20, the synthetic route is as follows:

the methylation product of compound Ic1 of the present application was deiodinated in the presence of potassium carbonate, xphos, potassium fluoride and palladium acetate to give a deiodinated product which was obtained at BBr ₃ Demethylation to give the Ic20 compound described above.

Route four

R in the general formula I is shown below ₁ And R ₂ At least one of which is not H, R ₃ Synthetic routes to compounds that are methyl (e.g., compounds Id8-Id44 above):

dissolving the compound of the general formula Id' in an organic solvent, and reacting with the compound with R as defined above under the action of alkali ₁ /R ₂ Corresponding radicalReacting the reactants of the group to obtain a compound of the general formula Id, in the above reaction formula, R ₁ 、R ₂ 、R ₄ -R ₈ Are as defined above, respectively.

In specific embodiments, the organic solvent is selected from acetonitrile, acetone, N-Dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), and N-methylpyrrolidone (NMP); the base is selected from triethylamine, potassium carbonate, N-diisopropylethylamine and pyridine; said has R ₁ /R ₂ Reactants for the corresponding group are selected from ethyl acetoacetonate, isopropyl acetoacetate, ethyl 3-methylbutyrate-2-isocyanate, isopropyl propionate-2-isocyanate, cyclohexyl isocyanatoacetate, cyclopentylacetoacetate, 4-chloromethyl-5-methyl-1,3-dioxol-2-one, isopropyl chlorocarbonate, bromoacetonitrile, chlorosulfonic acid, isobutyric anhydride, 2-methoxyethoxymethyl chloride, N' -dimethylcarbamoyl chloride, ethyl isocyanate and methylcarbamoyl chloride.

In the case of compounds of general formula I Id8 and Id9 above, the synthesis can be illustrated by the following equations:

the Id' compound is first reacted with acetic anhydride, followed by benzyl bromide to protect the hydroxyl group, followed by Pd (OH) ₂ In the presence of C, with H ₂ Reducing, reacting the reaction product with acetyl bromide-alpha-D-glucuronic acid methyl ester or acetyl bromide-alpha-D-glucose, and hydrolyzing ester group in the compound with alkali, wherein the synthesis conditions in the above reaction steps are well known in the art, and in the above reaction formula, R is ₄ -R ₈ Are as defined above, respectively.

In still another aspect, the present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of one or more selected from the group consisting of the baicalein derivatives, stereoisomers thereof, and pharmaceutically acceptable salts thereof as a pharmaceutically active ingredient, and optionally pharmaceutically acceptable excipients.

In still another aspect, the present invention provides the use of the baicalein derivative, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition for the preparation of a medicament for inhibiting a novel coronavirus main protease.

In still another aspect, the present invention further provides the use of the baicalein derivative, the stereoisomer or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a medicament, wherein the medicament has an activity of inhibiting coronavirus main protease, or the medicament has an activity against a novel coronavirus and a variant thereof (such as WIV strain or south african strain b.1.351), SARS virus and MERS virus, or the medicament has an activity against enterovirus EV71, coxsackie virus and norovirus.

The technical scheme of the invention at least has the following technical effects:

the compound of the invention has stronger SARS-CoV-2 3CLpro inhibitory activity, stronger anti-novel coronavirus replication function and better pharmacokinetic property, shows obvious anti-novel coronavirus drug effect in a mouse body, and can be used for preventing and treating diseases caused by the novel coronavirus.

The preparation method has the technical advantages of simple steps, high yield and easily obtained raw materials.

Drawings

FIG. 1: a plot of the inhibitory activity of compound Ia7 on SARS-CoV-2 3CLpro.

FIG. 2: a graph of the inhibitory activity of Compound Ib1 against SARS-CoV-2 3CLpro.

FIG. 3: a plot of the inhibitory activity of Compound Ib2 against SARS-CoV-2 3CLpro.

FIG. 4: a graph of the inhibitory activity of compound Ib3 on SARS-CoV-2 3CLpro.

FIG. 5 is a schematic view of: a plot of the inhibitory activity of compound Ic3 against SARS-CoV-2 3 CLpro.

FIG. 6: a plot of the inhibitory activity of compound Ic4 against SARS-CoV-2 3CLpro.

FIG. 7: a profile of inhibitory activity of compound Ic5 against SARS-CoV-2 3CLpro.

FIG. 8: the compound Ic5 has an in vitro inhibitory activity against SARS-CoV-2 WIV04 strain.

FIG. 9: compound Ic5 has inhibitory activity against SARS-CoV-2 south Africa strain B.1.351 in vitro.

FIG. 10: effect of compound Ic5 on the activity of Vero E6 cells.

FIG. 11: inhibitory Activity of Compounds Ic5 and Id16 on SARS-CoV-2 Virus replication in mice.

Detailed Description

In the following detailed description, numerous specific details are set forth in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details.

Radical definition

The term "alkyl" denotes straight or branched chain alkyl groups, e.g. "C1-Cx alkyl" denotes straight or branched chain alkyl groups containing 1-x carbon atoms. For example, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. For example, "C1-C6 alkyl" denotes straight-chain or branched alkyl having 1 to 6 carbon atoms.

The term "alkenyl" denotes straight or branched alkenyl groups, e.g., C2-C6 alkenyl denotes straight or branched alkenyl groups containing 2-6 carbon atoms, such as vinyl, 1-propenyl, 2-propenyl, allyl, 1-butenyl, 2-butenyl, isobutenyl and the like

The term "C2-C6 fatty acyl" denotes acyl groups having 2 to 6 carbon atoms, for example, including, but not limited to, acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, tert-hexanoyl, and the like.

The term "arylalkyl" denotes a substituent having a benzene ring and "C6-C12 arylalkyl" denotes a substituent having 6-12 carbon atoms, preferably "C6-C12 aryl", which denotes an aryl group having 6-12 carbon atoms. For example, including but not limited to phenyl, substituted phenyl, naphthyl, substituted naphthyl, and the like.

The term "C7-C12 aroyl" denotes an aroyl group having 7-12 carbon atoms, including, for example and without limitation, benzoyl, methylbenzoyl, ethylbenzoyl, naphthoyl, and the like.

The term "halogen" is selected from fluorine, chlorine, bromine, iodine.

For a further understanding of the present invention, the following examples are given to illustrate further features of the present invention and are not intended to limit the scope of the present invention or the scope of the claims. The invention is protected by the technical solution that those skilled in the art make simple substitutions or improvements to the invention.

I. Preparation of intermediates

Preparation example 1: synthesis of intermediate I1

3g of 3,4,5-trimethoxyphenol and 6mL of BH ₃ ·OEt ₂ Adding the mixture into a 50mL three-necked bottle, replacing the nitrogen for three times, slowly dropwise adding 9mL of acetic acid into the reaction solution, gradually heating to 80 ℃, and reacting for 2 hours. The reaction solution was slowly added to 30mL of ice water, stirred for 2 hours, filtered, and dried to obtain 2.8g of a yellow solid with a yield of 76%. ¹ H NMR(600MHz, Chloroform-d)δ7.26(s,1H),6.28(s,1H),4.10(s,3H),3.97(s,3H), 3.77(s,3H),2.81(s,3H).MS(ESI,ev):m/z＝227.2[M+H] ⁺ 。

Preparation example 2: synthesis of intermediate I2

Adding 1g of baicalein, 2.5g of potassium carbonate and 10mL of acetone into a 50mL two-neck bottle, adding 1.6g of dimethyl sulfate, gradually heating to 60 ℃, and reacting for 15h. Cooling the reaction solution to 25 deg.C, filtering the reaction solution, distilling under reduced pressure to remove acetone, adding 50mL ethyl acetate and 50mL saturated sodium bicarbonate solution, stirring for 5min, demixing, adding organic phase50mL and saline solution are stirred for 5min, then the layers are separated, the organic phase is dried for 1h by anhydrous sodium sulfate, and the mixture is concentrated to obtain a light yellow solid 920mg with the yield of 80%. ¹ H NMR(600MHz,DMSO-d6)δ 8.10-8.03(m,2H),7.62-7.54(m,3H),7.24(s,1H),6.82(s,1H),3.96(s, 3H),3.81(s,3H),3.77(s,3H).MS(ESI,ev):m/z＝313.1[M+H] ⁺ 。

Preparation example 3: synthesis of intermediate I3

1) 226mg of I1 and 5mL of methanol were put into a25 mL single-neck flask, and 140 mg of 2-chlorobenzaldehyde and 3mL of 33% aqueous sodium hydroxide solution were added to react at room temperature and 25 ℃ for 12 hours. Distilling under reduced pressure to remove methanol, adding 30mL diluted hydrochloric acid and 30mL ethyl acetate, stirring for 5min, demixing, adding 30mL saturated saline solution to the organic phase, stirring for 5min, demixing, drying the organic phase with anhydrous sodium sulfate for 1h, and performing column chromatography (SiO) ₂ Ethyl acetate = 10) to give 300mg of product as an orange solid in 82% yield.

2) 300mg of the reaction product in the previous step, 197mg of iodine simple substance and 5mL of dimethyl sulfoxide are added into a25 mL single-neck bottle, nitrogen is replaced for three times, the temperature is gradually increased to 110 ℃, and the reaction is carried out for 3 hours. Adding 10mL of ethyl acetate and 10mL of water, stirring for 5min, separating the layers, adding 10mL of organic phase and saline, stirring for 5min, separating the layers, and drying the organic phase with anhydrous sodium sulfate for 1h. By column chromatography (SiO) ₂ Ethyl acetate = 10) to yield 150mg of white solid product in 50% yield.

3) 150mg of the reaction product of the previous step and 2mL of methylene chloride were charged into a 10mL three-necked flask, nitrogen was purged three times, the temperature of the reaction solution was lowered to-78 deg.C, and 4mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Slowly adding methanol into the reaction solution under ice water bath condition for quenching, layering the system, concentrating the organic phase, and performing column chromatography (SiO) ₂ Dichloromethane methanol = 30) gave 50mg brown solid product in 37% yield. ¹ H NMR(500MHz,DMSO-d6)δ12.50(s,1H),10.66 (s,1H),8.89(s,1H),7.77(dd,J＝7.6,1.7Hz,1H),7.68(dd,J＝8.1,1.2 Hz,1H),7.61(td,J＝7.8,1.7Hz,1H),7.54(td,J＝7.5,1.3Hz,1H),6.53 (s,1H),6.52(s,1H).MS(ESI,ev):m/z＝305.01[M+H] ⁺ 。

Preparation of the Compounds

Example 1: synthesis of Compound Ia1

According to the method of preparation example 3 except for using I1 and 3-cyanobenzaldehyde as raw materials, the three-step reaction yield was 15%. ¹ H NMR(500MHz,DMSO-d6)δ12.61(s,1H), 10.60(s,1H),8.84(s,1H),8.49(d,J＝2.1Hz,1H),8.31(d,J＝7.9Hz, 1H),8.14(d,J＝7.8Hz,1H),7.71(s,1H),6.99(s,1H),6.64(s,1H).MS (ESI,ev):m/z＝296.05[M+H] ⁺ 。

Example 2: synthesis of Compound Ia2

The procedure of preparation example 3 was followed except that I1 and 3-iodobenzaldehyde were used as starting materials, and the three-step reaction yield was 17%. ¹ H NMR(500MHz,Chloroform-d)δ8.27(d,J ＝1.8Hz,1H),7.87(dd,J＝15.4,7.9Hz,2H),7.27(d,J＝7.8Hz,1H), 6.88(s,1H),6.79(d,J＝17.0Hz,1H).MS(ESI,ev):m/z＝297.13[M+ H] ⁺ 。

Example 3: synthesis of Compound Ia3

The procedure of preparation example 3 was followed except that I1 and 2-chloro-3-fluorobenzaldehyde were used as starting materials, and the three-step reaction yield was 19%. ¹ H NMR(500MHz,DMSO-d6)δ12.44(s, 1H),10.66(s,1H),8.88(s,1H),7.71–7.63(m,2H),7.59(td,J＝8.0,5.2 Hz,1H),6.58(s,1H),6.52(s,1H).MS(ESI,ev):m/z＝323.67[M+ H] ⁺ 。

Example 4: synthesis of Compound Ia4

According to the method of preparation example 3, except for using I1 and 2-chloro-5-fluorobenzaldehyde as raw materials, the three-step reaction yield was 13%. ¹ H NMR(500MHz,DMSO-d6)δ12.44(s, 1H),10.66(s,1H),8.88(s,1H),7.79–7.68(m,2H),7.57–7.41(m,1H), 6.58(s,1H),6.53(s,1H).MS(ESI,ev):m/z＝323.64[M+H] ⁺ 。

Example 5: synthesis of Compound Ia5

According to the method of preparation example 3 except for using I1 and 2-trifluoromethylbenzaldehyde as raw materials, the three-step reaction yield was 16%. ¹ H NMR(500MHz,DMSO-d6)δ12.46(s, 1H),10.64(s,1H),7.98(d,J＝7.6Hz,1H),7.87(d,J＝4.0Hz,2H),7.83 (dt,J＝8.9,4.6Hz,1H),6.53(s,1H),6.45(s,1H).MS(ESI,ev):m/z ＝339.04[M+H] ⁺ 。

Example 6: synthesis of Compound Ia6

According to the method of preparation example 3 except for using I1 and 2-ethylbenzaldehyde as raw materials, the three-step reaction yield was 14%. ¹ H NMR(500MHz,DMSO-d6)δ12.60(s,1H), 10.54(s,1H),7.57-7.46(m,2H),7.44–7.39(m,2H),7.36(td,J＝7.5,1.3 Hz,2H),6.50(s,1H),6.39(s,1H),2.76–2.65(m,2H),1.19(td,J＝7.5, 4.2Hz,3H).MS(ESI,ev):m/z＝299.08[M+H] ⁺ 。

Example 7: synthesis of Compound Ia7

The procedure of preparation example 3 was followed except that I1 and 2,6-dichlorobenzaldehyde were used as starting materials, and the three-step reaction yield was 20%. MS (ESI, ev) m/z =229.98[ M + H ]] ⁺ 。

Example 8: synthesis of Compound Ib1

1) 1g of I1 and 20mL of methanol were put into a 50mL single-neck flask, and 296mg of 3-nitrobenzaldehyde and 12mL of 33% aqueous sodium hydroxide were added to react at room temperature and 25 ℃ in 12h. Distilling under reduced pressure to remove methanol, adding 50mL diluted hydrochloric acid and 50mL ethyl acetate, stirring for 5min, separating layers, adding 100mL saturated saline solution to organic phase, stirring for 5min, separating layers, drying organic phase with anhydrous sodium sulfate for 1h, and performing column chromatography (SiO) ₂ Ethyl acetate = 10) to give 1.02g of product as an orange solid in 64% yield.

2) 100mg of the product prepared in the previous step, 159mg of anhydrous sodium acetate and 15mL of anhydrous ethanol were put into a25 mL single-necked flask, replaced with nitrogen three times, gradually warmed to 70 ℃ and reacted for 13 hours. Concentrating under reduced pressure, and purifying by column chromatography (SiO) ₂ Ethyl acetate = 10) to yield 50mg of product as an orange solid in 50% yield.

3) 50mg of the product of the above one-step preparation and 1mL of dichloromethane were charged into a 10mL three-necked flask, nitrogen was replaced three times, the temperature of the reaction mixture was lowered to-78 deg.C, and 0.5mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Slowly adding methanol into the reaction solution under ice water bath condition for quenching, layering the system, concentrating the organic phase, and performing column chromatography (SiO) ₂ Dichloromethane: methanol = 30) gave 25mg of the product as a brown solid in 56% yield. ¹ H NMR(500MHz,DMSO-d6)δ11.92(s,1H), 10.48(s,1H),8.38(t,J＝2.0Hz,1H),8.33–8.21(m,2H),7.99(d,J＝7.7 Hz,1H),7.74(t,J＝8.0Hz,1H),6.03(s,1H),5.71(dd,J＝12.7,3.1Hz, 1H),3.28–3.22(m,1H),2.87(dd,J＝17.1,3.1Hz,1H).MS(ESI,ev): m/z＝318.06[M+H] ⁺ 。

Example 9: synthesis of Compound Ib2

The procedure of example 8 was followed, except that I1 and 2-chloro-5-fluorobenzaldehyde were used as starting materials, and the three-step reaction yield was 18%. ¹ H NMR(500MHz,DMSO-d6)δ11.87(s, 1H),7.59(dt,J＝8.6,3.9Hz,2H),7.32(td,J＝8.5,3.1Hz,1H),6.01(s, 1H),5.72(dd,J＝13.2,2.8Hz,1H),3.27–3.21(m,1H),2.74(dd,J＝ 17.1,2.9Hz,1H).MS(ESI,ev):m/z＝325.68[M+H] ⁺ 。

Example 10: synthesis of Compound Ib3

According to the method of example 8 except for using I1 and 3-trifluoromethylbenzaldehyde as raw materials, the three-step reaction yield was 13%. ¹ H NMR(500MHz,DMSO-d6)δ11.93(s, 1H),10.45(s,1H),8.26(s,1H),7.91–7.80(m,2H),7.76(d,J＝7.8Hz, 1H),7.68(t,J＝7.5Hz,1H),6.01(s,1H),5.73–5.59(m,1H),3.24(s, 1H),2.81(dd,J＝17.1,2.9Hz,1H).MS(ESI,ev):m/z＝341.25[M+ H] ⁺ 。

Example 11: synthesis of Compound Ib4

According to the method of example 8, except for using I1 and 2-trifluoromethylbenzaldehyde as raw materials, the three-step reaction yield was 17%. ¹ H NMR(500MHz,DMSO-d6)δ11.88(s,1H), 10.49(s,1H),8.32(s,1H),8.02(d,J＝7.9Hz,1H),7.83(t,J＝8.0Hz, 3H),7.66(t,J＝7.8Hz,1H),5.98(s,1H),2.63(dd,J＝17.1,2.8Hz,2H). MS(ESI,ev):m/z＝341.20[M+H] ⁺ 。

Example 12: synthesis of Compound Ib5

According to the method of example 8 except for using I1 and 2-ethylbenzaldehyde as raw materials, the three-step reaction yield was 16%. ¹ H NMR(500MHz,DMSO-d6)δ12.62(s,1H), 10.56(s,1H),8.88(s,1H),7.59(dd,J＝7.4,2.0Hz,1H),7.38–7.26(m, 3H),6.51(s,1H),5.80–5.73(m,1H),3.16(dd,J＝16.7,13.5Hz,1H), 2.72(dd,J＝9.0,7.4Hz,2H),2.59(dd,J＝16.6,2.7Hz,1H),1.18(t,J＝ 7.5Hz,3H).MS(ESI,ev):m/z＝301.3[M+H] ⁺ 。

Example 13: synthesis of Compound Ib6

The procedure of example 8 was followed, except that I1 and 2-bromobenzaldehyde were used as starting materials, and the three-step reaction yield was 17%. ¹ H NMR(500MHz,DMSO-d6)δ12.50(s,1H), 11.43(s,1H),8.07–7.96(m,1H),7.78–7.66(m,1H),7.53–7.43(m,1H), 7.23–7.14(m,1H),5.94(s,1H).MS(ESI,ev):m/z＝399.1[M+H] ⁺ 。

Example 14: synthesis of Compound Ib7

The procedure of example 8 was followed, except that I1 and 2-iodobenzaldehyde were used as starting materials, to give a reaction yield in three steps of 18%. ¹ H NMR(500MHz,DMSO-d6)δ8.08–7.94(m, 3H),7.82(dd,J＝15.5,10.1Hz,1H),7.77–7.71(m,1H),7.50(t,J＝7.6 Hz,1H),7.22–7.12(m,1H),5.95(s,1H).MS(ESI,ev):m/z＝399.1[M +H] ⁺ 。

Example 15: synthesis of Compound Ic1

1) 62mg of I2 prepared in preparation example 2, 54mg of N-iodosuccinimideAnd 1mL of trifluoroacetic acid were added to a 10mL single-necked flask, and the temperature was gradually increased to 70 ℃ to react with 4h. Distilling the reaction solution under reduced pressure to small volume, adding 10mL ethyl acetate and 10mL saturated sodium bicarbonate solution, stirring for 5min, separating layers, adding 10mL saturated saline solution into organic phase, stirring for 5min, separating layers, drying organic phase with anhydrous sodium sulfate for 1h, and performing column chromatography (SiO) ₂ Ethyl acetate = 10) to give the product as a white solid.

2) 100mg of the reaction product of the previous step and 2mL of dichloromethane were added to a 10mL three-necked flask, replaced with nitrogen three times, the temperature of the reaction solution was lowered to-78 deg.C, and 1.4mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Slowly adding methanol into the reaction solution under ice water bath condition for quenching, layering the system, concentrating the organic phase, and performing column chromatography (SiO) ₂ Dichloromethane methanol = 30) gave 70mg yellow solid in 55% yield. ¹ H NMR(500MHz,DMSO-d6)δ8.23-8.12(m,2H), 7.66-7.54(m,3H),6.94(s,1H).MS(ESI,ev):m/z＝396.8[M+H] ⁺ 。

Example 16: synthesis of Compound Ic2

1) 200mg of the product of step 1) of example 15, 114mg of dichlorodicyanobenzoquinone (DDQ), 92mg of copper acetate, 190mg of silver carbonate and 6mL of NMP were added to a 10mL sealed tube, and the temperature was gradually raised to 120 ℃ to react for 20 hours. Cooling the reaction solution to 25 ℃, filtering the reaction solution, adding the filtrate into 20mL of water, stirring for 30min, filtering, and drying the solid to obtain 80mg.

2) 80mg of the reaction product prepared in the previous step and 2mL of dichloromethane were added to a 10mL three-necked flask, nitrogen was replaced three times, the temperature of the reaction solution was lowered to-78 deg.C, and 1.3mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Slowly adding methanol into the reaction solution under ice water bath condition for quenching, layering the system, concentrating the organic phase, and performing column chromatography (SiO) ₂ Dichloromethane methanol = 30) to give 50mg brown solid, which was collectedThe ratio was 71.4%. ¹ H NMR(500MHz,DMSO-d6)δ13.21(s,1H), 9.74(s,1H),8.07(s,2H),7.63(s,3H),7.15(s,1H).MS(ESI,ev):m/z ＝296.1[M+H] ⁺ 。

Example 17: synthesis of Compound Ic3

In a 10mL single-necked flask, 18mg of I3, 8mg of N-chlorosuccinimide and 2mL of acetonitrile were introduced, and the temperature was gradually raised to 40 ℃ to conduct a reaction for 24 hours. Distilling the reaction solution under reduced pressure, adding 3mL dichloromethane, pulping for 2h, filtering, and drying to obtain a yellow solid. ¹ H NMR(500MHz, DMSO-d6)δ12.59(s,1H),11.03(s,1H),9.66(s,1H),7.81(dt,J＝7.6, 2.3Hz,1H),7.70(dd,J＝8.0,1.3Hz,1H),7.64(td,J＝7.7,1.7Hz,1H), 7.56(dd,J＝7.5,1.3Hz,1H),6.68(s,1H).MS(ESI,ev):m/z＝321.1[M +H] ⁺ 。

Example 18: synthesis of Compound Ic4

The one-step reaction yield was 87% by the method of example 17, except that Ia7 was used as a starting material. MS (ESI, ev) m/z =373.93[ m + H ]] ⁺ 。

Example 19: synthesis of Compound Ic5

1) 4.02g of 3,4, 5-trimethoxyphenol and 50mL of methylene chloride were added to a 100mL two-necked flask, and 9.10g of TiCl was slowly added dropwise under ice-water bath conditions ₄ After stirring for 1h, 3.26g of CHCl were slowly added dropwise ₂ OMe, reaction for 1h. Adding saturated ammonium chloride solution for quenching, adding 100mL dichloromethane, stirring for 5min, demixing, adding 100mL saturated saline solution to the organic phase, stirring for 5min, demixing, drying the organic phase with anhydrous sodium sulfate for 1h, and purifying by column chromatography to obtain 3.58g solid product.

2) 3.57g of the reaction product of the previous step and 30mL of trifluoroacetic acid were added to a 50mL two-necked flask, and 89g of triethylsilane was slowly added dropwise thereto and reacted at room temperature for 1.5 hours. The reaction solution was distilled under reduced pressure and purified by column chromatography to obtain 3g of a solid product.

3) 2.72g of the reaction product of the previous step, 10mL of boron trifluoride diethyl etherate and 15mL of acetic acid were charged into a 50mL two-necked flask, and the temperature was gradually raised to 95 ℃ for 2 hours. The reaction solution was cooled to 25 ℃, slowly added to 150mL of ice water, stirred for 2 hours, filtered and dried to obtain 3.2g of a solid with a yield of 76%.

4) 3.17g of the reaction product of the previous step and 20mL of methanol were charged into a 50mL single-necked flask, 3.71g of 2-chlorobenzaldehyde was added, and then 33% NaOH aqueous solution was slowly dropped thereinto to conduct reaction for 1 hour. Distilling under reduced pressure to remove methanol, adding diluted hydrochloric acid to adjust pH to neutral, adding 50mL ethyl acetate, stirring for 5min, demixing, adding 100mL saturated saline solution to the organic phase, stirring for 5min, demixing, drying the organic phase with anhydrous sodium sulfate for 1h, and purifying by column chromatography to obtain 1.2g solid product.

5) 1.12g of the reaction product in the previous step, 709mg of iodine simple substance and 10mL of DMSO are gradually heated to 100 ℃ for reaction for 4 hours. Adding 60mL of dichloromethane and 60mL of water, stirring for 5min, demixing, adding 60mL of saturated saline solution into the organic phase, stirring for 5min, demixing, drying the organic phase for 1h by anhydrous sodium sulfate, and purifying by column chromatography to obtain a solid product of 500 mg.

6) 452mg of the reaction product of the previous step and 4mL of dichloromethane were added to a 10mL three-necked flask, replaced with nitrogen three times, the temperature of the reaction mixture was lowered to-78 deg.C, and 12.5mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Slowly adding methanol into the reaction solution under ice water bath condition for quenching, demixing the system, concentrating the organic phase, and performing column chromatography (SiO) ₂ Dichloromethane methanol = 30) to give 30mg brown solid. ¹ H NMR(500MHz,DMSO-d6)δ12.53(s,1H),10.06(s,1H),9.21 (s,1H),7.81(dd,J＝7.6,1.7Hz,1H),7.69(dd,J＝8.0,1.3Hz,1H),7.62 (td,J＝7.7,1.7Hz,1H),7.55(td,J＝7.5,1.3Hz,1H),6.56(s,1H),2.15 (s,3H).MS(ESI,ev):m/z＝319.68[M+H] ⁺ 。

Example 20: synthesis of Compound Ic6

1) 1.0g of 3,4, 5-trimethoxyphenol, 2.2g of acetic anhydride and 10mL of nitromethane were put into a 50mL three-necked flask, 2.2g of aluminum trichloride was added to the reaction mixture at 0 ℃ and reacted for 15 hours at 25 ℃. Adding 50mL ethyl acetate and 10mL 1N hydrochloric acid, separating, adding 10mL water into organic phase, adding 10mL saturated sodium chloride into organic phase, separating, drying organic phase with anhydrous sodium sulfate, concentrating organic phase, and performing column chromatography (SiO) ₂ Ethyl acetate = 5:1) gave 900mg of a white solid.

2) 800mg of the reaction product in the previous step, 210mg of o-chlorobenzaldehyde and 10mL of methanol are added into a 50mL three-necked flask, 4mL of 33% sodium hydroxide aqueous solution is added into the reaction solution at the temperature of 0 ℃, and the reaction is carried out for 10 hours at the temperature of 25 ℃. Concentrating organic phase, adding 50mL ethyl acetate and 10mL 1N hydrochloric acid, separating, adding 10mL water, 10mL saturated sodium chloride, separating, drying organic phase with anhydrous sodium sulfate, concentrating, and performing column chromatography (SiO) ₂ Ethyl acetate = 4:1) to yield 450mg as a white solid.

3) 400mg of the reaction product of the previous step, 240mg of iodine and 5mL of dimethyl sulfoxide were put into a 50mL three-necked flask and reacted at 100 ℃ for 4 hours. Cooling to 25 deg.C, adding 50mL dichloromethane and 10mL water, separating, adding 10mL saturated sodium chloride into organic phase, separating, drying organic phase with anhydrous sodium sulfate, and performing column chromatography (SiO) ₂ Dichloromethane methanol = 30) to give 100mg of a white solid.

4) 80mg of the reaction product in the previous step and 5mL of dimethyl sulfoxide are added into a25 mL single-neck bottle, 2mL of boron tribromide (1M in DCM) is added into the reaction solution at 0 ℃, and the reaction is carried out for 10h at 25 ℃.5mL of water was added to the reaction solution, a solid precipitated, filtered and slurried with water/methanol, filtered again and dried to give 15mg of a pale yellow product. ¹ H NMR(400MHz,DMSO-d6)δ13.75(s,1H),13.55(s,1H),9.22(s, 1H),7.88(dd,J＝7.6,1.8Hz,1H),7.72(dd,J＝8.0,1.3Hz,1H),7.65 (td,J＝7.7,1.8Hz,1H),7.58(td,J＝7.5,1.4Hz,1H),6.78(s,1H),2.66 (s,3H).MS(ESI,ev):m/z＝346.9[M+H] ⁺ 。

Example 21: synthesis of Compound Ic7

1) 450mg of 2' -chloro-5,6,7-trimethoxy baicalein and 321mg of N-iodosuccinimide were dissolved in 4mL of trifluoroacetic acid, and the mixture was replaced with nitrogen three times, gradually raised to 70 ℃ and reacted for 5 hours. The reaction solution is cooled to room temperature, most trifluoroacetic acid is removed by reduced pressure distillation, dichloromethane and saturated sodium bicarbonate solution are added for multiple times for layering, and organic phases are combined, dried and concentrated to obtain 500mg light purple solid.

2) 200mg of the above product and 2mL of DMF were put into a 10mL sealed tube, 160mg of methyl fluorosulfonyl difluoroacetate and 127mg of cuprous iodide were added, and the mixture was sealed and gradually warmed to 100 ℃ for reaction for 12 hours. The reaction mixture was cooled to room temperature, insoluble matter was removed by filtration, and ethyl acetate and water were added to the filtrate to separate them into an organic phase, which was then concentrated by drying to obtain 190mg of a pale yellow solid.

3) 190mg of the product of the above step and 2mL of dichloromethane were added to a 10mL three-necked flask, nitrogen was purged three times, the temperature of the reaction mixture was lowered to-78 deg.C, and 4.58mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Under the condition of ice-water bath, methanol is slowly added into the reaction solution for quenching, methanol and water are added after concentration for pulping, and 150mg of yellow solid product is obtained after filtration. ¹ H NMR(500MHz,DMSO-d6)δ13.31(s,1H),7.90(s, 1H),7.80–7.74(m,1H),7.69(d,J＝7.6Hz,1H),7.66–7.59(m,1H), 6.75(d,J＝2.0Hz,1H).MS(ESI,ev):m/z＝372.2[M+H] ⁺ 。

Example 22: synthesis of Compound Ic8

1) 450mg of 2' -chloro-5,6,7-trimethoxy baicalein and 321mg of N-iodosuccinimide (1.1 eq.) were added to 4mL of trifluoroacetic acid, and the mixture was replaced with nitrogen three times, gradually raised to 70 ℃ and reacted for 5 hours. The reaction solution was cooled to room temperature, most of the trifluoroacetic acid was removed by distillation under reduced pressure, methylene chloride and saturated sodium bicarbonate solution were added for several times for layering, and the organic phases were combined, dried and concentrated to obtain 500mg of a pale purple solid.

2) Adding 250mg of the reaction product of the previous step into 4mL of 1, 4-dioxane, and adding 250mg of PdCl in sequence ₂ (DPPF)-CH ₂ Cl ₂ (0.2 eq.), 115mg of potassium carbonate (2 eq.), 72mg of cyclopropylboronic acid ester (2 eq.), three times with nitrogen and gradually increased to 95 ℃ for 23h. Cooling the reaction solution to room temperature, distilling under reduced pressure to remove 1,4-dioxane, adding ethyl acetate and saturated ammonium chloride solution for multiple times of layering, mixing organic phases, drying, concentrating, and separating by column chromatography to obtain 150mg of brown oil.

3) 150mg of the reaction product of the previous step and 2mL of methylene chloride were charged into a 10mL three-necked flask, nitrogen was purged three times, the temperature of the reaction solution was lowered to-78 deg.C, and 3.88mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Under the condition of ice-water bath, slowly adding methanol into the reaction liquid for quenching, adding methanol and water after concentration for pulping, and filtering to obtain a 56 mg earthy yellow solid product. MS (ESI, ev) m/z =344.2[ M + H ]] ⁺ 。

Example 23: synthesis of Compound Ic9

1) 200mg of the starting material was added to 2mL of DCM, and 236 mg of TiCl was slowly added dropwise in an ice bath ₄ Stirring for 1h, and slowly dropping 85mg of CHCl ₂ OMe, reaction for 45min. Quenched with saturated aqueous ammonium chloride, the aqueous phase was extracted three times with DCM, the combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated to 150mg on the column.

2) And adding 100mg of the reaction product in the previous step into 2mL of tetrahydrofuran, replacing the nitrogen for three times, adding 20mg of sodium borohydride into ice-water bath, and reacting for 5 hours. And (3) dropwise adding 1M HCl in an ice water bath to quench the reaction until no bubbles are generated, adding dichloromethane and saturated sodium bicarbonate solution for multiple layering, and combining organic phases, drying and concentrating to obtain 80mg.

3) 80mg of the reaction product of the previous step and 2mL of methylene chloride were added to a 10mL three-necked flask, the flask was purged with nitrogen three times, the temperature of the reaction solution was lowered to-78 deg.C, and 2mL of BBr was added ₃ Slowly dropwise adding the mixture into the reaction solution, gradually heating to 25 ℃, and reacting for 15h. Under the condition of ice-water bath, methanol is slowly added into the reaction liquid for quenching, methanol and water are added after concentration for pulping, and the yellow solid product of 56 mg is obtained by filtration. MS (ESI, ev) m/z =335.9[ m + H ], [ m ], [] ⁺ 。

Example 24: synthesis of Compound Ic10

1) 3.0g of 3,4, 5-trimethoxyphenol and 6mL of boron trifluoride diethyl etherate were put into a 50mL three-necked flask, and after replacement with nitrogen, 9mL of acetic acid was added thereto, and the temperature was raised to 90 ℃ to react with 2.5 h. The reaction solution was slowly poured into 32mL of ice water and stirred for 0.5h, a large amount of solid precipitated, filtered, and the solid dried to give 3.5g of yellow product. MS (ESI, ev) M/z 249.02 [ M + Na ]] ⁺ .

2) 2.0g of the product of the previous step and 5mL of trifluoroacetic acid were added to a 50mL three-necked flask. After the external temperature was decreased to 0 deg.C, 3.5mL of triethylsilane was added dropwise to the reaction mixture, and the reaction was carried out at 25 deg.C for 12 hours. The concentrated organic phase was purified by column chromatography to give 1.0g of a white solid in 56.3% yield. MS (ESI, ev) 212.6[ 2 ] M + H] ⁺ .

3) 850mg of the product of the previous step, 2mL of boron trifluoride diethyl etherate and 4mL of acetic acid were put into a 50mL three-necked flask, and the temperature was raised to 90 ℃ for reaction for 10 hours. After cooling to 25 ℃, 4mL water and 20mL EA are added into the reaction solution, the mixture is kept stand and layered, the organic phase is washed by 5mL saturated sodium chloride, dried by anhydrous sodium sulfate, and the concentrated organic phase is purified by column chromatography to obtain 470mg of oily product. MS (ESI, ev) 254.7[ deg. ] M + H] ⁺ .

4) 440mg of the product of the previous step, 1mL of DBU and 5mL of acetone are added into a25 mL three-necked flask, 0.4mL of o-chlorobenzoyl chloride is slowly added dropwise into the reaction solution under ice bath, and the reaction is carried out for 3 hours at 25 ℃. Slowly dropwise adding 1N HCl into the reaction solution to adjust the pH =4-5 of the reaction solution, adding 10mL of EA into the reaction solution, standing for layering, layering an organic phase by using 2mL of saturated sodium bicarbonate, layering the organic phase by adding 2mL of saturated sodium chloride, drying with anhydrous sodium sulfate, and concentrating the organic phase to obtain 645mg of a crude product after esterification.

5) 645mg of crude esterification product, 104mg of sodium hydroxide, 5mL of acetone were added to a 50mL three-necked flask. Heating the external temperature to 65 ℃ for reaction for 2h, slowly dropwise adding 1N HCl into the reaction solution to adjust the pH =4-5 of the reaction solution, adding 10mL of EA into the reaction solution, standing for layering, adding 2mL of saturated sodium chloride into the organic phase for layering, drying with anhydrous sodium sulfate, and concentrating the organic phase to obtain 650mg of rearranged crude product.

6) Adding 650mg of the rearranged crude product and 6mL of acetonitrile into a25 mL three-necked bottle, heating the external temperature to 50 ℃, slowly dropwise adding 0.2mL of concentrated sulfuric acid into the reaction solution, and continuously reacting for 3h at 50 ℃. Concentrating dry reaction liquid, adding 10mL EA and 2mL water into the reaction liquid, standing for layering, adding 2mL saturated sodium chloride into an organic phase for layering, drying anhydrous sodium sulfate, concentrating the organic phase, pulping the solid with 3mL methyl tert-butyl ether, filtering, purifying the filtrate by column chromatography to obtain 348mg of white solid, and obtaining MS (ESI, ev): 374.6[ m ] +H ]] ⁺ .

7) Adding 280mg of the ring-closed product and 3mL of DCM into a25 mL three-necked bottle, slowly adding 6.7mL of boron tribromide into the reaction solution in an ice water bath, and heating to 25 ℃ at the external temperature for reaction for 3h. And slowly pouring the reaction liquid into 10mL of ice water, stirring for 0.5h to separate out a large amount of yellow solid, filtering the solid, pulping the solid by using 3mL of methanol, filtering, and drying a filter cake to obtain 92mg of a light yellow solid product Ic10. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.58 (s,1H),10.00(s,1H),9.21(s,1H),7.79(dd,J＝7.6,1.8Hz,1H),7.69 (dd,J＝8.0,1.3Hz,1H),7.62(td,J＝7.7,1.8Hz,1H),7.55(td,J＝7.5, 1.4Hz,1H),6.54(s,1H),2.70(q,J＝7.4Hz,2H),1.08(t,J＝7.4Hz, 3H).MS(ESI,ev):332.5[M+H] ⁺ .

Example 25: synthesis of Compound Ic11

1) 3.0g of 3,4,5-trimethoxyphenol, 1.9mL of isobutyryl chloride, 8.8mL of boron trifluoride diethyl etherate and 50mL of DCM were added into a 250mL three-necked flask, and the temperature was raised to 80 ℃ at the external temperature for reaction for 12 hours. The reaction solution was concentrated, 26mL of 3N HCl and 20mL of EA were added to the reaction flask, allowed to stand for layer separation, the organic phase was added with 2mL of saturated sodium chloride for layer separation, dried over anhydrous sodium sulfate, and the concentrated organic phase was purified by column chromatography to give 2.6g of an oily product.

Continuing with reference to the synthesis procedures for 2) to 7) for Ic10, ic11 was obtained. ¹ H NMR(400 MHz,DMSO-d6)δ12.62(s,1H),9.94(s,1H),9.40-9.00(m,1H),7.76 (dd,J＝7.6,1.8Hz,1H),7.69(dd,J＝8.0,1.3Hz,1H),7.61(td,J＝7.7, 1.8Hz,1H),7.55(td,J＝7.4,1.3Hz,1H),6.54(s,1H),2.57(d,J＝7.2 Hz,2H),1.90(hept,J＝6.8Hz,1H),0.82(d,J＝6.7Hz,6H).MS(ESI, ev):720.5[2M+H] ⁺ .

Example 26: synthesis of Compound Ic12

1) 2.0g of 3,4, 5-trimethoxyphenol and 1.6g of anhydrous MgCl were mixed ₂ And 16mL of ACN were added to a 100mL three-necked flask. After the external temperature is reduced to 0 ℃, 6mL of triethylamine is added into the reaction liquid, stirring is carried out for 30min at 25 ℃,1.2 g of paraformaldehyde is added into the reaction liquid, and the external temperature is increased to 70 ℃ for reaction for 1h. Cooling the external temperature to 25 ℃, slowly dropping 1N hydrochloric acid into the reaction solution, adjusting the pH of the reaction solution to be less than or equal to 5, adding 20mL ethyl acetate and 5mL water into the organic phase, stirring for 10min, standing for layering, adding 5mL saturated salt water into the organic phase for layering, and drying the organic phase by anhydrous sodium sulfate. The concentrated organic phase was purified by column chromatography to give 1.9g of a pale yellow solid product. MS (ESI, ev) 212.6[ 2 ], [ M ] +H] ⁺ 。

Continuing with reference to the syntheses of Ic10 from 2) to 7) to give Ic12, with the exception that o-fluorobenzoyl chloride was used instead of o-chlorobenzoyl chloride. ¹ H NMR(400MHz,DMSO-d6) δ12.50(s,1H),10.01(s,1H),9.16(s,1H),7.99(td,J＝7.8,1.7Hz,1H), 7.65(dddd,J＝8.7,7.1,5.1,1.8Hz,1H),7.49-7.38(m,2H),6.67(s,1H), 2.21(s,3H).MS(ESI,ev):302.8[M+H] ⁺ .

Example 27: synthesis of Compound Ic14

Reference is made to the synthesis of 4) to 7) for Ic10 to Ic14, except that 4' -chlorobiphenyl-3-carbonyl chloride is used instead of o-chlorobenzoyl chloride. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.56(s,1H),10.06(s,1H),9.21(s,1H),8.07(d,J＝2.3Hz,1H),7.91 (dd,J＝8.4,2.3Hz,1H),7.82–7.73(m,3H),7.51(t,J＝7.6Hz,2H), 7.46–7.40(m,1H),6.71(s,1H),2.17(s,3H).MS(ESI,ev):m/z＝394.2 [M+H] ⁺ 。

Example 28: synthesis of Compound Ic15

Reference is made to the synthesis of 4) to 7) for Ic10 to Ic15, except that 2-methyl-5-nitro-benzoyl chloride is used instead of o-chlorobenzoyl chloride. ¹ H NMR(500MHz, DMSO-d ₆ )δ12.58(s,1H),10.06(s,1H),9.22(s,1H),8.45(d,J＝2.5 Hz,1H),8.33(dd,J＝8.5,2.5Hz,1H),7.73(d,J＝8.5Hz,1H),6.63(s, 1H),2.59(s,3H),2.16(s,3H).MS(ESI,ev):m/z＝344.1[M+H] ⁺ 。

Example 29: synthesis of Compound Ic16

Ic15 (14mg, 0.041mmol) was added to methanol (2 mL), pd/C (5 mg) was added, stirred at room temperature for 1 hour, and the reaction was monitored by TLC for completion. Filtering, concentrating the filtrate, adding ethyl acetate, pulping, stirring, filtering, and drying the filter cake to obtain a solid of 8mg with a yield of 62%. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.70(s,1H),9.54(s,2H),7.03 (d,J＝8.2Hz,1H),6.82(d,J＝2.5Hz,1H),6.69(dd,J＝8.2,2.5Hz, 1H),6.29(s,1H),5.19(s,2H),2.28(s,3H),2.16(s,3H).MS(ESI,ev): m/z＝314.1[M+H] ⁺ 。

Example 30: synthesis of Compound Ic17

Reference is made to the synthesis of 4) to 7) in Ic10, with the difference that 3-chloro- [1,1-biphenyl is used to obtain Ic17]-4-acyl chloride instead of o-chlorobenzoyl chloride. ¹ H NMR(500MHz, DMSO-d ₆ )δ12.54(s,1H),10.05(s,1H),9.22–9.19(m,1H),7.98(d,J ＝1.8Hz,1H),7.96–7.83(m,2H),7.81(dd,J＝7.2,1.8Hz,2H),7.53 (dd,J＝8.4,6.8Hz,3H),7.49–7.44(m,1H),6.62(s,1H),2.18(s,3H). MS(ESI,ev):m/z＝394.2[M+H] ⁺ 。

Example 31: synthesis of Compound Ic18

1) Adding ethanol (10 mL) into nitro-containing raw material (200mg, 0.52mmol), adding stannous chloride dihydrate (720mg, 6.4mmol, 3.0eq) under nitrogen protection, dropwise adding 2 drops of acetic acid, heating to 70 ℃, and reacting for 3h. TLC: the raw material disappeared. And (3) post-treatment: after cooling, adding an aqueous solution of sodium bicarbonate to adjust the pH to 7, filtering, concentrating, diluting with water, extracting with EA, washing the organic phase with salt, drying, filtering, concentrating to a smaller volume, adding a hydrogen chloride dioxane solution (1.1 eq) while stirring, precipitating a large amount of solids, and filtering to obtain 173mg of the amino compound. ¹ H NMR(500MHz,DMSO-d ₆ )δ10.27(s,2H),7.65(d, J＝2.2Hz,1H),7.52(d,J＝8.2Hz,1H),7.47(dd,J＝8.2,2.3Hz,1H), 6.34(s,1H),3.91(s,3H),3.85(s,3H),3.82(s,3H),2.46(s,3H),2.27(s, 3H).

2) DCM (1 mL) was added to the amino compound (50mg, 0.13mmol), and acetic anhydride (16mg, 0.15mmol, 1.2eq) and triethylamine (26mg, 0.26mmol, 2eq) were added under nitrogen for 3h at ambient temperature (20 ℃ C.). TLC: the raw materials remained in small amounts. And (3) post-treatment: adding ammonium chloride aqueous solution to adjust pH to 7, EA extracting, salt washing organic phase, drying, filtering, and separating by thin layer chromatography to obtain amide compound 51mg. ¹ H NMR(500 MHz,DMSO-d ₆ )δ10.09(s,1H),7.90(d,J＝2.3Hz,1H),7.61(dd,J＝ 8.3,2.3Hz,1H),7.31(d,J＝8.4Hz,1H),6.27(s,1H),3.91(s,3H),3.85 (s,3H),3.81(s,3H),2.39(s,3H),2.27(s,3H),2.06(s,3H).

3) The product obtained in the previous step (20mg, 0.05mmol) and DCM (2 mL) were added dropwise with boron tribromide solution (1M, 0.45mmol,9 eq) under nitrogen protection at 0 ℃ and then removed from the ice bath, and the reaction was carried out at ambient temperature (20 ℃) for 3 hours. TLC: the raw material disappeared. And (3) post-treatment: adding 1 drop of methanol to quench, concentrating, adding water into the residue, pulping, and filtering to obtain a crude product. DCM and PE were added for slurry and filtration gave compound Ic18, a total of 7mg. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.64(s,1H),10.09(s,1H),10.00(s,1H), 9.17(s,1H),7.89(d,J＝2.3Hz,1H),7.63(dd,J＝8.3,2.3Hz,1H),7.32 (d,J＝8.4Hz,1H),6.37(s,1H),2.39(s,3H),2.16(s,3H),2.06(s,4H). MS(ESI,ev):m/z＝356.1[M+H] ⁺ 。

Example 32: synthesis of Compound Ic19

1) Substituted phenol (528mg, 2.2mmol,1.1 eq), dichloromethane (6 ml), triethylamine (555. Mu.l, 4mmol, 2eq), and DMAP (6 mg,1mmol, 0.05eq) were added sequentially to a reaction flask, 4' -methylbiphenyl-2-carbonyl chloride was added under ice bath, and after the addition, the ice bath was removed, and the mixture was stirred at ambient temperature. After the reaction is completed, washing the mixture by using a 1M HCl solution, a saturated sodium bicarbonate solution and a saturated sodium chloride solution in sequence, drying the mixture by using anhydrous sodium sulfate, concentrating and mixing the mixture, and performing column chromatography to obtain 520mg of the esterified compound.

2) Potassium tert-butoxide (58mg, 0.52mmol, 1.3eq) is added to a solution of the product of the previous step (173mg, 0.4mmol, 1eq) in THF, N ₂ Protecting and stirring, precipitating a large amount of yellow solid, after 5 hours, adding 0.5eq of potassium tert-butoxide, after the reaction is completed, stopping the reaction, adding water, dropwise adding 1M HCl to adjust the pH value to 6, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating, stirring, and performing column chromatography to obtain a golden yellow oily matter rearrangement compound of 143mg.

3) The product of the previous step and glacial acetic acid (2 ml) were added to a reaction flask,stirring for dissolving, dripping 3 drops of concentrated hydrochloric acid for catalysis, and adding N ₂ And (4) protecting, heating and refluxing at 110 ℃, and stopping the reaction after the reaction is completed after two hours. Adding water, adding 1M NaOH dropwise to adjust pH to 6, extracting with ethyl acetate, drying with anhydrous sodium sulfate, concentrating, purifying with thick plate, and separating to obtain 50mg of ring-closed compound. ¹ H NMR(500MHz,DMSO-d ₆ ):δ7.77(d,J＝7.4Hz,1H),7.66 (t,J＝7.3Hz,1H),7.53(dd,J＝17.1,7.7Hz,2H),7.24(d,J＝7.8Hz, 2H),7.17(d,J＝7.7Hz,2H),6.34(s,1H),3.79(d,J＝15.7Hz,9H),2.29 (s,3H),1.54(s,3H).

4) Dissolving the closed-loop compound obtained in the previous step in dichloromethane, and adding BBr under ice bath ₃ (1ml, 1mmol, 10eq), after the addition, the ice bath was removed, and the reaction mixture was stirred at room temperature to give an orange-red reaction solution. After the reaction is completed, adding methanol for quenching, carrying out spin drying, pulping by EA, purifying impurities, pulping again by acetone and water, and purifying to obtain 20mg of a product Ic19, ¹ H NMR(500MHz,DMSO-d ₆ ):δ 12.61(s,1H),9.87(s,1H),9.11(s,1H),7.78(d,J＝7.7,1.1Hz,1H), 7.67(dd,J＝7.6,1.3Hz,1H),7.58–7.48(m,2H),7.18(dd,J＝23.5,8.1 Hz,4H),6.48(s,1H),2.28(s,3H),1.42(s,3H).MS(ESI,ev):m/z＝ 375.1[M+H] ⁺ 。

example 33: synthesis of Compound Ic20

1) Iodine-containing raw material (300mg, 0.684mmol), potassium carbonate (189mg, 1.369 mmol), xphos (98mg, 0.2052 mmol), potassium fluoride (6mg, 0.2052 mmol), palladium acetate (31mg, 0.1398mmol), DMF (5 mL), nitrogen protection, reaction at 105 ℃ for 12 hours, TLC the next day shows the generation of new points of deiodinated product, and isopropenyl compound at 8-position. And (3) post-treatment: diatomite for filtration assistance, ethyl acetate/water extraction, water washing, brine washing, drying, filtration and column chromatography to obtain the 8-position isopropenyl compound 150mg.

2) The product of the previous step (75 mg) was dissolved in dichloromethane (2 mL) and 1M BBr was added in an ice bath ₃ After the addition of the solution (0.6 mL), the reaction was carried out at room temperature for 3 hours, and the TLC starting material was completely reacted. And (3) post-treatment: the reaction solution was added dropwise to methanol (2 mL) and stirredAfter 30min, concentrate, add acetone: water =2:1 (3 mL) and filtered to give 50mg of yellow solid. The nuclear magnetization showed a small amount of impurities. A small amount of ethanol dissolved the solid, water was added to precipitate a solid, which was stirred and filtered to obtain 37mg total Ic 20. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.85(s,1H),9.91(s,1H), 9.22(s,1H),8.08–8.03(m,1H),8.00(dd,J＝7.4,2.3Hz,1H),7.58(p, J＝5.0Hz,4H),6.99–6.91(m,1H),5.48(t,J＝2.0Hz,1H),5.04(s, 1H),2.08(s,3H).

Example 34: synthesis of Compound Id1

270mg of baicalein and 4mL of acetonitrile were put into a 10mL two-necked flask, and after nitrogen substitution, 129mg of acetoacetyl isocyanate and 101mg of triethylamine were added to the reaction solution, followed by reaction at 25 ℃ for 12 hours. The acetonitrile was distilled off under reduced pressure, 15mL of ethyl acetate and 10mL water were added, and after stirring for 5min, layers were separated, and 10mL of saturated saline was added to the organic phase, and after stirring for 5min, layers were separated, and the organic phase was dried over anhydrous sodium sulfate for 1h. Finally, purification by column chromatography (SiO 2, dichloromethane: methanol = 40) gave 330mg of the product as a white solid in 83.1% yield. 1H NMR (500MHz, DMSO-d 6) delta 12.94 (s, 1H), 11.28 (s, 1H), 8.17 (t, J =6.1Hz, 1H), 8.12-8.08 (m, 2H), 7.67-7.54 (m, 3H), 7.02 (s, 1H), 6.67 (s, 1H), 4.13 (q, J =7.2Hz, 2H), 3.85 (d, J =6.1Hz, 2H), 1.22 (t, J =7.1Hz, 3H). MS (m/z =400.1[ ESI ] M H ] +.

Example 35: synthesis of Compound Id3

152mg of chloromethyl isopropyl carbonate is added into 3mL of acetone, 149mg of NaI is added, the temperature is raised to 60 ℃, the reaction is carried out for 3h, and the mixture is concentrated for standby. 272mg of baicalein and 276mg of potassium carbonate are added into 5mL of acetone, a little DMF is added for dissolving assistance, and then the oily substance for standby concentration is added, and the reaction is carried out for 12h at the ambient temperature of 12 ℃. The reaction was concentrated to small volume and EA and H were added ₂ Separating by O layer, concentrating by organic phase column chromatography50mg of a yellow solid. ¹ H NMR(500MHz,DMSO-d6)δ12.66(s,1H),9.15(s,1H),8.17– 8.11(m,2H),7.68–7.57(m,3H),7.09(d,J＝10.0Hz,2H),5.93(s,2H), 4.85(hept,J＝6.2Hz,1H),1.26(d,J＝6.3Hz,6H).MS(ESI,ev):m/z＝ 387.1[M+H] ⁺ 。

Example 36: synthesis of Compounds Id5 and Id6

Baicalein (300mg, 1.11mmol, 1eq) and anhydrous potassium carbonate (153mg, 1.11mmol, 1eq) were put into DMF (3 ml), bromoacetonitrile (76. Mu.l, 1.11mmol, 1.1eq) was slowly added under ice bath, and stirred at room temperature for 10 hours. DCM/H was used for the reaction system ₂ O extraction (aqueous layer adjusted to weakly acidic pH with 1M HCl, aqueous layer viscous), organic phase washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated. Pulping and purifying by DCM, DCM and PE in turn, filtering to obtain a filter cake as a compound Id5, ¹ H NMR(400MHz,DMSO-d ₆ )δ 13.08(s,1H),8.15–8.12(m,2H),7.67–7.60(m,3H),7.25(s,1H),7.16 (s,1H),5.39(s,2H),5.05(s,2H).MS(ESI,ev):m/z＝383.3[M-H] ^- 。

concentrating the filtrate, purifying by column chromatography to obtain compound Id6, ¹ H NMR (500MHz,DMSO-d ₆ )δ13.13(s,1H),11.33(s,1H),8.12–8.08(m,2H), 7.65–7.56(m,3H),7.03(s,1H),6.68(s,1H),5.00(s,2H).MS(ESI,ev): m/z＝308.2[M+H] ⁺ 。

example 37: synthesis of Compound Id8

1) 2.0g of Ic5, 15.0mL of pyridine, and 3.5mL of acetic anhydride were put into a 100mL three-necked flask, and the temperature was raised to 70 ℃ to react for 10 hours. After concentrating the dry reaction solution, 50mL of ethyl acetate and 10mL of water were added to the crude product, the mixture was stirred for 10min and then allowed to stand for layer separation, 10mL of water was added to the organic phase, 10mL of saturated sodium chloride was added to the organic phase, and the organic phase was dried over anhydrous sodium sulfate. Concentrating the organic phase byPurification was carried out by column chromatography to give 1.7g of trihydroxyacetylated product. MS (ESI, ev) m/z =445.0[ M + H ]] ⁺ 。

2) 1.2g of trihydroxy acetylated compound, 3.0g of potassium carbonate, 220mg of potassium iodide, 20mL of acetone and 1.6mL of benzyl bromide were put into a 100mL three-necked flask, and the temperature was raised to 60 ℃ to react for 10 hours. Filtration and concentration of the organic phase followed by column chromatography gave 420mg of a white product having 7-acetoxy groups instead of benzyloxy groups. MS (ESI, ev) m/z = 492.9[ M + H ]] ⁺ 。

3) 400mg of the product of the previous step, 40mg of Pd (OH) ₂ The reaction mixture was reacted at 25 ℃ for 15 hours after adding/C and 5mL of tetrahydrofuran to a25 mL single-neck flask and replacing hydrogen three times with a hydrogen balloon. The reaction solution was filtered through celite, the dry organic phase was concentrated and the crude product was slurried with methanol, filtered and the solid dried to yield 250mg of the debenzylated compound. MS (ESI, ev) m/z =402.7[ M + H ]] ⁺ 。

4) 150mg of a debenzylation compound, 296mg of acetyl bromo-alpha-D-glucuronic acid methyl ester, 216mg of silver oxide, 250mg of

Molecular sieves and 3mL of quinoline were added to a25 mL single-necked flask and reacted at 25 ℃ for 10h. The reaction solution was filtered, and the concentrated organic phase was purified by column chromatography to give 135mg of a white glycosidated product. MS (ESI, ev) m/z = 719.7[ M + H ]] ⁺ 。

5) Adding 80mg of intermediate 4 and 2mL of acetone into a 10mL single-mouth bottle, cooling the outer temperature to 0 ℃, dropwise adding 0.6mL of 2.5N sodium hydroxide solution into the reaction solution, and reacting for 0.5h. Slowly dripping 1N hydrochloric acid into the reaction liquid at the external temperature of 0 ℃, adjusting the pH of the reaction liquid to be less than or equal to 3, generating yellow solid, filtering the solid, and drying to obtain 30mg of yellow product Id8. ¹ H NMR(400MHz,Methanol-d4)δ7.75(dd,J＝7.5,1.8Hz, 1H),7.63(dd,J＝8.0,1.4Hz,1H),7.58(td,J＝7.6,1.6Hz,1H),7.52(td, J＝7.4,1.5Hz,1H),6.55(s,1H),5.04(d,J＝7.9Hz,1H),3.86(d,J＝ 9.7Hz,1H),3.68-3.61(m,2H),3.53(d,J＝9.1Hz,1H),2.39(s,3H). MS(ESI,ev):m/z＝494.5[M+H] ⁺

Example 38: synthesis of Compound Id9

The same procedure as in example Id8 was followed, except that acetyl bromide- α -D-glucose was used instead of acetyl bromide- α -D-glucuronic acid methyl ester, to give Id9. ¹ H NMR(400 MHz,DMSO-d6)δ12.38(s,1H),9.15(s,1H),7.83(dd,J＝7.6,1.8Hz, 1H),7.71(dd,J＝8.1,1.3Hz,1H),7.67-7.61(m,1H),7.57(td,J＝7.5, 1.4Hz,1H),6.66(s,1H),4.77(dd,J＝7.8,4.6Hz,1H),3.67(d,J＝11.5 Hz,1H),3.48(dd,J＝11.6,4.7Hz,1H),3.37(t,J＝8.4Hz,1H),3.32- 3.25(m,1H),3.23-3.18(m,2H),2.34(s,3H).MS(ESI,ev):m/z＝480.5 [M+H] ⁺ 。

Example 39: synthesis of Compounds Id14 and Id15

Compound Ic5 (477mg, 1.5mmol, 1eq) was charged into DCM (20 mL), undissolved and clear, and NEt was added under ice bath ₃ (0.6 mL,4.5mmol,3 eq) and the system was clarified, replaced with nitrogen, 10-fold diluted chlorosulfonic acid (1.5 mL,2.25mmol, 1.5 eq) was added with DCM, and after stirring in an ice bath for 30 minutes, the mixture was stirred overnight at room temperature, the next day TLC indicated that a small amount of starting material remained. DCM/H was used for the reaction system ₂ Extracting with water, adjusting pH of water layer to weak acidity with 1M HCl, drying organic phase with anhydrous sodium sulfate, filtering, concentrating, separating prepared liquid phase to obtain two compounds, and respectively sending hydrogen spectra. ¹ H NMR(400MHz,DMSO-d ₆ ) δ 12.32 (s, 1H), 8.84 (s, 1H), 8.78 (s, 1H), 7.84 (dt, J =7.7,2.0hz, 1h), 7.70 (dd, J =8.0, 1.3hz, 1h), 7.63 (td, J =7.7,1.8hz, 1h), 7.58-7.53 (m, 1H), 6.65 (s, 1H), 2.25 (s, 3H) and ¹ H NMR(400MHz,DMSO-d ₆ )δ12.63(s,1H), 10.38(s,1H),8.84(s,1H),7.84(dt,J＝7.7,2.0Hz,1H),7.70(dd,J＝ 8.0,1.3Hz,1H),7.63(td,J＝7.7,1.8Hz,1H),7.60–7.52(m,1H),6.62 (s,1H),2.15(s,3H).

example 40: synthesis of Compounds Id16 and Id20

Compound Ic5 (0.8g, 2.52mmol, 1eq) and DMF (6.5 mL) were added in this order to a two-necked flask, and after dissolution and clarification, NEt was added under ice bath ₃ (0.4mL, 1.1eq), after stirring for 15mins, ethyl isocyanatoacetate (0.31mL, 1.1eq) was added dropwise, and after 3h ice-bath reaction, TLC showed the starting material was essentially completely reacted. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of a water layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, purifying and concentrating by using column chromatography to obtain 720mg of the compound Id16 and 212mg of the compound Id20, wherein the compounds are yellow solids. Id16: ¹ H NMR(500MHz,DMSO-d6)δ12.71(s,1H),10.68(s,1H),8.17(t,J＝ 6.1Hz,1H),7.83(dd,J＝7.6,1.8Hz,1H),7.70(dd,J＝8.0,1.3Hz,1H), 7.63(td,J＝7.8,1.8Hz,1H),7.56(td,J＝7.5,1.3Hz,1H),6.64(s,1H), 4.13(q,J＝7.1Hz,2H),3.86(d,J＝6.1Hz,2H),2.17(s,3H),1.21(t,J＝ 7.1Hz,3H).MS(ESI,ev):m/z＝432.1[M+H] ⁺ 。Id20： ¹ H NMR(500 MHz,DMSO-d ₆ )δ12.67(s,1H),8.51(t,J＝6.2Hz,1H),8.34(t,J＝6.1 Hz,1H),7.87(dd,J＝7.7,1.7Hz,1H),7.71(dd,J＝8.1,1.3Hz,1H), 7.65(td,J＝7.7,1.7Hz,1H),7.57(td,J＝7.5,1.3Hz,1H),6.81(s,1H), 4.13(q,J＝7.1Hz,4H),3.88(d,J＝6.1Hz,,4H),2.20(s,3H),1.21(t,J ＝7.1Hz,6H).MS(ESI,ev):m/z＝576.1[M+H] ⁺

example 41: synthesis of Compound Id18

Compound Ic5 (0.1g, 0.3mmol, 1eq) is dissolved in 4mL of tetrahydrofuran, NEt ₃ (60mg, 2eq) was then added and isobutyric anhydride (96mg, 2 eq) was added dropwise under ice bath and stirred for one hour under ice bath, TLC showed the starting material was essentially completely reacted. And (3) post-treatment: adding EA/H into the reaction solution ₂ Adjusting pH of water layer to weak acidity with 1M HCl, extracting with EA, washing organic phase with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, purifying with column chromatography, and concentrating to obtain compoundThe drug Id18 is 25mg in total, MS (ESI, ev) m/z =389.1[ m ] +H ]] ⁺ 。

Example 42: synthesis of Compounds Id21 and Id22

Sequentially adding compound Ic5 (1g, 3.14mmol, 1eq) and DMF (6 mL) into a double-neck bottle, replacing by nitrogen after the compound Ic5 and the DMF are dissolved and clarified, and adding K under ice bath ₂ CO ₃ (651mg, 4.71mmol, 1.5eq), the reaction solution became turbid, after stirring for 10mins, 2-methoxyethoxymethyl chloride (538. Mu.L, 4.71mmol, 1.5eq) diluted 10-fold with DMF was further added dropwise, the reaction solution gradually clarified, after 2h in ice bath, TLC showed about 40% of the raw material remained, 0.5eq of 2-methoxyethoxymethyl chloride was added, the reaction was carried out overnight at room temperature, and TLC the next day showed 10% of the raw material remained. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ Extraction with O, pH adjustment of the aqueous layer to weak acidity with 1M HCl, liquid separation, washing of the organic phase with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtration, concentration, sample stirring with silica gel, column chromatography (DCM: meOH = 500). Id21: ¹ H NMR (500MHz,DMSO-d ₆ )δ12.86(s,1H),10.27(s,1H),7.82(dd,J＝7.6, 1.7Hz,1H),7.70(dd,J＝8.1,1.3Hz,1H),7.63(td,J＝7.8,1.8Hz,1H), 7.56(d,J＝1.3Hz,1H),6.62(s,1H),5.18(s,2H),3.91(t,J＝4.6Hz, 2H),3.49(t,J＝4.6Hz,3H),3.24(s,3H),2.16(s,3H).MS(ESI,ev): m/z＝429.20[M+Na] ⁺ ,Id22: ¹ H NMR(500MHz,DMSO-d ₆ )δ12.49(s, 1H),9.27(s,1H),7.83(d,J＝7.6Hz,1H),7.70(d,J＝8.0Hz,1H),7.63 (t,J＝7.7Hz,1H),7.56(t,J＝7.5Hz,1H),6.65(s,1H),5.31(s,2H), 3.83(t,J＝4.6Hz,2H),3.47(t,J＝4.6Hz,3H),3.22(s,3H),2.25(s, 3H).MS(ESI,ev):m/z＝428.61[M+Na] ⁺ 。

example 43: synthesis of Compounds Id23 and Id24

The compound isIc5 (1g, 3.14mmol, 1eq) and THF (5 mL) were added sequentially to a two-necked flask, undissolved and clear, pyridine (585. Mu.L, 2.3mmol, 2.3eq) was added under ice-cooling, after stirring for 10 minutes, N' -dimethylcarbamoyl chloride (637. Mu.L, 6.9mmol,2.2 eq) was added dropwise, after 2 hours of ice-cooling, TLC showed almost reaction of the starting material, supplemented with 3.7eq pyridine, and after about 22 hours of continued reaction, TLC showed that about 30% of the starting material remained. And (3) post-treatment: EA/H for reaction solution ₂ Extraction with O, pH adjustment of the aqueous layer to weak acidity with 1M HCl, liquid separation, washing of the organic phase with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtration, sample mixing, concentration, column chromatography, thin layer chromatography (DCM: meOH = 40) to give compounds Id23 and Id24. Hydrogen spectrum of Id23 ¹ H NMR(500MHz,DMSO-d ₆ )δ12.61(s,1H), 7.87(d,J＝7.7Hz,1H),7.71(d,J＝7.9Hz,1H),7.65(t,J＝7.8Hz,1H), 7.58(t,J＝7.7Hz,1H),6.81(s,1H),3.02(d,J＝4.7Hz,6H),2.93(d,J ＝8.8Hz,6H),2.18(s,3H).MS(ESI,ev):m/z＝461.3[M+H] ⁺ (ii) a Hydrogen spectrum of Id 24: ¹ H NMR(500MHz,DMSO-d ₆ )δ12.68(s,1H),10.64(s,1H), 7.83(dd,J＝7.6,1.8Hz,1H),7.70(dd,J＝8.1,1.3Hz,1H),7.63(td,J＝ 7.7,1.8Hz,1H),7.56(td,J＝7.6,1.3Hz,1H),6.63(s,1H),3.07(s,3H), 2.92(s,3H),2.17(s,3H).MS(ESI,ev):m/z＝390.3[M+H] ⁺

example 44: synthesis of Compound Id25

Compound Ic5 (1g, 3.14mmol, 1eq) and DMF (6 mL) were added sequentially to a two-necked flask, and after dissolution and clarification, NEt was added under ice bath ₃ (873. Mu.L, 6.28mmol, 2eq) and after stirring for 10 minutes, ethyl isocyanate (513. Mu.L, 6.28mmol, 2eq) diluted 10 times with DMF was added dropwise and after 1 hour of ice-bath reaction TLC showed the raw materials were essentially reacted to completion, giving a primary fluorescence spot. And (3) post-treatment: EA/H for reaction solution ₂ And (3) extracting, adjusting the pH of a water layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, concentrating, pulping by using acetonitrile, methanol and ethyl acetate in sequence, and filtering to obtain a compound Id25. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.61 (s,1H),7.99(q,J＝4.6Hz,1H),7.86(dd,J＝7.7,1.8Hz,1H),7.82(d,J ＝4.6Hz,1H),7.71(d,J＝1.2Hz,1H),7.65(td,J＝7.7,1.7Hz,1H), 7.58(td,J＝7.5,1.3Hz,1H),6.80(s,1H),3.10(qd,J＝7.2,5.5Hz,4H), 2.15(s,3H).1.10(t,J＝7.2Hz,6H).MS(ESI,ev):m/z＝482.85[M+ Na] ⁺

Example 45: synthesis of Compound Id27

Compound Ic5 (500mg, 1.57mmol, 1eq) and DMF (5 mL) were added sequentially to a two-necked flask, after dissolution and clarification, NEt was added under ice bath ₃ (219. Mu.L, 1.57mmol,1 eq), after stirring for 10 minutes, ethyl isocyanate (107. Mu.L, 1.65mmol, 1.05eq) diluted 10-fold with DMF was added dropwise and after 1 hour of ice-bath reaction TLC indicated substantial completion of the reaction. And (3) post-treatment: EA/H for reaction solution ₂ And (3) extracting, adjusting the pH of a water layer to be weakly acidic by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, concentrating, performing column chromatography, and concentrating to obtain a compound Id27. ¹ H NMR(500MHz, DMSO-d ₆ )δ12.68(s,1H),10.61(s,1H),7.83(dd,J＝7.6,1.7Hz,1H), 7.75–7.69(m,2H),7.63(td,J＝7.7,1.7Hz,1H),7.56(td,J＝7.5,1.3 Hz,1H),6.63(s,1H),3.10(qd,J＝7.2,5.5Hz,2H),2.17(s,3H),1.10(t, J＝7.2Hz,3H).MS(ESI,ev):m/z＝411.83[M+Na] ⁺

Example 46: synthesis of Compounds Id28 and Id29

Compound Ic5 (500mg, 1.57mmol, 1eq) and DMF (5 mL) were added sequentially to a two-necked flask, after dissolution and clarification, NEt was added under ice bath ₃ (656. Mu.L, 4.72mmol,3 eq) and after stirring for 10min, methylaminocarbonyl chloride (294mg, 3.14mmol,2 eq) was added dropwise, after 2h ice-bath reaction, TLC showed the basic reaction of the starting material was complete, producing two main fluorescent spots, and the reaction was continued for 2hAfter that, TLC showed no significant change. And (3) post-treatment: EA/H for reaction solution ₂ And (3) extracting, adjusting the pH of an aqueous layer to subacidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, mixing a sample by using silica gel, performing column chromatography, and concentrating to obtain compounds Id28 and Id29. Hydrogen spectrum of Id 28: ¹ H NMR(500 MHz,DMSO-d ₆ )δ12.61(s,1H),7.99(q,J＝4.6Hz,1H),7.86(dd,J＝ 7.7,1.8Hz,1H),7.82(q,J＝4.4Hz,1H),7.72(dd,J＝8.1,1.2Hz,1H), 7.65(td,J＝7.7,1.7Hz,1H),7.58(td,J＝7.5,1.3Hz,1H),6.80(s,1H), 2.66(d,4.6Hz,6H),2.15(s,3H).MS(ESI,ev):m/z＝454.87[M+ Na] ⁺ . Hydrogen spectrum of Id 29: ¹ H NMR(500MHz,DMSO-d ₆ )δ12.68(s,1H), 10.61(s,1H),7.83(dd,J＝7.7,1.7Hz,1H),7.70(dd,J＝8.0,1.2Hz, 1H),7.65–7.60(m,2H),7.56(td,J＝7.6,1.3Hz,1H),6.63(s,1H),2.67 (d,J＝4.6Hz,3H),2.16(s,3H).MS(ESI,ev):m/z＝397.79[M+Na] ⁺

example 47: synthesis of Compound Id30

The compound Ic17 (1.5g, 3.79mmol, 1eq) and DMF (13 mL) were sequentially added to a two-necked flask, and the mixture was left undissolved and clear, replaced with nitrogen, and NEt was added dropwise in ice bath ₃ (581. Mu.l, 4.17mmol, 1.1eq), after stirring for 10 minutes, ethyl isocyanatoacetate (492. Mu.L, 4.17mmol, 1.1eq) diluted 10-fold with DCM was added dropwise and after reaction in an ice bath for about 1.5h, TLC showed almost complete reaction of the starting material. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (2) extracting, adjusting the pH of an aqueous layer to weak acidity by using 1M hydrochloric acid, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying anhydrous sodium sulfate, filtering, concentrating, stirring a sample by using silica gel, performing column chromatography (DCM: meOH =600, and outputting a product), concentrating to obtain a crude compound Id30, and sequentially pulping and purifying by using PE: EA =1:1 and acetonitrile + a small amount of methanol to obtain the compound Id30: yellow solid, 643mg. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.73(s,1H),10.68(s, 1H),8.16(t,J＝6.1Hz,1H),8.00(d,J＝1.8Hz,1H),7.92(d,J＝8.1Hz, 1H),7.87(dd,J＝8.1,1.8Hz,1H),7.83–7.80(m,2H),7.53(td,J＝7.2, 6.3,1.3Hz,3H),7.50–7.45(m,1H),6.70(s,1H),4.13(q,J＝7.1Hz, 2H),3.86(d,J＝6.1Hz,2H),2.20(s,3H),1.22(t,J＝7.1Hz,3H).MS (ESI,ev):m/z＝524.4[M+H] ⁺

Example 48: synthesis of Compound Id31

Sequentially adding the compound Ic14 (200mg, 0.5mmol, 1eq) and DMF (2 mL) into a double-neck bottle, and dropwise adding NEt under ice bath when the clear solution is not dissolved ₃ (77. Mu.L, 0.56mmol,1.1 eq), after stirring for 10mins, a 10-fold dilution of methylaminocarbonyl chloride (50mg, 0.53mmol, 1.05eq) with DCM was added dropwise and the reaction was carried out in an ice bath for about 6 h. EA/H for reaction solution ₂ And (3) extracting, adjusting the pH of an aqueous layer to weak acidity by using 1M hydrochloric acid, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, concentrating, and purifying by using column chromatography to obtain a compound Id31: yellow solid, 76mg. ¹ H NMR(400MHz,DMSO-d ₆ )δ 12.70(s,1H),10.63(s,1H),7.99(s,1H),7.94–7.77(m,3H),7.65– 7.42(m,5H),6.68(s,1H),2.68(s,3H),2.19(s,3H).MS(ESI,ev):m/z＝ 452.3[M+H] ⁺ 。

Example 49: synthesis of Compound Id32

The compound Ic14 (200mg, 0.5mmol, 1eq) and DMF (2 mL) were added in sequence to a two-necked flask, undissolved and clear, and NEt was added dropwise in ice bath ₃ (77. Mu.L, 0.56mmol,1.1 eq), stirring for 10mins, continuing to add N, N' -dimethylaminochloride (49. Mu.L, 0.53mmol, 1.05eq) diluted 10 times with DCM dropwise, reacting for about 1h in ice bath, TLC showed no significant change in the starting material, and 2eqNEt was added ₃ The reaction was continued for 9 h. The reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of an aqueous layer to weak acidity by using 1M hydrochloric acid, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, concentrating, and purifying by using column chromatography to obtain a compound Id32: yellowish brown solid, 30mg. ¹ H NMR(500MHz,DMSO-d ₆ )δ 12.71(s,1H),10.65(s,1H),8.00(d,J＝1.8Hz,1H),7.93(d,J＝8.1Hz, 1H),7.87(dd,J＝8.1,1.8Hz,1H),7.83–7.80(m,2H),7.53(dd,J＝8.4, 6.8Hz,3H),7.49–7.45(m,1H),6.70(s,1H),3.08(s,3H),2.92(s,3H), 2.20(s,3H).MS(ESI,ev):m/z＝466.3[M+H] ⁺ 。

Example 50: synthesis of Compound Id33

Adding compound Ic5 (636mg, 2mmol, 1eq) and NMP (8 mL) into a two-necked flask in sequence, adding powdered potassium carbonate (276mg, 2mmol) after dissolution and clarification, continuing to dropwise add isopropyl isocyanate (314mg, 2.2mmol,1.1 eq) after stirring for 15mins in an ice bath, and after reacting for 3h in the ice bath, TLC shows that the raw materials are basically completely reacted. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of a water layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, purifying by using column chromatography, and concentrating to obtain 530mg of a yellow compound Id 33. ¹ H NMR(500MHz,DMSO-d6)δ 12.71(s,1H),10.67(s,1H),8.14(t,J＝6.1Hz,1H),7.82(dd,J＝7.6,1.8 Hz,1H),7.70(dd,J＝8.1,1.3Hz,1H),7.63(td,J＝7.7,1.7Hz,1H), 7.56(td,J＝7.6,1.3Hz,1H),6.63(s,1H),4.95(dq,J＝12.5,6.5Hz,1H), 3.82(d,J＝6.1Hz,2H),2.17(s,3H),1.21(d,J＝6.3Hz,6H).MS(ESI, ev):m/z＝462.3[M+H] ⁺ 。

Example 51: synthesis of Compounds Id35 and Id36

Adding the compound Ic5 (636mg, 2mmol, 1eq) and NMP (8 mL) into a double-neck flask in sequence, adding powdered potassium carbonate (276mg, 2mmol) after the compound Ic5 and the NMP are dissolved and clarified, continuing to dropwise add the isocyanato-2-isopropyl ethyl valerate (376mg, 2.2mmol, 1.1eq) after stirring for 15mins in an ice bath, reacting for 3 hours in the ice bath, and then showing that the raw materials are basically reacted completely by TLC. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ O extraction, aqueous layerAdjusting pH to weak acidity with 1M HCl, separating liquid, washing an organic phase with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, purifying by column chromatography and concentrating to obtain a yellow compound Id35 of 630mg totally, and Id36 of 100mg totally. Id35: ¹ H NMR(500MHz,DMSO-d6)δ12.70(s,1H),10.69(s,1H),8.18(d,J ＝8.4Hz,1H),7.83(dd,J＝7.7,1.7Hz,1H),7.70(dd,J＝8.0,1.3Hz, 1H),7.63(td,J＝7.8,1.7Hz,1H),7.56(td,J＝7.5,1.3Hz,1H),6.64(s, 1H),3.94(dd,J＝8.5,6.6Hz,1H),3.07(s,1H),2.18(s,3H),2.09(h,J＝ 6.8Hz,1H),1.22(t,J＝7.1Hz,3H),1.00–0.90(m,6H).MS(ESI,ev): m/z＝490.3[M+H] ⁺ ，Id36： ¹ H NMR(500MHz,DMSO-d6)δ12.66 (s,1H),8.55(d,J＝8.3Hz,1H),8.38(d,J＝8.4Hz,1H),7.86(dd,J＝ 7.7,1.7Hz,1H),7.71(dd,J＝8.1,1.3Hz,1H),7.65(td,J＝7.7,1.8Hz, 1H),7.57(td,J＝7.5,1.3Hz,1H),4.21–4.03(m,4H),3.97–3.90(m, 2H),2.17(s,3H),2.19–2.03(m,2H),1.20(q,J＝7.1Hz,6H),1.01– 0.91(m,12H).

example 52: synthesis of Compound Id37

Compound Ic5 (636mg, 2mmol, 1eq) and NMP (8 mL) were sequentially added to a two-necked flask, after dissolution and clarification, powdered potassium carbonate (276mg, 2mmol) was added, after stirring for 15mins in an ice bath, isocyanto 2-isopropyl valeryl ester (407mg, 2.2mmol, 1.1eq) was added dropwise, and after 3h in an ice bath reaction, TLC showed that the starting material was substantially reacted completely. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of a water layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, purifying by using column chromatography, and concentrating to obtain the yellow compound Id37 of 200mg in total. ¹ H NMR(500MHz, DMSO-d6)δ12.71(s,1H),10.64(s,1H),8.13(d,J＝8.3Hz,1H),7.82 (dd,J＝7.7,1.8Hz,1H),7.70(dd,J＝8.0,1.3Hz,1H),7.63(td,J＝7.7, 1.8Hz,1H),7.56(td,J＝7.5,1.3Hz,1H),6.63(s,1H),4.94(tt,J＝13.2, 6.3Hz,1H),3.90(dd,J＝8.4,6.4Hz,1H),2.17(s,3H),2.09(q,J＝6.7 Hz,1H),1.22(dd,J＝6.3,1.2Hz,6H),1.00–0.90(m,6H).MS(ESI, ev):m/z＝504.3[M+H] ⁺ 。

Example 53: synthesis of Compounds Id38 and Id39

Adding the compound Ic5 (636mg, 2mmol, 1eq) and NMP (8 mL) into a double-neck flask in sequence, adding powdered potassium carbonate (276mg, 2mmol) after the compound Ic5 and the NMP are dissolved and clarified, continuing to dropwise add isopropyl isocyanate (345mg, 2.2mmol,1.1 eq) after stirring for 15mins in an ice bath, and after reacting for 3 hours in the ice bath, TLC shows that the raw materials are basically reacted completely. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of an aqueous layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, purifying and concentrating by using column chromatography to obtain 450mg of the yellow compound Id38 and 120mg of the yellow compound Id39. Id38: ¹ H NMR(500MHz,DMSO-d6)δ12.71(s,1H),10.65(s,1H),8.25(d,J＝ 7.3Hz,1H),7.83(dd,J＝7.6,1.7Hz,1H),7.70(dd,J＝8.0,1.3Hz,1H), 7.63(td,J＝7.7,1.8Hz,1H),7.56(td,J＝7.5,1.3Hz,1H),6.64(s,1H), 4.97–4.85(m,1H),4.07(p,J＝7.2Hz,1H),2.17(s,3H),1.34(d,J＝ 7.2Hz,3H),1.20(dd,J＝6.3,5.2Hz,6H).MS(ESI,ev):m/z＝498.3[M +18] ⁺ 。Id39： ¹ H NMR(500MHz,DMSO-d6)δ12.67(s,1H),8.57(d,J ＝7.2Hz,1H),8.40(d,J＝7.3Hz,1H),7.87(dd,J＝7.7,1.8Hz,1H), 7.72(dd,J＝8.0,1.2Hz,1H),7.65(td,J＝7.8,1.7Hz,1H),7.58(td,J＝ 7.5,1.3Hz,1H),6.81(s,1H),4.91(pd,J＝6.3,1.1Hz,2H),4.08(h,J＝ 7.0Hz,2H),2.18(s,3H),1.33(dd,J＝7.3,5.2Hz,6H),1.21–1.17(m, 12H).

example 54: synthesis of Compound Id40

Adding the compound Ic5 (636mg, 2mmol, 1eq) and NMP (8 mL) into a double-neck flask in sequence, adding powdered potassium carbonate (276mg, 2mmol) after the compound Ic5 and the NMP are dissolved and clarified, continuing to dropwise add isopropyl isocyanate (402mg, 2.2mmol,1.1 eq) after stirring in ice bath for 15mins, reacting in ice bath for 3h, and then TLC shows that the raw materials are basically and completely reacted. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of a water layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, purifying by using column chromatography, and concentrating to obtain the yellow compound Id40 of 450mg in total. ¹ H NMR(500MHz,DMSO-d6)δ 12.71(s,1H),10.65(s,1H),8.15(t,J＝6.1Hz,1H),7.83(dd,J＝7.7,1.8 Hz,1H),7.70(dd,J＝8.1,1.3Hz,1H),7.67–7.62(m,1H),7.56(td,J＝ 7.5,1.3Hz,1H),6.63(s,1H),4.72(tt,J＝8.9,3.9Hz,1H),3.83(d,J＝ 6.0Hz,2H),2.17(s,3H),1.79(tt,J＝7.4,3.5Hz,2H),1.73–1.64(m, 2H),1.56–1.20(m,6H).MS(ESI,ev):m/z＝502.3[M+H] ⁺

Example 55: synthesis of Compound Id41

Sequentially adding the compound Ic5 (636 mg,2mmol, 1eq) and NMP (8 mL) into a double-neck bottle, adding powdered potassium carbonate (276 mg, 2mmol) after the compound Ic5 and NMP are dissolved and clarified, continuing to dropwise add cyclopentyl isocyanate acetate (372mg, 2.2mmol,1.1 eq) after stirring for 15mins in ice bath, reacting for 3h in ice bath, and then displaying that the raw materials are basically reacted completely by TLC. And (3) post-treatment: the reaction solution is treated with DCM/H ₂ And (3) extracting, adjusting the pH of a water layer to weak acidity by using 1M HCl, separating liquid, washing an organic phase by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, purifying by using column chromatography, and concentrating to obtain the yellow compound Id41 of 450mg in total. ¹ H NMR(500MHz,DMSO-d6)δ 12.71(s,1H),10.67(s,1H),8.15(t,J＝6.1Hz,1H),7.83(dd,J＝7.6,1.7 Hz,1H),7.70(dd,J＝8.3,1.4Hz,1H),7.63(td,J＝7.7,1.7Hz,1H), 7.56(td,J＝7.5,1.3Hz,1H),6.64(s,1H),5.12(tt,J＝5.9,2.7Hz,1H), 3.81(d,J＝6.0Hz,2H),2.17(s,3H),1.87–1.78(m,2H),1.73–1.49(m, 6H).MS(ESI,ev):m/z＝488.3[M+H] ⁺

Example 56: synthesis of Compounds Id42, id43 and Id44

Compound Ic5 (1g, 3)14mmol and NMP (10 mL) were added to the clear solution in the eggplant-shaped bottle in sequence, and K was added ₂ CO ₃ (434mg, 3.14mmol, 1eq), nitrogen replacement, adding 4-chloromethyl-5-methyl-1,3-dioxol-2-one (171 uL, 3.14mmol,1 eq) under ice bath, reacting for 2h in ice bath, TLC shows no obvious change, adding 0.5eq NaI, converting to room temperature reaction for 2h, keeping 40% of TLC raw material, heating to 40 ℃ for 2h without obvious change, continuing to add 1eq K ₂ CO ₃ After reacting for 2h at 40 ℃, TLC shows that the newly generated two small polar points have light fluorescence and the raw materials are concentrated. And (3) post-treatment: filtrate with DCM/H ₂ Extracting with O for three times (adjusting pH of water layer to subacidity with 1M hydrochloric acid), separating, washing organic phase with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying by column chromatography to obtain compounds:

Id41： ¹ H NMR(500MHz,DMSO-d6)δ12.89(s,1H),10.41(s, 1H),7.67(dd,J＝82.6,49.3Hz,4H),6.63(s,1H),4.89(s,2H),2.15(s, 3H),2.04(s,3H).

Id42： ¹ H NMR(500MHz,DMSO-d6)δ12.51(s,1H),9.49(s,1H), 7.83(d,J＝7.7Hz,1H),7.70(d,J＝7.7Hz,1H),7.63(t,J＝7.4Hz,1H), 7.57(d,J＝7.6Hz,1H),6.67(s,1H),5.07(s,2H),2.19(s,3H),2.07(s, 3H).

Id43： ¹ H NMR(500MHz,DMSO-d6)δ12.79(s,1H),7.85(dt,J＝ 7.8,2.3Hz,1H),7.71(t,J＝5.4Hz,1H),7.65(tt,J＝5.2,2.0Hz,1H), 7.57(t,J＝7.6Hz,2H),6.77(s,1H),5.03(s,2H),4.93(s,2H),2.21(d,J ＝3.2Hz,3H),2.10(s,3H),1.99(s,3H).

pharmacological examples

The main reagents, instruments, cells and virus information used in the following pharmacological examples are as follows:

primary reagent

Main instrument

Enzyme-linked immunosorbent assay (SYNERGY-H1);

a microanalysis balance (XP 26, mettler-toledo instruments shanghai ltd);

vortex shaker (SI-A256, scientific Industries, inc.);

centrifuges (5417R, eppendorf);

pipette guns (Eppendorf);

LC/MS-MS LC (Waters), mass (Applied Biosystems);

biological safety cabinets (AC 2-3S1, ESCO);

a carbon dioxide incubator (Thermo Scientific herracell 150i, thermo Scientific);

a water purifier (seepage source SYS ultrapure water machine);

StepOne Plus Real-time PCR system(4376600，ABI)；

TC20 ^TM an automated cell counter (1450102, BIO-RAD);

T100 ^TM Thermal Cycler(1861096，BIO-RAD)；

centrifuge (Micro 21/21R Saimei fly, thermo Scientific).

Enzymes, cells and viruses

3CL protease: the recombinant full-length coronavirus 3CL protease is self-prepared according to the genome sequence of the coronavirus, the GenBank numbers of SARS-CoV-2, SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV and OC43-CoV genomes are respectively MN908947.3, AAP13442.1, MT387202.1, AF304460.1, AY597011.2, AY567487.2 and AY903459.1, and the DNA sequences required by expression of seven coronavirus 3CL protease proteins are synthesized by Nanjing Kinsrui biology GmbH;

vero E6, hepG2, HEK293 and L02 cells were purchased from ATCC;

SARS-CoV-2 is obtained from national viral resource library application.

Pharmacological example 1: test of inhibitory Activity of Baicalein derivatives Ia1-7, ib1-7 and Ic1-8 on SARS-CoV-2 3CLpro

Evaluating and measuring the inhibitory activity of baicalein derivatives Ia1-7, ib1-7 and Ic1-8 on the activity of SARS-CoV-2 3CLpro enzyme by using a fluorescence resonance energy transfer method. The volume of the whole enzymatic reaction system was 120. Mu.L, the final concentration of protease was 30nM and the final concentration of substrate was 20. Mu.M. The buffer of the reaction system included 50mM Tris pH7.3, 1mM EDTA. SARS-CoV-2 3CLpro protease and compounds of different concentrations were added to a 96-well plate, incubated at 30 ℃ for 10min, substrate was added and read quickly in a microplate reader. The excitation and emission light were 320nM and 405nM, respectively. The test time was 3.5min, and fluorescence was read every 35 s. The final result is obtained by fitting the reading value of the previous 2min to obtain the reaction rate, and the reaction rate is compared with the control group (DMSO) to calculate the inhibition rate, wherein the calculation formula is as follows: inhibition = [1- (test group reaction rate/control group reaction rate)]X 100%. IC was obtained by fitting with the software GraphPad Prism 8 ₅₀ Values and inhibition rate curves. FIGS. 1 to 7 are graphs showing the inhibition of SARS-CoV-2 3CLpro by baicalein derivatives Ia7, ib1-3 and Ic3-5, respectively.

The inhibition rate and IC of baicalein derivatives Ia1-7, ib1-7 and Ic1-8 on SARS-CoV-2 3CLpro ₅₀ The values are listed in table 1 below.

Table 1: inhibition of SARS-CoV-2 3CLpro by baicalein derivatives Ia1-7, ib1-7 and Ic1-8

"-" indicates not measured.

Pharmacological example 2: evaluation of replication inhibitory Activity of Compound Ic5 against SARS-CoV-2 WIV04 Strain and south African Strain B.1.351

Test compound Ic5 was dissolved in DMSO at 40mM stock, and on the day of the test, the compound stock was serially diluted 10-fold with DMEM, i.e., 1 μ L of the compound stock was added to 9 μ L of DMSO, and after 2 10-fold dilutions, 0.4mM dilutions were obtained, 20-fold dilutions were made to 20 μ M, and then three-fold gradient dilutions were continued to obtain 6.7, 2.2, 0.74, 0.25, and 0.08 μ M in this order.

Vero E6 cells were used, vero E6 cells (50000 cells/well) were added to a 48-well plate, 100. Mu.L/well of a medium containing the compound at a gradient concentration was added, and one hour later, SARS-CoV-2 WIV04 strain or south Africa strain B.1.351 was added, with a multiplicity of infection (MOI) of 0.01. After 1 hour of co-incubation, the supernatant was aspirated, washed and 200. Mu.L/well of medium containing compounds at a gradient concentration was added again and incubated at 37 ℃ for 24 hours. After 24 hours, collecting cell supernatant, extracting supernatant virus RNA, detecting the copy number of the supernatant virus by using a real-time fluorescent quantitative PCR method, calculating the inhibition rate of the compound according to the copy number of the virus, and calculating the EC of the compound ₅₀ 。

The data analysis process used GraphPad Prism 6 and Excel software. The degree of inhibition of SARS-CoV-2 replication by different concentrations of compound was calculated using the following formula:

suppression% = [1- (I/I) ₀ )]×100％

Wherein% inhibition represents the percent inhibition of SARS-CoV-2 replication by the compound, I and I ₀ The copy number of SARS-CoV-2 viral RNA in cell supernatants in compound and control wells (DMSO group) respectively is indicated.

Compound EC ₅₀ Calculated using GraphPad Prism 6 software by equation fitting:

Y＝Bottom+(Top-Bottom)/(1+10^((LogIC ₅₀ -X)*HillSlope)

wherein X is the Log value of the detected concentration of the test sample, Y is the inhibition percentage under the corresponding concentration, and Bottom and Top are respectively the minimum and maximum inhibition percentages.

The test result shows that Ic5 inhibits WIV strain half effective concentration EC in Vero E6 cells ₅₀ 0.72. + -. 0.18. Mu.M, see FIG. 8.

The test result shows that Ic5 can obviously inhibit the replication of SARS-CoV-2 south Africa strain B.1.351 in Vero E6 cell, EC ₅₀ 1.21 + -0.44, see FIG. 9.

Pharmacological example 3: examination of the Effect of Compound Ic5 on cell viability

Removing supernatant from cells in a 96-well plate, adding diluted compound Ic5 to each well, incubating for 24 hours, and detecting the cells by using a CCK8 detection kitAnd (4) vitality. The test results show that Ic5 is very weak in cytotoxicity and CC for Vero E6 cells ₅₀ Greater than 500. Mu.M, see FIG. 10.

Pharmacological example 4: ICR mouse PK property test experiment for detecting compound Ic5

ICR mice (purchased from Ministry of birth control science institute experimental animals, shanghai) were administered with 5mg/Kg of intravenous injection, vehicle: 5% dimethyl sulfoxide +5% ethanol +40% polyethylene glycol 300+50% normal saline; the dosage of the group for intragastric administration is 100mg/Kg, and the solvent: 5% dimethylsulfoxide +95%0.5% hydroxypropylmethylcellulose solution. Blood was collected via the inframandibular vein at 0.03 mL/time point. Samples were placed in tubes containing K2-EDTA and stored on ice until centrifugation. Blood samples were centrifuged at 6800g for 6 minutes at 2-8 ℃ within 1 hour after collection and stored frozen at about-80 ℃. A20. Mu.L plasma sample containing 10ng/mL IS was precipitated with 400. Mu.L methanol. The mixture was spun for 1 minute and centrifuged for 7 minutes. Transfer 400 μ L of supernatant to 96-well plate. A10. Mu.L aliquot of the supernatant was injected for LC-MS/MS analysis.

When the dose of the gavage administration group is 100mg/Kg, the test results are shown in the following table 2:

TABLE 2

Pharmacological example 5: test of inhibitory Activity of Baicalein derivatives Ic10-21, id5-6, id8-9, id14-16, id18 and Id30-32 on SARS-CoV-2 3CLpro

The inhibition activity of partial baicalein derivatives on the activity of SARS-CoV-2 3CLpro enzyme is evaluated and determined by a fluorescence resonance energy transfer method. The volume of the whole enzymatic reaction system was 120. Mu.L, the final concentration of protease was 30nM and the final concentration of substrate was 20. Mu.M. The buffer of the reaction system included 50mM Tris pH7.3, 1mM EDTA. SARS-CoV-2 3CLpro protease and compounds of different concentrations were added to a 96-well plate, incubated at 30 ℃ for 10min, substrate was added and read quickly in a microplate reader. The excitation and emission light were 320nM and 405nM, respectively. The test time was 3.5min, and fluorescence was read every 35 s. The final result is obtained by taking the reading of the first 2min to fit the reaction rate, and comparing with a control group (DMSO) to calculate the inhibition rate.

Table 3: inhibition of SARS-CoV-2 3CLpro by baicalein derivatives Ic10-21, id5-6, id8-9, id14-16, id18 and Id30-32

"-" indicates not measured.

Pharmacological example 6: part of baicalein derivatives was evaluated for the replication inhibitory activity of SARS-CoV-2 WIV04 strain.

Test compounds were dissolved in DMSO to 40mM stock solution, and on the day of testing, the stock solution of compounds was serially diluted with DMEM to give 1. Mu.M.

Vero E6 cells were used, vero E6 cells (50000 cells/well) were added to a 48-well plate, 100. Mu.L/well of medium containing compounds at a gradient concentration was added, and after one hour SARS-CoV-2 WIV04 strain was added or the multiplicity of infection (MOI) was 0.01. After 1 hour of co-incubation, the supernatant was aspirated, washed and 200. Mu.L/well of medium containing 1. Mu.M compound was added again and incubated at 37 ℃ for 24 hours. After 24 hours, collecting cell supernatant, extracting supernatant virus RNA, detecting the copy number of the supernatant virus by using a real-time fluorescent quantitative PCR method, and calculating the compound inhibition rate according to the virus copy number, wherein the result is respectively represented by A, B or C, wherein A is 70-100%, B is 30-69% and C is 10-29%.

Table 4: baicalein derivatives Ic 13-18, id8, id16, id18, id21 and Id 28-32 having activity of inhibiting replication of SARS-CoV-2 WIV04 strain

Pharmacological example 7 ICR mouse PK Property testing experiment for Compound Id16

The intravenous injection dosage of ICR mice is 10mg/kg, and the solvent: 40% PEG400+10% Solutol HS 15+50% distilled water; the dose of an ICR mouse gavage administration group is 100mg/kg, and the ratio of a solvent: 5% dimethyl sulfoxide +5% solutol HS 15+90% normal saline, sodium hydroxide adjusted to pH 8. Blood was collected via the inframandibular vein at 0.03 mL/time point. Samples were placed in tubes containing K2-EDTA and stored on ice until centrifugation. Blood samples were centrifuged at 6800g for 6 minutes at 2-8 ℃ within 1 hour after collection and stored frozen at about-80 ℃. A20 μ L sample of plasma containing 10ng/mL IS was precipitated with 400 μ L methanol (1 mL methanol: 0.1mL (1M HCl +1% VC)). The mixture was spun for 1 minute and centrifuged for 7 minutes. Transfer 400 μ L of supernatant to 96-well plate. A10. Mu.L aliquot of the supernatant was injected for LC-MS/MS analysis to detect Id16. The test results shown in table 5 indicate that the compound has a high oral bioavailability of 57.5% in mice and a high blood exposure.

TABLE 5 pharmacokinetic parameters of Id16 ICR mice at 10mg/kg for intravenous injection and 100mg/kg for gastric gavage

The dosage of an ICR mouse gastric perfusion administration group is 200mg/kg, and the ratio of a solvent: 10% PEG400+90% (0.5% hydroxypropyl methylcellulose). According to the same administration and plasma treatment method when the dose of the Id16 intragastric administration group is 100mg/kg, LC-MS/MS detects Id16 and a hydrolysis metabolite Ic5 and a post-hydrolysis glucuronidation metabolite Id8 respectively. As shown in the following Table 6, the metabolites Ic5 and Id8 of the compound Id16 in the mice still have good anti-neocoronaviral activity, and not only a high plasma exposure of Id16 but also Ic5 and Id8 was detected in the plasma of the mice when the mice were gavaged with 200 mg/kg.

TABLE 6 pharmacokinetic parameters of Id16 ICR mice administered 200mg/kg by gavage

Pharmacological example 8 cynomolgus monkey PK Property testing experiment of Compound Id16

Cynomolgus monkey (purchased from asahi biotechnology limited, guangzhou) was administered at an intravenous dose of 10mg/kg, vehicle: 5% DMSO +5% Solutol HS 15+ pH adjusted to 8 with sodium hydroxide; the dosage of the gavage administration group of the cynomolgus monkeys is 100mg/kg, and the solvent: 10% PEG400+90% (0.5% hydroxypropyl methylcellulose). Blood will be collected via the femoral vein or other suitable vein at 1.0 mL/time point. The samples will be placed in tubes containing K2-EDTA and stored on ice until centrifugation. Blood samples were centrifuged at 2200g for 10 minutes at 2-8 ℃ within 1 hour after collection and stored frozen at about-80 ℃. A40 μ L sample of plasma, containing 10ng/mL IS, was precipitated with 400 μ L of methanol (1 mL methanol: 0.1mL (1M HCl +1% VC)). The mixture was spun for 1 minute and centrifuged at 18000 g for 7 minutes. Transfer 400 μ L of supernatant to 96-well plate. A1. Mu.L aliquot of the supernatant was injected for LC-MS/MS analysis. Id16 and metabolites Ic5 and Id8 were detected, respectively. The test results are shown in table 7 below, where compound Id16 and metabolites Ic5 and Id8 both had higher plasma exposure and good PK properties in cynomolgus monkeys.

TABLE 7 pharmacokinetic parameters of Id16 cynomolgus monkey at 10mg/kg for intravenous administration and 100mg/kg for intragastric administration

Pharmacological example 9 liver and Kidney cytotoxicity test experiments at cell level of Compounds Ic5, id8, id16

The stock solutions of each compound (100. Mu.M in dimethylsulfoxide) were diluted to 200, 100, 50, 25, 10, 5, 2.5, 1, 0.5. Mu.M with cell culture medium. The negative control was 0.5% dimethylsulfoxide in cell culture medium. Human hepatoma cells HepG2, human embryonic kidney cells HEK293 and human normal liver cells L02 at 1X 10 per well ⁴ Density of individual cells plated in 96-well plates for 48 hours. Different concentrations of compound were then added to the cells and incubated for 48 hours (n = 6). The cytotoxicity was detected by using CCK8 detection kit. The absorbance was measured by an automated microplate reader (Biotek, winooski, VT, USA) at a wavelength of 450 nm. Half Inhibitory Concentration (IC) was calculated for each compound using GraphPad Prism 6 software ₅₀ ) The value is obtained. The results are shown in Table 8, and the IC of the three compounds tested on different hepatorenal cells ₅₀ Are all larger than 25 μ M.

TABLE 8 inhibitory Activity of Compounds Ic5, id8, id16 on liver and Kidney related cells.

Pharmacological example 10. Effect of Compounds Ic5 and Id16 against Neocoronaviruses in New coronal mouse model prepared by Adenoviral vector transduction of hACE2

H11-K18-hACE2 mice, 7-8 weeks old, were transferred to the institute BSL-3 laboratory. Each mouse was infected with 1X 10 of the drug by nasal drip ³ PFU SARS-CoV-2 virus, day 0. After 2 hours of SARS-CoV-infection, the mice were treated with the drug by gavage. Mice were divided into 3 groups. Control group (BID, oral vehicle), group 10 (BID, oral vehicle) with 200mg/kg Ic5 and 200mg/kg Id16 (BID, oral vehicle). Wherein the administration is once on

day

0, 2 times on

days

1, 2 and 3, respectively, at 9h intervals, and the body weight of the mice is recorded daily.

On day 2, 5 mice were sacrificed per group, and on day 4,5 mice were sacrificed per group. The lung tissue of the mice was then extracted, with the right lung being triturated with DMEM, and a portion of the homogenate taken for RNA extraction, the remainder being stored at-80 ℃.

Virus copy number determination: extracting RNA from the ground lung tissue by using TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0, and performing PrimeScript according to the Kit TaKaRa PrimeScript ^TM RT reagent Kit with gDNA Eraser instructions for RNA extraction and reverse transcription, followed by TaKaRa

Premix Ex Taq ^TM II (Tli RNaseH Plus) absolute quantitative detection of viral RNA copy number in tissues using real-time fluorescent quantitative PCR technology. Sample copy number was calculated by standard plasmid concentration. The results are shown in FIG. 11, where Ic5 exhibited some anti-neocoronaviral effect on day 4, and Id16 exhibited significant anti-neocoronaviral effect on both day 2 and day 4.

Pharmacological example 11. Sub-acute toxicity test of Compound Id16 in mice

The male ICR mice are provided with a control group, a 200mg/kg group, a 400mg/kg group and a 800 mg/kg group, 10 mice in each group are administrated once a day, the food intake, the body weight and symptoms are observed, the result shows that the administration is repeated for 7 days, the mice in each dose group have no obvious symptoms, no obvious difference in body weight and no obvious difference in food intake, and in combination, the compound Id16 has no obvious toxicity in the sub-acute toxicity test of the mice, and the detailed tables are 9a, 9b and 9c.

TABLE 9a observation of symptoms in mice with Compound Id16 on 7-day repeat dosing

TABLE 9b Compound Id16 changes in body weight of mice on 7-day repeat dosing

TABLE 9c Compound Id16 intake Change in mice on 7-day repeat dosing

Pharmacological example 12 testing of the inhibitory Activity of Compound Ic5 on the different 3CLpro enzymes

And evaluating and determining the inhibitory activity of Ic5 on the enzyme activity of 3CLpro from other sources by using a fluorescence resonance energy transfer method. The experimental procedure was the same as in pharmacological example 1, except that the 3CLpro enzyme was from other coronaviruses as shown in table 10 below. The results of the experiments are shown in table 10 below,

TABLE 10 inhibitory Activity of Compound Ic5 on different 3CLpro enzymes

As shown in Table 10, compound Ic5 has good inhibitory activity on different 3CLpro enzymes tested, IC ₅₀ Are all less than 0.5. Mu.M.

The compound of the invention has stronger SARS-CoV-2 3CLpro inhibitory activity and stronger cell level anti-novel coronavirus replication function, can be orally absorbed, and is expected to be used for preventing and treating diseases caused by novel coronavirus.

Claims

1. A baicalein derivative represented by the following general formula I, a stereoisomer or a pharmaceutically acceptable salt thereof:

-SO ₂ OH、

Wherein the substituent of the C1-C6 alkyl is selected from cyano,

-O(CH ₂ ) _m OR ₁₂ 、-OC(＝O)OR ₁₃ And

R ₁₁ selected from C1-C6 alkyl, C3-C10 cycloalkyl; in particular from C1-C4 alkyl;

R ₁₂ selected from C1-C6 alkyl, C3-C10 cycloalkyl; in particular from C1-C4 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ selected from C1-C6 alkyl; in particular from C1-C4 alkyl;

in particular, R ₉ And R ₁₀ Each independently selected from H, C-C4 alkyl, C1-C4 alkoxy formyl substituted C1-C4 alkyl, C5-C6 cycloalkoxy formyl substituted C1-C4 alkyl;

in particular, it is possible to use, for example,

is selected from

In particular, it is possible to use, for example,

is selected from

In particular, it is possible to use, for example,

is selected from

In particular, R ₁ And R ₂ Are all H;

in particular, R ₃ A substituent selected from H, cyano, halogen, unsubstituted or substituted C1-C4 alkyl, unsubstituted or substituted C2-C4 alkenyl, unsubstituted or substituted C3-C4 cycloalkyl, or unsubstituted or substituted C2-C4 fatty acyl, wherein C1-C4 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl, or C2-C4 fatty acyl is selected from halogen and hydroxy;

more particularly, R ₃ Selected from H, cyano, F, cl, br, I, unsubstituted or substituted C1-C3 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl, or C2-C4 fatty acyl, wherein the substituents of C1-C3 alkyl are selected from F, cl and hydroxyl, and the number of substituents can be one or more, for example, 1, 2 or 3;

more particularly, R ₃ Selected from the group consisting of H, cyano, F, cl, br, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, acetyl, trifluoromethyl, cyclopropyl, hydroxymethyl, vinyl, 1-propenyl, 2-propenyl, allyl, and nitro;

in particular, R ₄ -R ₈ Each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ Cyano, C6-C10 aryl which is unsubstituted or substituted by methyl or ethyl, wherein the substituent for C1-C6 alkyl is halogen, R ₁₅ And R ₁₆ Each independently selected from H and C2-C4 fatty acyl;

more particularly, R ₄ -R ₈ Each independently selected from H, halogen, C1-C3 alkyl substituted with one or more (e.g. 1, 2 or 3) F, nitro, amino, acetamido, cyano, phenyl, tolyl and ethylphenyl;

more particularly, R ₄ -R ₈ Each independently selected from the group consisting of H, cyano, halogen, trifluoromethyl, methyl, ethyl, nitro, amino, acetamido, phenyl, and tolyl;

represents a single or double bond;

provided that R ₁ -R ₈ Not both being H, and compounds of formula I excluding compounds

2. The baicalein derivative, a stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein the baicalein derivative of the general formula I is represented by the following general formulae II to IV:

in the above general formulas II to III, R ₄ -R ₈ Are as defined in claim 1, respectively; in the above formula IV, except for R ₃ Not being other than H, R ₃ And R ₄ -R ₈ Are each as defined in claim 1.

3. The baicalein derivative, its stereoisomer or pharmaceutically acceptable salt according to claim 1, wherein R ₁ And R ₂ Each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aralkyl, C7-C12 aralkyl,

-SO ₂ OH、

Wherein the substituent of the C1-C6 alkyl is selected from cyano,

-O(CH ₂ ) _m OR ₁₂ 、-OC(＝O)OR ₁₃ And

R ₁₁ selected from C1-C6 alkyl;

R ₁₂ selected from C1-C6 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ is selected from C1-C6 alkyl;

wherein R is ₉ And R ₁₀ Each independently selected from H, C-C6 alkyl, by R ₁₄ OC (= O) -substituted C1-C10 alkyl; r is ₁₄ Selected from C1-C6 alkyl, C3-C8 cycloalkyl; in particular from C1-C4 alkyl, C5-C6 cycloalkyl;

represents a single bond or a double bond.

4. The baicalein derivative, a stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein R ₁ And R ₂ Each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aralkyl, C7-C12 aralkyl,

-SO ₂ OH、

Wherein the substituents of the C1-C6 alkyl are selected from cyano,

-O(CH ₂ ) _m OR ₁₂ 、-OC(＝O)OR ₁₃ And

R ₁₁ selected from C1-C6 alkyl;

R ₁₂ selected from C1-C6 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ selected from C1-C6 alkyl;

wherein R is ₉ And R ₁₀ Each independently selected from H, C-C6 alkyl, by R ₁₄ OC (= O) -substituted C1-C10 alkyl; r ₁₄ Selected from C1-C6 alkyl, C3-C8 cycloalkyl; in particular from C1-C4 alkyl, C5-C6 cycloalkyl;

R ₃ selected from H, cyano, F, cl, br, I, unsubstituted or substituted C1-C3 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl, or C2-C4 fatty acyl, wherein the substituents of C1-C3 alkyl are selected from F, cl and hydroxy, and the number of substituents may be one or more, for example 1, 2 or 3;

represents a single bond or a double bond.

5. The baicalein derivative, its stereoisomer or pharmaceutically acceptable salt according to claim 1, wherein R ₁ And R ₂ Are all H;

R ₃ selected from H, cyano, halogen, nitro, unsubstitutedOr substituted C1-C6 alkyl, unsubstituted or substituted C2-C6 alkenyl, unsubstituted or substituted C3-C6 cycloalkyl, unsubstituted or substituted C2-C6 fatty acyl, C6-C12 aralkyl or C7-C12 aroyl, wherein the substituents of C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl or C2-C6 fatty acyl are selected from halogen and hydroxy;

represents a single bond or a double bond.

6. The baicalein derivative, a stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein R ₁ And R ₂ Are all H;

R ₃ selected from the group consisting of H, cyano, F, cl, br, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, acetyl, trifluoromethyl, cyclopropyl, hydroxymethyl, vinyl, 1-propenyl, 2-propenyl, allyl, and nitro;

R ₄ -R ₈ each independently selected from the group consisting of H, cyano, halogen, trifluoromethyl, methyl, ethyl, nitro, amino, acetamido, phenyl, and tolyl;

represents a single bond or a double bond.

7. The baicalein derivative, a stereoisomer or pharmaceutically acceptable salt thereof according to claim 1, wherein the baicalein derivative is selected from one of the following compounds:

8. a process for the preparation of a baicalein derivative as claimed in any one of claims 1-7, said process is carried out by one of the routes comprising the steps of:

route one:

wherein R is ₁ 、R ₂ Is C1-C6 alkyl, R ₃ -R ₈ Each as defined in any one of claims 1 to 7;

or

And a second route:

d) Compound I' -2 and BBr ₃ In dichloromethane to produce compound I-2, wherein R ₁ 、R ₂ Is C1-C6 alkyl, R ₃ -R ₈ Each as defined in any one of claims 1 to 7;

or

When R in the formula I ₃ Selected from CN, CF ₃ Cyclopropyl and halogen, prepared by the following route:

and a third route:

g) A compound of formula I' -4 with BBr ₃ Mixing in dichloromethane under an inert atmosphere such as nitrogen at-78 deg.C and reacting at 0-50 deg.C to produce the compound of formula I-3,

wherein R is ₁ 、R ₂ Is C1-C6 alkyl, R ₄ -R ₈ Are as defined in any one of claims 1 to 7, respectivelyDefined as R ₃ Selected from trifluoromethyl, cyclopropyl;

or

f1 A compound of a general formula I '-3 reacts for 1-28h at 0-150 ℃ in the presence of dichlorodicyano benzoquinone (DDQ), copper acetate, silver carbonate and NMP to obtain a compound of a general formula I' -5,

wherein R is ₁ 、R ₂ And R ₄ -R ₈ Each as defined in any one of claims 1 to 7, R ₃ Is cyano;

or

wherein R is ₁ 、R ₂ Is C1-C6 alkyl, R ₄ -R ₈ Each as defined in any one of claims 1 to 7, R ₃ Is a halogen, and the halogen is a halogen,

in particular, formula I' -1 is obtained by the following reaction:

reacting the compound of the general formula I-A with the compound of the general formula I-B in methanol under alkaline conditions, such as 10-50% sodium hydroxide or potassium hydroxide aqueous solution, at-20-50 ℃ for 0.1-24h to obtain a compound of the general formula I' -1;

in particular, it is possible to provide a device,

when R is ₃ When methyl, the compounds of formula I-A are obtained using the following reaction scheme:

j) Reacting the compound I-C with boron trifluoride diethyl etherate and acetic acid at 0-100 ℃ to obtain a compound I-A,

when R is ₃ In the case of acetyl, the compounds of formula I-A are obtained using the following reaction scheme:

mixing the compound I-F, acetic anhydride and nitromethane with aluminum trichloride at the temperature of-20-20 ℃, and reacting for 1-24h at the temperature of 0-50 ℃ to obtain a compound I-A;

in particular, the synthetic routes for the specific compounds Ic10 and Ic11 are as follows:

the compound 3,4,5-trimethoxyphenol and acetic acid or isobutyryl chloride are subjected to acylation reaction in the presence of boron trifluoride ethyl ether; then, carrying out reduction reaction under the action of trifluoroacetic acid and triethylsilane; the obtained reaction product is subjected to acetylation reaction in the presence of boron trifluoride diethyl etherate and acetic acid, and then reacts with o-chlorobenzoyl chloride to generate corresponding ester; the generated ester is subjected to rearrangement reaction under the action of alkali, the obtained rearrangement crude product is further subjected to ring closure reaction under the action of strong acid (such as concentrated sulfuric acid), and the ring-closed product is demethylated under the action of boron tribromide to obtain a final product Ic10 or Ic11, wherein R is methyl or isopropyl;

in particular, the synthetic route for the specific compound Ic18 is shown below:

the methylation product of the compound Ic15 is subjected to a reduction reaction under the action of stannous chloride dihydrate to generate a corresponding amino compound, acetic anhydride is added into the reaction product to perform an acetylation reaction to generate a corresponding amide compound, and the amide compound is subjected to a demethylation reaction under the action of boron tribromide to generate a compound Ic18;

in particular, the synthetic route for the specific compound Ic20 is as follows:

the methylation product of compound Ic1 was deiodinated in the presence of potassium carbonate, xphos, potassium fluoride and palladium acetate to give a deiodinated product which was taken up at BBr ₃ Demethylation to give compound Ic20;

route four

R in the general formula I is shown below ₁ And R ₂ At least one of which is not H, R ₃ Synthetic route to compounds that are methyl:

dissolving a compound of the general formula Id' in an organic solvent, reacting with a compound having R as defined in claims 1 to 7, respectively, under the action of a base ₁ /R ₂ To give a compound of the general formula Id in which R is a hydrogen atom ₁ 、R ₂ 、R ₄ -R ₈ Are as defined in claims 1 to 7, respectively, and R ₁ And R ₂ At least one of which is not H,

preferably, in said scheme four, said organic solvent is selected from acetonitrile, acetone, N-Dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF) and N-methylpyrrolidone (NMP); the base is selected from triethylamine, potassium carbonate, N-diisopropylethylamine and pyridine; said has R ₁ /R ₂ The corresponding non-hydrogen radical reactant is selected from isocyanoacetoacetateEthyl acylate, isopropyl isocyanate acetate, ethyl 3-methylbutyrate-2-isocyanate, isopropyl propionate-2-isocyanate, cyclohexyl isocyanate acetate, cyclopentyl isocyanate acetate, 4-chloromethyl-5-methyl-1,3-dioxol-2-one, isopropyl chlorocarbonate, bromoacetonitrile, chlorosulfonic acid, isobutyric anhydride, 2-methoxyethoxymethyl chloride, N' -dimethylcarbamoyl chloride, ethyl isocyanate and methylaminocarbonyl chloride;

in particular, the synthesis of specific compounds Id8 and Id9 is shown in the following reaction scheme:

the Id' compound is first reacted with acetic anhydride, followed by benzyl bromide to protect the hydroxyl group, followed by Pd (OH) ₂ In the presence of C, with H ₂ Reducing, reacting the reaction product with acetyl bromide-alpha-D-glucuronic acid methyl ester or acetyl bromide-alpha-D-glucose, and hydrolyzing ester group in the compound with alkali to obtain the final product, wherein in the reaction formula, R is ₄ -R ₈ Are as defined in claims 1-7, respectively.

9. A pharmaceutical composition comprising a pharmaceutically effective amount of one or more selected from the group consisting of the baicalein derivative, the stereoisomer thereof, and the pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 as a pharmaceutically active ingredient, and optionally a pharmaceutically acceptable adjuvant.

10. Use of a baicalein derivative, a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 9, in the manufacture of a medicament having an activity of inhibiting a coronavirus main protease, or an activity against a novel coronavirus and a variant thereof (such as WIV strain or south african strain b.1.351), SARS virus, MERS virus, or an activity against enterovirus EV71, coxsackie virus and norovirus.