CN115819391A - A class of baicalein derivatives, its preparation method and use - Google Patents

Abstract

The present invention relates to a class of baicalein derivatives, their preparation method and application. The structure of the baicalein derivative of the present invention is shown in the following general formula I. After pharmacological experiments, the baicalein derivatives of the present invention exhibit inhibitory activity against novel coronaviruses, and can be used as anti-new coronavirus drugs or potential drugs for treating related diseases caused by new coronaviruses.

Description

A kind of baicalein derivative, its preparation method and use

technical field

The invention belongs to the field of medicinal chemistry, and relates to a class of baicalein derivatives, a preparation method thereof and an antiviral application thereof.

Background technique

Scutellaria baicalensis, also known as camellia root and Tujincha root, is used as medicine from the roots. It is bitter in taste and cold in nature. Hemoptysis embolism. Scutellaria baicalensis is an active ingredient in various heat-clearing and detoxifying Chinese patent medicines such as Qingfei Paidu Decoction, Jinhua Qinggan Granules, Shuanghuanglian Oral Liquid, and Lanqin Oral Liquid.

Baicalein, one of the main active ingredients of Scutellaria baicalensis, has antiviral effects. According to literature reports, the EC ₅₀ of baicalein inhibiting the replication of SARS virus is 12.5～25 μg/mL (Journal of Clinical Virology, 2004,31,69-75); the EC ₅₀ of inhibiting the replication of influenza A virus (H1N1) is 0.018 μM (Evidence -Based Complementary and Alternative Medicine, 2013, 750803); meanwhile, baicalein also exhibits 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 inhibitory effect. On the Vero E6 cell model infected with SARS-Cov-2, the EC ₅₀ of anti-SARS-CoV-2 replication is 2.94μM (Acta Pharmacologica Sinica, 2020, 41, 1167- 1177).

Baicalein is well absorbed in the gastrointestinal tract and is mainly distributed in tissues and organs such as liver, kidney, and lung. The metabolite of baicalein in rats is mainly the 7-hydroxyglucuronide of baicalein, which can be transported to the intestinal cavity through bile and intestinal mucosa to form reabsorption. After oral administration of baicalein to rats, the original form of baicalein was hardly detected in the blood, and the elimination half-life was 0.1 hour. of Pharmaceutical Sciences, 2014, 103, 2330-2337). It is of great significance to search for baicalein derivatives with antiviral effects and better pharmacokinetic properties in vivo.

In view of this, the present invention is proposed.

Contents of the invention

One object of the present invention is to provide a new class of baicalein derivatives.

Another object of the present invention is to provide an application of the baicalein derivative in the preparation of anti-coronavirus drugs, especially anti-new coronavirus drugs.

Another object of the present invention is to provide an application of the baicalein derivative in the preparation of drugs against enterovirus EV71, Coxsackie virus and norovirus.

In order to accomplish the above object, the present invention provides a baicalein derivative as shown in the following general formula I, its stereoisomer or pharmaceutically acceptable salt:

In the above general formula I, R _and R _are each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aromatic hydrocarbon, C7-C12 aroyl,

- O(CH₂)_mOR₁₂、-OC(＝O)OR₁₃和

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

-O(CH ₂ ) _m OR ₁₂ , -OC(=O)OR ₁₃ and

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

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

m means 1, 2 or 3;

R ₁₃ is selected from C1-C6 alkyl; especially selected from C1-C4 alkyl;

Wherein, R ₉ and R ₁₀ are each independently selected from H, C1-C6 alkyl, C1-C10 alkyl substituted by R ₁₄ OC (=O) (such as C1 substituted by R ₁₄ OC (=O) - C6 alkyl); R ₁₄ is selected from C1-C6 alkyl, C3-C8 cycloalkyl, especially selected from C1-C4 alkyl, C5-C6 cycloalkyl;

In particular, R ₉ and R ₁₀ are each independently selected from H, C1-C4 alkyl, C1-C4 alkoxyformyl substituted C1-C4 alkyl, C5-C6 cycloalkyloxyformyl substituted C1 -C4 alkyl;

可选自

In particular,

can be selected from

可选自

In particular,

can be selected from

可选自

In particular,

can be selected from

In particular, R ₁ and R ₂ may both be H;

_R3 is 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 group, C6-C12 aromatic hydrocarbon group or C7-C12 aroyl group, wherein, the substituent of C1-C6 alkyl group, C2-C6 alkenyl group, C3-C6 cycloalkyl group or C2-C6 fatty acyl group selected from halogen and hydroxyl; in particular, _R3 is 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 the substituents of C1-C4 alkyl, C2-C4 alkenyl, C3-C4 cycloalkyl or C2-C4 fatty acyl are selected from halogen and hydroxyl ; more particularly, R _is 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 substituent of C1-C3 alkyl is selected from F, Cl and hydroxyl, and the number of substituents can be one or more, such as 1, 2 or 3; more particularly, _R3 is 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 are each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ , cyano, C1-C4 alkyl substituted or unsubstituted C6- C10 aryl, C2-C6 fatty acyl, wherein, the substituent of C1-C6 alkyl is halogen, R ₁₅ and R ₁₆ are each independently selected from H and C2-C6 fatty acyl; especially, R ₄ -R ₈ are each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ , cyano, methyl or ethyl substituted or unsubstituted C6-C10 aryl, wherein, The substituent of C1-C6 alkyl is halogen, R ₁₅ and R ₁₆ are each independently selected from H and C2-C4 fatty acyl; more particularly, R ₄ -R ₈ are each independently selected from H, halogen, C1-C3 Alkyl, C1-C3 alkyl substituted by one or more (eg 1, 2 or 3) F, nitro, amino, acetamido, cyano, phenyl, tolyl, ethylphenyl; more particularly , R ₄ -R ₈ are each independently selected from H, cyano, halogen, trifluoromethyl, methyl, ethyl, nitro, amino, acetamido, phenyl, tolyl;

表示单键或双键；

Indicates a single or double bond;

The premise is that R ₁ -R ₈ are not H at the same time, and the compound of general formula I excludes the compound

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

In the above general formula II-III, the definitions of R ₄ -R ₈ are as above respectively; in the above general formula IV, except that R ₃ is not H, the definitions of R ₃ and R ₄ -R ₈ are respectively as above .

For example,

In a specific embodiment,

- SO₂OH、

R ₁ and R ₂ are each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aromatic hydrocarbon group, C7-C12 aroyl,

- SO ₂ OH,

- O(CH₂)_mOR₁₂、-OC(＝O)OR₁₃和

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

-O(CH ₂ ) _m OR ₁₂ , -OC(=O)OR ₁₃ and

R ₁₁ is selected from C1-C6 alkyl;

R ₁₂ is selected from C1-C6 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ is selected from C1-C6 alkyl;

Wherein, R ₉ and R ₁₀ are each independently selected from H, C1-C6 alkyl, C1-C10 alkyl substituted by R ₁₄ OC (=O)-; R ₁₄ is selected from C1-C6 alkyl, C3-C8 Cycloalkyl, in particular, R ₁₄ is selected from C1-C4 alkyl, C5-C6 cycloalkyl;

_R3 is 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 group, C6-C12 aromatic hydrocarbon group or C7-C12 aroyl group, wherein, the substituent of C1-C6 alkyl group, C2-C6 alkenyl group, C3-C6 cycloalkyl group or C2-C6 fatty acyl group selected from halogen and hydroxyl;

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

表示单键或双键。

Indicates a single or double bond.

In a specific embodiment,

- SO₂OH、

R ₁ and R ₂ are each independently selected from H, substituted or unsubstituted C1-C6 alkyl, C2-C6 fatty acyl, C6-C12 aromatic hydrocarbon group, C7-C12 aroyl,

- SO ₂ OH,

- O(CH₂)_mOR₁₂、-OC(＝O)OR₁₃和

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

-O(CH ₂ ) _m OR ₁₂ , -OC(=O)OR ₁₃ and

R ₁₁ is selected from C1-C6 alkyl;

R ₁₂ is selected from C1-C6 alkyl;

m is selected from 1, 2 or 3;

R ₁₃ is selected from C1-C6 alkyl;

Wherein, R ₉ and R ₁₀ are each independently selected from H, C1-C6 alkyl, C1-C10 alkyl substituted by R ₁₄ OC (=O)-; R ₁₄ is selected from C1-C6 alkyl, C3-C8 Cycloalkyl, especially selected from C1-C4 alkyl, C5-C6 cycloalkyl;

_R is 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 substituent of C1-C3 alkyl is selected from F, Cl and hydroxyl, and the number of substituents can be one or more, such as 1, 2 or 3;

R ₄ -R ₈ are each independently selected from H, halogen, substituted or unsubstituted C1-C6 alkyl, nitro, -NR ₁₅ R ₁₆ , cyano, methyl or ethyl substituted or unsubstituted C6- C10 aryl, wherein, the substituent of C1-C6 alkyl is halogen, R ₁₅ and R ₁₆ are each independently selected from H and C2-C4 fatty acyl;

表示单键或双键。

Indicates a single or double bond.

In a specific embodiment,

_{Both R1} and _R2 are H;

_R3 is 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 group, C6-C12 aromatic hydrocarbon group or C7-C12 aroyl group, wherein, the substituent of C1-C6 alkyl group, C2-C6 alkenyl group, C3-C6 cycloalkyl group or C2-C6 fatty acyl group selected from halogen and hydroxyl;

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

表示单键或双键。

Indicates a single or double bond.

In a specific embodiment,

_{Both R1} and _R2 are H;

_R is 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 are each independently selected from H, cyano, halogen, trifluoromethyl, methyl, ethyl, nitro, amino, acetamido, phenyl, tolyl;

表示单键或双键。

Indicates a single or double bond.

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

On the other hand, the present invention also provides the preparation method of the baicalein derivative shown in the above-mentioned general formula I, and described method is carried out by one of the route that comprises the following steps:

Route 1:

a) Compound I'-1 reacts with iodine element in dimethyl sulfoxide under an inert atmosphere such as a nitrogen atmosphere at 0-150°C for 1-36h to generate compound I"-1,

B) compound I "-1 reacts with BBr ₃ in dichloromethane to generate compound I-1,

Wherein R ₁ and R ₂ are C1-C6 alkyl, and R ₃ -R ₈ are as defined above;

or

Route two:

c) React intermediate I'-1 with anhydrous sodium acetate in absolute ethanol under an inert atmosphere such as nitrogen atmosphere at 0-100°C for 1-24h to generate compound I"-2;

d) Compound I"-2 reacts with BBr ₃ in dichloromethane to generate compound I-2, wherein R ₁ and R ₂ are C1-C6 alkyl, and R ₃ -R ₈ are as defined above;

or

When R ₃ in the general formula I is selected from CN, CF ₃ , cyclopropyl and halogen, it can alternatively be prepared by the following route:

Route three:

e) a compound of general formula I"'-2 and a halogenating reagent (such as N-chlorosuccinimide (NCS) or N-iodosuccinimide (NIS)) in acetonitrile or trifluoroacetic acid , react at 0-100°C for 1-24h to obtain a compound of general formula I"'-3;

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

g) the compound of the general formula I"'-4 and BBr ₃ are mixed in methylene chloride under an inert atmosphere such as a nitrogen atmosphere at -78 ° C and reacted at 0-50 ° C to generate a compound of the general formula I-3,

Wherein R ₁ and R ₂ are C1-C6 alkyl, R ₄ -R ₈ are respectively as defined above, and R ₃ is selected from trifluoromethyl and cyclopropyl;

or

f1) The compound of the general formula I"'-3 reacts at 0-150°C for 1-28h to obtain the compound of the general formula I"'-5 in the presence of dichlorodicyanobenzoquinone (DDQ), copper acetate, silver carbonate and NMP compound,

g1) the compound of the general formula I"'-5 and BBr ₃ are mixed in methylene chloride under an inert atmosphere such as a nitrogen atmosphere at -78° C. and reacted at 0-50° C. to generate a compound of the general formula I-4,

Wherein R ₁ , R ₂ and R ₄ -R ₈ are respectively as defined above, and R ₃ is cyano;

or

f2) the compound of the general formula I"'-3 and BBr ₃ are mixed in methylene chloride under an inert atmosphere such as a nitrogen atmosphere at -78 ° C and reacted at 0-50 ° C to generate a compound of the general formula I-5,

Wherein R ₁ and R ₂ are C1-C6 alkyl, R ₄ -R ₈ are as defined above respectively, R ₃ is halogen,

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

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

In a specific embodiment, when R ₃ is a methyl group, the compound of general formula IA is obtained by the following reaction formula:

h) reacting compound IE with CHCl ₂ OMe in dichloromethane in the presence of TiCl ₄ under ice-water bath conditions to obtain compound ID,

i) react compound I-D and trifluoroacetic acid in the presence of triethylsilane at 0-50°C to obtain compound I-C,

j) react compound I-C with boron trifluoride diethyl ether and acetic acid at 0-100°C to obtain compound I-A.

In a specific embodiment, when _R3 is an acetyl group, the compound of general formula IA is obtained by the following reaction formula:

Compound I-F is mixed with acetic anhydride, nitromethane and aluminum chloride at -20-20°C and reacted at 0-50°C for 1-24h to obtain compound I-A.

Preferably, the present invention also provides the preparation method of the above-mentioned baicalein derivatives of general formula II-IV,

As shown in the above reaction formula, the method includes:

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

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

3) the compound of general formula II' reacts with _BBr in dichloromethane to generate the compound of general formula II;

or

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

2) Intermediate I1' reacts with anhydrous sodium acetate in absolute ethanol to generate a compound of general formula III';

3) the compound of general formula III' reacts with _BBr in dichloromethane to generate the compound of general formula III;

Further, the compound of general formula II or II' and reaction reagent (such as halogenated reagent (such as N-chlorosuccinimide (NCS) or N-iodosuccinimide (NIS)), dichloro Dicyanobenzoquinone (DDQ), cyclopropyl borate or methyl fluorosulfonyl difluoroacetate (FSO ₂ CF ₂ COOCH ₃ )) react to form formula IV' or formula IV substituted at the R ₃ position The compound of the general formula IV' reacts with BBr ₃ in dichloromethane to generate the compound of the general formula IV.

In a specific embodiment, when preparing a compound of the general formula IV in which R _is a methyl group, the product prepared according to the above reaction scheme is used to replace I1 in the above reaction formula and the same steps as the synthesis of the compound of the general formula II are further carried out.

Specifically, as shown in the above reaction scheme:

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

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

3) reacting the reaction product of step 2), boron trifluoride diethyl ether and acetic acid at, for example, 95° C., and the obtained reaction product is used for subsequent preparation of a compound of general formula IV in which R ₃ is a methyl group;

In a specific embodiment, when preparing R ₃ is the compound of the general formula IV of acetyl group, the product prepared according to the following reaction scheme is used to replace I1 in the above reaction formula and further carry out the same steps as the synthesis of the compound of the general formula II:

Specifically, as shown in the above reaction scheme:

Mix 3,4,5-trimethoxyphenol, acetic anhydride, and nitromethane with aluminum trichloride at 0°C, react at 0-50°C for 1-24h, and the resulting reaction product is used for the subsequent preparation of _R3 as acetyl The compound of general formula IV of group.

In a specific embodiment, when preparing R ₃ is the compound of the general formula IV of ethyl or isobutyl, taking compounds Ic10 and Ic11 as examples, the synthetic route is as follows:

The acylation reaction of compound 3,4,5-trimethoxyphenol with acetic acid or isobutyryl chloride in the presence of boron trifluoride ether; after that, a reduction reaction occurs under the action of trifluoroacetic acid and triethylsilane; the obtained reaction The product undergoes an acetylation reaction in the presence of boron trifluoride ether and acetic acid, and then reacts with o-chlorobenzoyl chloride to generate the corresponding ester; the resulting ester subsequently undergoes a rearrangement reaction under the action of a base, and the obtained heavy The crude product is further subjected to a ring-closing reaction under the action of strong acid (such as concentrated sulfuric acid), and after the ring-closing, the product is demethylated under the action of boron tribromide to obtain the final product Ic10 or Ic11, wherein R is methyl or isopropyl.

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

The methylation product of the compound Ic15 of the present application undergoes a reduction reaction under the effect of stannous chloride dihydrate to generate the corresponding amino compound, and acetylation reaction occurs by adding acetic anhydride to the reaction product to generate the corresponding amide compound, and the amide The compound undergoes a demethylation reaction under the action of boron tribromide to generate compound Ic18.

In a specific embodiment, when preparing R ₃ is the compound of general formula IV of isopropenyl, taking compound Ic20 as an example, the synthetic route is as follows:

The methylated product of the compound Ic1 of the present application is deiodinated in the presence of potassium carbonate, Xphos, potassium fluoride and palladium acetate to generate a deiodinated product, which is demethylated under the action of _BBr to obtain the above-mentioned Ic20 compound.

route four

The following shows the synthetic route of the compound (such as the above-mentioned compound Id8-Id44) of R1 _{and R2} in the general formula I, at least one of which is not H, and _R3 is a methyl group:

The compound of the general formula Id' is dissolved in an organic solvent, and reacted with reactants having the corresponding groups of R ₁ /R ₂ as defined above under the action of a base to obtain the compound of the general formula Id. In the above reaction formula, R ₁ , The definitions of R ₂ , R ₄ -R ₈ are as defined above, respectively.

In a specific embodiment, the organic solvent is selected from acetonitrile, acetone, N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF) and N-methylpyrrolidone (NMP); The base is selected from triethylamine, potassium carbonate, N,N-diisopropylethylamine and pyridine; the reactant with corresponding groups of R ₁ /R ₂ is selected from ethyl isocyanatoacetate, Isopropyl isocyanatoacetate, ethyl 3-methylbutyrate-2-isocyanate, isopropyl 3-methylbutyrate-2-isocyanate, isopropyl propionate-2-isocyanate, isocyanatoacetate ring Hexyl ester, cyclopentyl isocyanatoacetate, 4-chloromethyl-5-methyl-1,3-dioxol-2-one, chloromethyl isopropyl carbonate, bromoacetonitrile, chlorosulfur acid, isobutyric anhydride, 2-methoxyethoxymethyl chloride, N,N'-dimethylcarbamoyl chloride, ethyl isocyanate and methylcarbamoyl chloride.

In the case where the compound of general formula I is the above-mentioned Id8 and Id9, the synthesis process can be exemplified by the following reaction formula:

The Id' compound was first reacted with acetic anhydride, then reacted with benzyl bromide in order to protect the hydroxyl group, followed by reduction with H ₂ in the presence of Pd(OH) ₂ /C, and then reacted the reaction product with acetyl bromide-α-D- Methyl glucuronate or acetyl bromide-α-D-glucose is reacted, and then the ester group in the compound is hydrolyzed with alkali to obtain the product. The synthesis conditions in the above reaction steps are well known in the art. In the above reaction formula, The definitions of R ₄ -R ₈ are respectively as defined above.

In another aspect, the present invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of one or more selected from the above-mentioned baicalein derivatives, their stereoisomers, and pharmaceutically acceptable salts as Pharmaceutically active ingredients, and optionally pharmaceutically acceptable excipients.

In another aspect, the present invention provides the above-mentioned baicalein derivatives, their stereoisomers or pharmaceutically acceptable salts thereof, or the use of the above-mentioned pharmaceutical composition in the preparation of a drug for inhibiting the main protease of the new coronavirus.

In another aspect, the present invention also provides the above-mentioned baicalein derivatives, their stereoisomers or pharmaceutically acceptable salts thereof, or the use of the above-mentioned pharmaceutical composition in the preparation of the following medicine, wherein the medicine has the ability to inhibit coronavirus The activity of the main protease, or the drug has anti-new coronavirus and its mutant strains (such as WIV04 strain or South African strain B.1.351), SARS virus, MERS virus, or the drug has anti-enterovirus EV71, Ke Saatchie virus and norovirus activity.

The technical solution of the present invention has at least the following technical effects:

The compound of the present invention has stronger SARS-CoV-2 3CLpro inhibitory activity, stronger anti-new coronavirus replication and better pharmacokinetic properties, and exhibits significant anti-new coronavirus efficacy in mice , can be used for the prevention and treatment of diseases caused by novel coronaviruses.

The preparation method of the invention has the technical advantages of simple steps, high yield and readily available raw materials.

Description of drawings

Figure 1: Curve of the inhibitory activity of compound Ia7 on SARS-CoV-2 3CLpro.

Figure 2: Curve of the inhibitory activity of compound Ib1 on SARS-CoV-2 3CLpro.

Figure 3: Curve of the inhibitory activity of compound Ib2 against SARS-CoV-2 3CLpro.

Figure 4: Curve of the inhibitory activity of compound Ib3 on SARS-CoV-2 3CLpro.

Figure 5: Curve of the inhibitory activity of compound Ic3 on SARS-CoV-2 3CLpro.

Figure 6: Curve of the inhibitory activity of compound Ic4 on SARS-CoV-2 3CLpro.

Figure 7: Curve of the inhibitory activity of compound Ic5 on SARS-CoV-2 3CLpro.

Figure 8: In vitro inhibitory activity of compound Ic5 on SARS-CoV-2 WIV04 strain.

Figure 9: In vitro inhibitory activity of compound Ic5 on SARS-CoV-2 South African strain B.1.351.

Figure 10: The effect of compound Ic5 on the viability of Vero E6 cells.

Figure 11: Inhibitory activity of compounds Ic5 and Id16 on SARS-CoV-2 viral replication in mice.

Detailed ways

In order to better illustrate the present invention, numerous specific details are given in the following specific embodiments. It will be understood by those skilled in the art that the present invention may be practiced without certain of the specific details.

Group definition

The term "alkyl" means a straight chain or branched chain alkyl group, for example "C1-Cx alkyl" means a straight chain or branched chain alkyl group containing 1-x carbon atoms. For example, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl wait. For example, "C1-C6 alkyl" means a linear or branched alkyl group having 1-6 carbon atoms.

The term "alkenyl" means a straight-chain or branched alkenyl group, for example, C2-C6 alkenyl means a straight-chain or branched alkenyl group containing 2-6 carbon atoms, such as vinyl, 1-propenyl, 2 -propenyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, etc.

The term "C2-C6 fatty acyl" means an acyl group with 2-6 carbon atoms, for example, including but not limited to, acetyl, propionyl, isopropionyl, butyryl, isobutyryl, tert-butyryl, pentanoyl, Isovaleryl, pivaloyl, hexanoyl, tert-hexanoyl and the like.

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

The term "C7-C12 aroyl" means an aryl acyl group having 7-12 carbon atoms, for example, including but not limited to, benzoyl, methylbenzoyl, ethylbenzoyl, naphthoyl and the like.

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

In order to further understand the present invention, the present invention is further described below in conjunction with embodiment, and these embodiment descriptions are only for further specifying the feature of the present invention, rather than limiting the scope of the present invention or the scope of claims of the present invention. Simple replacements or improvements made by those skilled in the art to the invention all fall within the technical solutions protected by the present invention.

I. Preparation of Intermediates

Preparation Example 1: Synthesis of Intermediate I1

Add 3g of 3,4,5-trimethoxyphenol and 6mL of BH ₃ ·OEt ₂ into a 50mL three-necked flask, replace with nitrogen three times, slowly add 9mL of acetic acid dropwise into the reaction solution, gradually raise the temperature to 80°C, and react for 2h. The reaction solution was slowly added to 30 mL of ice water, stirred for 2 hours, filtered, and dried to obtain 2.8 g 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

Add 1g baicalein, 2.5g potassium carbonate and 10mL acetone into a 50mL two-necked bottle, add 1.6g dimethyl sulfate, gradually raise the temperature to 60°C, and react for 15h. Cool the reaction solution to 25°C, filter the reaction solution, distill off the acetone under reduced pressure, add 50 mL of ethyl acetate and 50 mL of saturated sodium bicarbonate solution, stir for 5 min, and then separate layers, add 50 mL of organic phase and saline, stir for 5 min, and then separate the layers. Dry the organic phase with sodium sulfate water for 1 h, and concentrate to obtain 920 mg of light yellow solid, with a 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) Add 226mg I1 and 5mL methanol into a 25mL single-necked bottle, add 140 mg 2-chlorobenzaldehyde and 3mL 33% sodium hydroxide aqueous solution, and react at room temperature 25°C for 12h. Distilled off methanol under reduced pressure, added 30mL dilute hydrochloric acid and 30mL ethyl acetate and stirred for 5min, then layered, added 30mL saturated brine to the organic phase and stirred for 5min, then layered, dried the organic phase with anhydrous sodium sulfate for 1h, passed column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 10:1) for purification to obtain 300 mg of orange solid product with a yield of 82%.

2) Add 300mg of the reaction product of the previous step, 197mg of iodine element and 5mL of dimethyl sulfoxide into a 25mL single-necked bottle, replace with nitrogen three times, gradually raise the temperature to 110°C, and react for 3h. Add 10 mL of ethyl acetate and 10 mL of water, stir for 5 min, and then separate the layers. Add 10 mL and brine to the organic phase, stir for 5 min, and then separate the layers. Dry the organic phase over anhydrous sodium sulfate for 1 h. Purification by column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 10:1) gave 150 mg of a white solid product with a yield of 50%.

3) Add 150mg of the reaction product from the previous step and 2mL of dichloromethane into a 10mL three-neck flask, replace with nitrogen three times, the temperature of the reaction solution drops to -78°C, slowly add 4mL of BBr ₃ to the reaction solution dropwise, and gradually raise the temperature to 25°C , Reaction 15h. Under the condition of ice-water bath, slowly add methanol to the reaction solution to quench, the system was separated, the organic phase was concentrated, and purified by column chromatography (SiO ₂ , dichloromethane:methanol=30:1) to obtain 50 mg of brown solid product , yield 37%. 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] ⁺ .

II. Preparation of compounds

Embodiment 1: the synthesis of compound Ia1

In addition to using I1 and 3-cyanobenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction 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] ⁺ .

Embodiment 2: the synthesis of compound Ia2

Except using I1 and 3-iodobenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction 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 = 17.0 Hz, 1H). MS (ESI, ev): m/z = 297.13 [M+H] ⁺ .

Embodiment 3: the synthesis of compound Ia3

Except using I1 and 2-chloro-3-fluorobenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction 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] ⁺ .

Embodiment 4: the synthesis of compound Ia4

Except using I1 and 2-chloro-5-fluorobenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction 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] ⁺ .

Embodiment 5: the synthesis of compound Ia5

Except using I1 and 2-trifluoromethylbenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction 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, 4.6 Hz, 1H), 6.53 (s, 1H), 6.45 (s, 1H). MS (ESI, ev): m/z = 339.04 [M+H] ⁺ .

Embodiment 6: the synthesis of compound Ia6

Except using I1 and 2-ethylbenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction 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] ⁺ .

Embodiment 7: the synthesis of compound Ia7

Except using I1 and 2,6-dichlorobenzaldehyde as raw materials, according to the method of Preparation Example 3, the yield of the three-step reaction was 20%. MS (ESI, ev): m/z = 229.98 [M+H] ⁺ .

Embodiment 8: the synthesis of compound Ib1

1) Add 1g of I1 and 20mL of methanol into a 50mL single-necked bottle, add 296 mg of 3-nitrobenzaldehyde and 12mL of 33% aqueous sodium hydroxide solution, and react at room temperature for 12 h at 25°C. Distilled off methanol under reduced pressure, added 50mL dilute hydrochloric acid and 50mL ethyl acetate and stirred for 5min, then layered, added 100mL saturated brine to the organic phase, stirred for 5min, then layered, dried the organic phase with anhydrous sodium sulfate for 1h, passed column chromatography (SiO ₂ , petroleum ether: ethyl acetate=10:1) for purification to obtain 1.02 g of orange solid product, yield 64%.

2) Add 100mg of the product prepared in the previous step, 159mg of anhydrous sodium acetate and 15mL of anhydrous ethanol into a 25mL single-necked bottle, replace with nitrogen three times, gradually raise the temperature to 70°C, and react for 13h. It was concentrated under reduced pressure and purified by column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 10:1) to obtain 50 mg of an orange solid product with a yield of 50%.

3) Add 50mg of the product prepared in the previous step and 1mL of dichloromethane into a 10mL three-necked flask, replace with nitrogen three times, and the temperature of the reaction solution drops to -78°C. Slowly add 0.5mL of BBr ₃ to the reaction solution, and gradually raise the temperature to 25°C. °C, react for 15h. Under the condition of ice-water bath, slowly add methanol to the reaction solution to quench, the system was separated, the organic phase was concentrated, and purified by column chromatography (SiO ₂ , dichloromethane:methanol=30:1) to obtain 25 mg of brown solid product , yield 56%. 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=7.7Hz,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, 3.1 Hz, 1H). MS (ESI, ev): m/z = 318.06 [M+H] ⁺ .

Embodiment 9: the synthesis of compound Ib2

Except using I1 and 2-chloro-5-fluorobenzaldehyde as raw materials, according to the method of Example 8, the yield of the three-step reaction 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] ⁺ .

Embodiment 10: the synthesis of compound Ib3

Except using I1 and 3-trifluoromethylbenzaldehyde as raw materials, according to the method of Example 8, the yield of the three-step reaction 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] ⁺ .

Embodiment 11: the synthesis of compound Ib4

Except using I1 and 2-trifluoromethylbenzaldehyde as raw materials, according to the method of Example 8, the yield of the three-step reaction 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] ⁺ .

Embodiment 12: the synthesis of compound Ib5

Except using I1 and 2-ethylbenzaldehyde as raw materials, according to the method of Example 8, 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, 2.7 Hz, 1H), 1.18 (t, J = 7.5 Hz, 3H). MS (ESI, ev): m/z = 301.3 [M+H] ⁺ .

Embodiment 13: the synthesis of compound Ib6

Except using I1 and 2-bromobenzaldehyde as raw materials, according to the method of Example 8, the yield of the three-step reaction 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] ⁺ .

Embodiment 14: the synthesis of compound Ib7

Except using I1 and 2-iodobenzaldehyde as raw materials, according to the method of Example 8, the yield of the three-step reaction was 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] ⁺ .

Embodiment 15: the synthesis of compound Ic1

1) Add 62 mg of I2 prepared in Preparation Example 2, 54 mg of N-iodosuccinimide and 1 mL of trifluoroacetic acid into a 10 mL single-necked bottle, gradually raise the temperature to 70°C, and react for 4 h. Distill the reaction solution to a small volume under reduced pressure, add 10 mL of ethyl acetate and 10 mL of saturated sodium bicarbonate solution and stir for 5 min, and then separate the layers. Add 10 mL of saturated brine to the organic phase and stir for 5 min, and then separate the layers. Purification by column chromatography (SiO ₂ , petroleum ether:ethyl acetate=10:1) gave a white solid product.

2) Add 100mg of the reaction product from the previous step and 2mL of dichloromethane into a 10mL three-necked flask, replace with nitrogen three times, and the temperature of the reaction solution drops to -78°C. Slowly add 1.4mL of BBr ₃ to the reaction solution dropwise, and gradually raise the temperature to 25°C, react for 15h. Under the condition of ice-water bath, methanol was slowly added to the reaction solution to quench the reaction solution. After the system was separated, the organic phase was concentrated and purified by column chromatography (SiO ₂ , dichloromethane:methanol=30:1) to obtain 70 mg of a yellow solid. Yield 55%. 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] ⁺ .

Embodiment 16: the synthesis of compound Ic2

1) Add 200mg of the product in step 1) of Example 15, 114mg of dichlorodicyanobenzoquinone (DDQ), 92mg of copper acetate, 190mg of silver carbonate and 6mL of NMP into a 10mL sealed tube, and gradually raise the temperature to 120°C, Reaction 20h. Cool the reaction solution to 25°C, filter the reaction solution, add the filtrate to 20 mL of water, stir for 30 min and then filter, the solid is 80 mg after drying.

2) Add 80 mg of the reaction product prepared in the previous step and 2 mL of dichloromethane into a 10 mL three-neck flask, replace with nitrogen three times, and the temperature of the reaction solution drops to -78°C. Slowly add 1.3 mL of BBr ₃ to the reaction solution dropwise, gradually Raise the temperature to 25°C and react for 15h. Under the condition of ice-water bath, methanol was slowly added to the reaction solution to quench the reaction solution. After the system was separated, the organic phase was concentrated and purified by column chromatography (SiO ₂ , dichloromethane:methanol=30:1) to obtain 50 mg of a brown solid. Yield 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] ⁺ .

Embodiment 17: the synthesis of compound Ic3

Add 18mg of I3, 8mg of N-chlorosuccinimide and 2mL of acetonitrile into a 10mL single-necked bottle, gradually raise the temperature to 40°C, and react for 24h. The reaction solution was distilled under reduced pressure, 3 mL of dichloromethane was added to make slurry for 2 h, filtered, and dried to obtain a yellow solid. ¹ HNMR(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] ⁺ .

Embodiment 18: the synthesis of compound Ic4

Except using Ia7 as raw material, according to the method of Example 17, the yield of one-step reaction was 87%. MS (ESI, ev): m/z = 373.93 [M+H] ⁺ .

Embodiment 19: the synthesis of compound Ic5

1) Add 4.02g of 3,4,5-trimethoxyphenol and 50mL of dichloromethane into a 100 mL two-necked flask, slowly add 9.10g of TiCl ₄ dropwise under ice-water bath conditions, stir for 1 hour, then slowly add 3.26g dropwise CHCl ₂ OMe, reacted for 1h. Add saturated ammonium chloride solution to quench, then add 100mL dichloromethane and stir for 5min, then separate layers, add 100mL saturated brine and stir for 5min, then separate layers, dry the organic phase with anhydrous sodium sulfate for 1h, and purify by column chromatography , 3.58 g of solid product were obtained.

2) Add 3.57g of the reaction product of the previous step and 30mL of trifluoroacetic acid into a 50mL two-necked bottle, slowly add .89g of triethylsilane dropwise, and react at room temperature for 1.5h. The reaction solution was distilled off under reduced pressure and purified by column chromatography to obtain 3 g of a solid product.

3) Add 2.72g of the reaction product of the previous step, 10mL of boron trifluoride diethyl ether and 15mL of acetic acid into a 50mL two-necked flask, gradually raise the temperature to 95°C, and react for 2h. The temperature of the reaction solution was lowered to 25° C., and the reaction solution was slowly added into 150 mL of ice water, stirred for 2 hours, filtered, and dried to obtain 3.2 g of solids, with a yield of 76%.

4) Add 3.17g of the reaction product of the previous step and 20mL of methanol into a 50mL single-necked bottle, add 3.71g of 2-chlorobenzaldehyde, and then slowly add 33% NaOH aqueous solution dropwise, and react for 1h. Remove methanol by distillation under reduced pressure, add dilute hydrochloric acid to adjust the pH to neutral, then add 50 mL of ethyl acetate and stir for 5 min, and then separate the layers. Add 100 mL of saturated saline to the organic phase, stir for 5 min, and then separate the layers. Dry the organic phase with anhydrous sodium sulfate for 1 h. Purification by column chromatography afforded 1.2 g of solid product.

5) Put 1.12g of the reaction product of the previous step, 709mg of iodine element and 10mL of DMSO into 100°C gradually, and react for 4h. Add 60mL of dichloromethane and 60mL of water and stir for 5min, then separate the layers, add 60mL of saturated brine to the organic phase, stir for 5min, then separate the layers, dry the organic phase with anhydrous sodium sulfate for 1h, and purify by column chromatography to obtain 500 mg of solid product.

6) Add 452mg of the reaction product of the previous step and 4mL of dichloromethane into a 10mL three-necked flask, replace with nitrogen three times, and the temperature of the reaction solution drops to -78°C, slowly add 12.5mL of BBr ₃ dropwise into the reaction solution, and gradually raise the temperature to 25 °C, react for 15h. Under the condition of ice-water bath, methanol was slowly added to the reaction solution to quench the reaction solution. After the system was separated, the organic phase was concentrated and purified by column chromatography (SiO ₂ , dichloromethane:methanol=30:1) to obtain 30 mg of a 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) Add 1.0g of 3,4,5-trimethoxyphenol, 2.2g of acetic anhydride, and 10mL of nitromethane into a 50mL three-neck flask, and add 2.2g of aluminum trichloride into the reaction solution at 0°C, 25 Under the condition of ℃, react for 15h. Add 50mL of ethyl acetate and 10mL of 1N hydrochloric acid for stratification, add 10mL of water to the organic phase, add 10mL of saturated sodium chloride to the organic phase, dry the organic phase with anhydrous sodium sulfate, and concentrate the organic phase by column chromatography ( _SiO2 , petroleum ether:ethyl acetate=5:1) to obtain 900 mg of white solid.

2) Add 800 mg of the reaction product of the previous step, 210 mg of o-chlorobenzaldehyde, and 10 mL of methanol into a 50 mL three-neck flask, add 4 mL of 33% sodium hydroxide aqueous solution to the reaction solution at 0°C, and react at 25°C for 10 h. Concentrate the organic phase, add 50mL ethyl acetate and 10mL 1N hydrochloric acid for stratification, add 10mL water to the organic phase, add 10mL saturated sodium chloride to the organic phase, dry the organic phase with anhydrous sodium sulfate, concentrate the organic phase and pass through column chromatography (SiO ₂ , petroleum ether: ethyl acetate = 4:1) to obtain 450 mg of white solid.

3) Add 400mg of the reaction product of the previous step, 240mg of iodine, and 5mL of dimethyl sulfoxide into a 50mL three-neck flask, and react at 100°C for 4h. After cooling down to 25°C, add 50mL of dichloromethane and 10mL of water for layering, add 10mL of saturated sodium chloride to the organic phase for layering, dry the organic phase over anhydrous sodium sulfate, and wet the organic phase through column chromatography (SiO ₂ , dichloro Methane:methanol=30:1) was purified to obtain 100 mg of white solid.

4) Add 80 mg of the reaction product of the previous step and 5 mL of dimethyl sulfoxide into a 25 mL single-necked bottle, add 2 mL of boron tribromide (1M in DCM) into the reaction solution at 0°C, and react at 25°C for 10 h. 5 mL of water was added to the reaction solution, and solids were precipitated. The solids were filtered and slurried with water/methanol, filtered again, and dried to obtain 15 mg of a light 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) Dissolve 450mg of 2'-chloro-5,6,7-trimethoxybaicalein and 321mg of N-iodosuccinimide in 4mL of trifluoroacetic acid, replace with nitrogen three times, and gradually raise the temperature to 70°C for 5 hours . The reaction solution was cooled to room temperature, most of the trifluoroacetic acid was distilled off under reduced pressure, dichloromethane and saturated sodium bicarbonate solution were added to separate layers, and the organic phases were combined, dried and concentrated to obtain 500 mg of lavender solid.

2) Add 200 mg of the product from the previous step and 2 mL of DMF into a 10 mL sealed tube, add 160 mg of methyl fluorosulfonyl difluoroacetate and 127 mg of cuprous iodide, and gradually raise the temperature to 100°C for 12 hours after sealing. Cool the reaction solution to room temperature, remove insoluble matter by filtration, add ethyl acetate and water to the filtrate, and dry and concentrate the organic phase to obtain 190 mg of light yellow solid.

3) Add 190mg of the product from the previous step and 2mL of dichloromethane into a 10mL three-necked flask, replace with nitrogen three times, the temperature of the reaction solution drops to -78°C, slowly add 4.58mL of BBr ₃ to the reaction solution, and gradually raise the temperature to 25°C , Reaction 15h. Under the condition of ice-water bath, methanol was slowly added to the reaction liquid to quench, after concentration, methanol and water were added to make a slurry, and 150 mg of yellow solid product was obtained by 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 = 2.0 Hz, 1H). MS (ESI, ev): m/z = 372.2 [M+H] ⁺ .

Example 22: Synthesis of Compound Ic8

1) Add 450mg 2'-chloro-5,6,7-trimethoxybaicalein and 321mg N-iodosuccinimide (1.1eq.) into 4mL trifluoroacetic acid, replace with nitrogen three times, gradually increase Reaction at 70°C for 5h. The reaction solution was cooled to room temperature, most of the trifluoroacetic acid was distilled off under reduced pressure, dichloromethane and saturated sodium bicarbonate solution were added to separate layers, and the organic phases were combined, dried and concentrated to obtain 500 mg of a lavender solid.

2) Add 250mg of the reaction product from the previous step to 4mL of 1,4-dioxane, add 250mg of PdCl ₂ (DPPF)-CH ₂ Cl ₂ (0.2eq.), 115mg of potassium carbonate (2eq.), 72mg of cyclic Propyl borate (2eq.) was replaced with nitrogen three times, and gradually raised to 95°C for 23h. The reaction solution was cooled to room temperature, 1,4-dioxane was distilled off under reduced pressure, ethyl acetate and saturated ammonium chloride solution were added to separate layers, and the organic phases were combined, dried and concentrated by column chromatography to obtain 150 mg of a yellow-brown oil.

3) Add 150 mg of the reaction product of the previous step and 2 mL of dichloromethane into a 10 mL three-necked flask, replace with nitrogen three times, and the temperature of the reaction liquid drops to -78 ° C. Slowly add 3.88 mL of BBr ₃ to the reaction liquid, and gradually raise the temperature to 25 °C. °C, react for 15h. Under the condition of an ice-water bath, slowly add methanol to the reaction liquid to quench, concentrate, add methanol and water to make a slurry, and filter to obtain 56 mg of a khaki solid product. MS (ESI, ev): m/z = 344.2 [M+H] ⁺ .

Example 23: Synthesis of Compound Ic9

1) Add 200 mg of raw materials into 2 mL of DCM, slowly add 236 mg of TiCl ₄ dropwise under ice bath, stir for 1 hour, then slowly add 85 mg of CHCl ₂ OMe dropwise, and react for 45 minutes. Quenched with saturated ammonium chloride aqueous solution, 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 through the column to obtain 150 mg.

2) Add 100 mg of the reaction product of the previous step into 2 mL of tetrahydrofuran, replace with nitrogen three times, add 20 mg of sodium borohydride in an ice-water bath, and react for 5 h. 1M HCl was added dropwise in an ice-water bath to quench the reaction until no bubbles formed, dichloromethane and saturated sodium bicarbonate solution were added to separate layers, and the organic phases were combined, dried and concentrated to obtain 80 mg.

3) Add 80mg of the reaction product of the previous step and 2mL of dichloromethane into a 10mL three-necked flask, replace with nitrogen three times, the temperature of the reaction solution drops to -78°C, slowly add 2mL of BBr ₃ into the reaction solution, and gradually raise the temperature to 25°C , Reaction 15h. Under the condition of ice-water bath, methanol was slowly added to the reaction solution to quench, after concentration, methanol and water were added to make a slurry, and 56 mg of yellow solid product was obtained by filtration. MS (ESI, ev): m/z = 335.9 [M+H] ⁺ .

Example 24: Synthesis of Compound Ic10

1) Add 3.0g of 3,4,5-trimethoxyphenol and 6mL of boron trifluoride diethyl ether into a 50mL three-necked flask, replace with nitrogen and add 9mL of acetic acid, and raise the external temperature to 90°C for 2.5 h. The reaction solution was slowly poured into 32 mL of ice water and stirred for 0.5 h, a large amount of solids precipitated, filtered, and dried to obtain 3.5 g of a yellow product. MS(ESI,ev):m/z 249.02 [M+Na] ⁺ .

2) Add 2.0 g of the product from the previous step and 5 mL of trifluoroacetic acid into a 50 mL three-necked flask. After the external temperature dropped to 0°C, 3.5 mL of triethylsilane was added dropwise to the reaction liquid, and reacted at 25°C for 12 hours. The concentrated organic phase was purified by column chromatography to obtain 1.0 g of white solid with a yield of 56.3%. MS(ESI,ev):212.6[M+H] ⁺ .

3) Add 850 mg of the product from the previous step, 2 mL of boron trifluoride diethyl ether and 4 mL of acetic acid into a 50 mL three-necked flask, and raise the external temperature to 90°C for 10 h. After cooling down to 25°C, add 4 mL of water and 20 mL of EA to the reaction solution, let stand to separate layers, wash the organic phase with 5 mL of saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate the organic phase for purification by column chromatography. 470 mg of oily product were obtained. MS(ESI,ev):254.7[M+H] ⁺ .

4) Add 440mg of the product from the previous step, 1mL DBU and 5mL acetone into a 25mL three-neck flask, slowly add 0.4mL o-chlorobenzoyl chloride dropwise into the reaction solution under ice cooling, and react at 25°C for 3h. Slowly add 1N HCl to the reaction solution dropwise to adjust the pH of the reaction solution to 4-5, add 10mL EA to the reaction solution, let stand to separate, the organic phase is layered with 2mL saturated sodium bicarbonate, and the organic phase is added with 2mL saturated chloride The sodium layer was separated, dried over anhydrous sodium sulfate, and the organic phase was concentrated to obtain 645 mg of the crude product after esterification.

5) Add 645mg of the crude esterified product, 104mg of sodium hydroxide, and 5mL of acetone into a 50 mL three-necked flask. Raise the external temperature to 65°C and react for 2 hours, slowly add 1N HCl to the reaction solution dropwise to adjust the pH of the reaction solution to 4-5, add 10mL EA to the reaction solution, let stand to separate layers, add 2mL saturated sodium chloride to the organic phase to separate layer, dried over anhydrous sodium sulfate, and concentrated the organic phase to obtain 650 mg of rearranged crude product.

6) Add 650 mg of the rearrangement crude product and 6 mL of acetonitrile into a 25 mL three-neck flask, and slowly add 0.2 mL of concentrated sulfuric acid dropwise to the reaction solution after the external temperature is raised to 50 °C, and continue the reaction at 50 °C for 3 h. Concentrate the dry reaction solution, add 10mL EA and 2mL water to the reaction solution, let stand to separate layers, add 2mL saturated sodium chloride to the organic phase, dry over anhydrous sodium sulfate, concentrate the organic phase and beat the solid with 3mL methyl tert-butyl ether , filtered, and the filtrate was purified by column chromatography to obtain 348 mg of white solid, MS (ESI, ev): 374.6[M+H] ⁺ .

7) Add 280 mg of the product after ring closure and 3 mL of DCM into a 25 mL three-necked flask, slowly add 6.7 mL of boron tribromide dropwise to the reaction solution in an ice-water bath, and raise the external temperature to 25°C for 3 hours. The reaction solution was slowly poured into 10 mL of ice water and stirred for 0.5 h. A large amount of yellow solid precipitated out. The solid was filtered and slurried with 3 mL of methanol, filtered, and the filter cake was dried to obtain 92 mg 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) Add 3.0g of 3,4,5-trimethoxyphenol, 1.9mL of isobutyryl chloride, 8.8mL of boron trifluoride ether and 50mL of DCM into a 250mL three-necked flask, and raise the external temperature to 80°C for 12 hours. Concentrate the reaction solution, add 26mL 3N HCl and 20mL EA to the reaction flask, let it stand for stratification, add 2mL saturated sodium chloride to the organic phase, dry it over anhydrous sodium sulfate, and purify the concentrated organic phase by column chromatography to obtain 2.6 g of oily product.

Continue referring to the synthesis method of 2)-7) in Ic10 to obtain Ic11. 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) Add 2.0g of 3,4,5-trimethoxyphenol, 1.6g of anhydrous MgCl ₂ and 16mL of ACN into a 100mL three-necked flask. After the external temperature was lowered to 0°C, 6 mL of triethylamine was added to the reaction liquid, after stirring at 25°C for 30 min, 1.2 g of paraformaldehyde was added to the reaction liquid, and the external temperature was raised to 70°C for 1 h. After the external temperature is lowered to 25°C, slowly add 1N hydrochloric acid dropwise to the reaction solution to adjust the pH of the reaction solution to ≤5, add 20mL ethyl acetate and 5mL water to the organic phase, stir for 10min and let it stand for stratification, add 5mL saturated salt to the organic phase The water layers were separated, and the organic phase was dried over anhydrous sodium sulfate. The concentrated organic phase was purified by column chromatography to obtain 1.9 g of the product as a pale yellow solid. MS (ESI, ev): 212.6 [M+H] ⁺ .

Continue referring to the synthesis method of 2)-7) in Ic10 to obtain Ic12, the difference is that o-fluorobenzoyl chloride is 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

Ic14 was obtained by referring to the synthesis method of 4)-7) in Ic10, except that 4'-chlorobiphenyl-3-acyl chloride was 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

Ic15 was obtained by referring to the synthesis method of 4)-7) in Ic10, except that 2-methyl-5-nitro-benzoyl chloride was 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 (2mL), Pd/C (5mg) was added, stirred at room temperature for 1 hour, and the reaction was complete as monitored by TLC. Filtrate, concentrate the filtrate, add ethyl acetate to make a slurry and stir, filter, and dry the filter cake to obtain 8 mg of solid, 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

Ic17 was obtained by referring to the synthesis method of 4)-7) in Ic10, except that 3-chloro-[1,1-biphenyl]-4-acyl chloride was used 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) Add ethanol (10 mL) to the nitro-containing raw material (200mg, 0.52mmol), under nitrogen protection, add stannous chloride dihydrate (720mg, 6.4mmol, 3.0eq), add 2 drops of acetic acid dropwise, and heat to 70°C, react for 3h. TLC: disappearance of starting material. Post-processing: After cooling, add aqueous sodium bicarbonate solution to adjust the pH to 7, filter, concentrate, dilute with water, extract with EA, wash the organic phase with salt, dry and filter, concentrate to a smaller volume, add hydrogen chloride dioxane solution (1.1 eq), a large amount of solid precipitated, filtered to obtain 173mg of 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) Add DCM (1mL) to amino compound (50mg, 0.13mmol), under nitrogen protection, add acetic anhydride (16mg, 0.15mmol, 1.2eq) and triethylamine (26mg, 0.26mmol, 2eq). ) to react for 3h. TLC: Small amount of starting material remained. Post-processing: add ammonium chloride aqueous solution to adjust the pH to 7, extract with EA, wash the organic phase with salt, dry and filter, and separate by thin layer chromatography to obtain 51 mg of amide compound in total. 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 (20mg, 0.05mmol) and DCM (2mL) obtained in the previous step were protected with nitrogen, and a boron tribromide solution (1M, 0.45mmol, 9 eq) was added dropwise in an ice bath (0°C), and the ice bath was removed. React at ambient temperature (20° C.) for 3 h. TLC: disappearance of starting material. Post-treatment: add 1 drop of methanol to quench, concentrate, add water to make a slurry of the residue, and filter to obtain a crude product. Add DCM and PE to make a slurry, and filter to obtain a total of 7 mg of compound Ic18. 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) Add substituted phenol (528mg, 2.2mmol, 1.1eq), dichloromethane (6ml), triethylamine (555μl, 4mmol, 2eq), DMAP (6mg, 1mmol, 0.05eq) successively in the reaction flask, Add 4'-methylbiphenyl-2-acid chloride under ice bath. After the addition is complete, remove the ice bath and stir at ambient temperature. After the reaction was complete, it was successively washed with 1M HCl solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated and mixed, and column chromatographed to obtain a total of 520 mg of the esterified compound.

2) Potassium tert-butoxide (58mg, 0.52mmol, 1.3eq) was added to the THF solution of the product from the previous step (173mg, 0.4mmol, 1eq), under N ₂ protection, stirring, a large amount of yellow solids precipitated, and a large amount remained after 5h If the raw material is not fully reacted, add 0.5eq potassium tert-butoxide. After the reaction is complete, stop the reaction, add water, add dropwise 1M HCl to adjust the pH to 6, extract with ethyl acetate, dry over anhydrous sodium sulfate, concentrate and mix the sample, and column layer A total of 143 mg of the rearranged compound was obtained as a golden yellow oil.

3) Add the product from the previous step and glacial acetic acid (2ml) into the reaction flask, stir to dissolve, add 3 drops of concentrated hydrochloric acid to catalyze, protect with _N2 , heat to reflux at 110°C, the reaction is complete after two hours, stop the reaction. Water was added, 1M NaOH was added dropwise to adjust the pH to 6, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, purified on a thick preparative plate, and a total of 50 mg of ring-closed compounds were isolated. 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) The ring-closed compound obtained in the previous step was dissolved in dichloromethane, and BBr ₃ (1ml, 1mmol, 10eq) was added in an ice bath. After the addition was complete, the ice bath was removed and stirred at room temperature. The reaction solution was orange. After the reaction is complete, add methanol to quench, spin dry, EA beating, impurity, acetone and water again beating and purifying to obtain 20 mg of 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 materials (300mg, 0.684mmol), potassium carbonate (189mg, 1.369 mmol), Xphos (98mg, 0.2052mmol), potassium fluoride (6mg, 0.2052mmol), palladium acetate (31mg, 0.1398mmol), add DMF (5mL), protected by nitrogen, reacted at an external temperature of 105°C for 12 hours, TLC on the next day showed that there were deiodinated products and 8-position isopropenyl compounds in the new spots. Post-processing: Diatomaceous earth filter, ethyl acetate/water extraction, water washing, brine washing, drying, filtration, column chromatography, to obtain 150 mg of the 8-position isopropenyl compound.

2) The product from the previous step (75mg) was dissolved in dichloromethane (2mL), and 1M _BBr3 solution (0.6mL) was added in an ice bath. After the addition was completed, the reaction was carried out at room temperature for 3 hours, and the TLC raw materials were completely reacted. Post-processing: Add the reaction solution dropwise to methanol (2 mL), stir for 30 minutes, concentrate, add acetone: water = 2:1 (3 mL), and filter to obtain 50 mg of a yellow solid. NMR showed a small amount of impurities. A small amount of ethanol was used to dissolve the solid, and water was added to precipitate the solid, which was stirred and filtered to obtain a total of 37 mg of Ic20. 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

Add 270mg of baicalein and 4mL of acetonitrile into a 10mL two-necked flask, and after nitrogen replacement, add 129mg of ethyl isocyanatoacetate and 101mg of triethylamine into the reaction liquid, and react at room temperature for 12 hours at 25°C. Acetonitrile was distilled off under reduced pressure, 15 mL of ethyl acetate and 10 mL of water were added and stirred for 5 min, and then layered. The organic phase was added with 10 mL of saturated saline, stirred for 5 min, and then layered. The organic phase was dried over anhydrous sodium sulfate for 1 h. Finally, it was purified by column chromatography (SiO2, dichloromethane:methanol=40:1) to obtain 330 mg of white solid product with a yield of 83.1%. 1H NMR (500MHz, DMSO-d6) δ12.94(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=7.1 Hz, 3H). MS (ESI, ev): m/z = 400.1 [M+H]+.

Example 35: Synthesis of Compound Id3

Add 152 mg of chloromethyl isopropyl carbonate to 3 mL of acetone, add 149 mg of NaI, raise the temperature to 60° C. for 3 h, and concentrate for use. Add 272mg of baicalein and 276mg of potassium carbonate to 5mL of acetone, add a little DMF to aid dissolution, then add the previously concentrated oil for use, and react at an ambient temperature of 12°C for 12h. The reaction solution was concentrated to a small volume, EA and H ₂ O were added to separate layers, and the organic phase was dried and concentrated by column chromatography to obtain 50 mg 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

Put baicalein (300mg, 1.11mmol, 1eq), anhydrous potassium carbonate (153mg, 1.11mmol, 1eq) into DMF (3ml), slowly add bromoacetonitrile (76μl, 1.11mmol, 1.1eq) under ice-cooling, room temperature Stir for 10h. The reaction system was extracted with DCM/H ₂ O (the pH of the aqueous layer was adjusted to weak acidity with 1 M HCl, and the aqueous phase was viscous). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. Slurry and purification with DCM, DCM+PE in turn, filtration, the filter cake is 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 [MH] ^- .

After the filtrate was concentrated, it was purified 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) Add 2.0g of Ic5, 15.0mL of pyridine, and 3.5mL of acetic anhydride into a 100mL three-necked flask, and raise the temperature to 70°C for 10h. After concentrating the dry reaction solution, add 50mL of ethyl acetate and 10mL of water to the crude product, stir for 10min and let it stand for stratification, add 10mL of water to the organic phase, add 10mL of saturated brine to the organic phase, dry the organic Mutually. The concentrated organic phase was purified by column chromatography to obtain 1.7 g of trihydroxyacetylated product. MS (ESI, ev): m/z = 445.0 [M+H] ⁺ .

2) Add 1.2g of trihydroxyacetylated compound, 3.0g of potassium carbonate, 220mg of potassium iodide, 20mL of acetone and 1.6mL of benzyl bromide into a 100mL three-necked flask, and heat up to 60°C for 10 hours. After filtration, the concentrated organic phase was purified by column chromatography to obtain 420 mg of a white product in which the acetoxy group at position 7 was replaced by benzyloxy group. MS (ESI, ev): m/z = 492.9 [M+H] ⁺ .

3) Add 400mg of the product from the previous step, 40mg of Pd(OH) ₂ /C and 5mL of tetrahydrofuran into a 25mL single-necked bottle, replace the hydrogen with a hydrogen balloon three times, and react at 25°C for 15h. The reaction was filtered through celite, the dry organic phase was concentrated and the crude was slurried with methanol, filtered, and the solid was dried to yield 250 mg of the debenzylated compound. MS (ESI, ev): m/z = 402.7 [M+H] ⁺ .

分子筛和3mL喹啉加入至 25mL单口瓶中，25℃反应10h。过滤反应液，浓缩有机相通过柱层析法进行纯化，得到135mg白色糖苷化产物。MS(ESI,ev):m/z＝ 719.7[M+H]⁺。4) 150mg debenzylated compound, 296mg acetyl bromide-α-D-glucuronic acid methyl ester, 216mg silver oxide, 250mg

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

5) Add 80mg of Intermediate 4 and 2mL of acetone into a 10mL single-necked bottle, drop the external temperature to 0°C and add 0.6mL of 2.5N sodium hydroxide solution dropwise into the reaction solution, and react for 0.5h. At an external temperature of 0°C, 1N hydrochloric acid was slowly added dropwise to the reaction solution to adjust the pH of the reaction solution to ≤3. A yellow solid was formed, which was filtered and dried to obtain 30 mg of the 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

Following the same method as in Example Id8, except that acetyl bromide-α-D-glucose was used instead of acetyl bromide-α-D-glucuronide methyl ester to obtain 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, 1.3 Hz,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 put into DCM (20mL), and it was not dissolved and clarified, and NEt ₃ (0.6mL, 4.5mmol, 3eq) was added under ice-cooling, the system became clear, replaced with nitrogen, added and diluted with DCM 10 times of chlorosulfonic acid (1.5ml, 2.25mmol, 1.5eq), stirred in ice bath for 30 minutes, then stirred at room temperature overnight, TLC showed a small amount of raw material remaining the next day. The reaction system was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the preparative liquid phase was separated to obtain two compounds, which were sent to hydrogen spectrum respectively. ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.32(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 (6.5mL) were sequentially added to the double-necked flask, and after being dissolved and clarified, NEt ₃ (0.4mL, 1.1eq) was added under the ice bath, and after stirring for 15mins, continue Ethyl isocyanatoacetate (0.31ml, 1.1eq) was added dropwise and reacted in an ice bath for 3h. TLC showed that the reaction of the raw materials was basically complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 720 mg of compound Id16 and a total of 212 mg of compound Id20 were obtained, both of which were 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) was dissolved in 4mL of tetrahydrofuran, NEt ₃ (60mg, 2eq) was added subsequently, isobutyric anhydride (96mg, 2eq) was added dropwise under ice-cooling, and stirred for one hour under ice-cooling, TLC showed that the reaction of starting material was basically complete. Post-treatment: Add EA/H ₂ O to the reaction solution, adjust the pH of the aqueous layer to weak acidity with 1M HCl, extract with EA, wash the organic phase with saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, and purify and concentrate by column chromatography to obtain A total of 25 mg of compound Id18, MS (ESI, ev): m/z=389.1 [M+H] ⁺ .

Example 42: Synthesis of Compounds Id21 and Id22

Compound Ic5 (1g, 3.14mmol, 1eq) and DMF (6mL) were sequentially added into a two-neck flask, and after being dissolved and clarified, replaced with nitrogen, and K ₂ CO ₃ (651mg, 4.71 mmol, 1.5eq) was added under an ice bath, The reaction solution became turbid. After stirring for 10 mins, continue to dropwise add 2-methoxyethoxymethyl chloride (538 μL, 4.71 mmol, 1.5 eq) diluted 10 times with DMF. The reaction solution gradually became clear. TLC showed that about 40% of the raw material remained, and 0.5eq 2-methoxyethoxymethyl chloride was added, and the reaction was carried out at room temperature overnight, and TLC showed that 10% of the raw material still remained the next day. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, the liquid was separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and mixed with silica gel , column chromatography (DCM:MeOH=500:1), after concentration, compound Id21 and compound Id22 were respectively obtained. 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

Compound Ic5 (1g, 3.14mmol, 1eq) and THF (5mL) were sequentially added to the double-neck flask, and if it was not dissolved and clarified, pyridine (585μL, 2.3mmol, 2.3eq) was added under ice cooling, and after stirring for 10 minutes, continued to drop Add N,N'-dimethylcarbamoyl chloride (637μl, 6.9mmol, 2.2 eq), react in ice bath for 2 hours, TLC shows that the raw material is almost reacted, add 3.7eq pyridine, and continue the reaction for about 22 hours, TLC shows that the raw material There are still about 30% left. Post-processing: the reaction solution was extracted with EA/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, the liquid was separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, mixed, concentrated, Column chromatography and thin layer chromatography (DCM:MeOH=40:1) gave compounds Id23 and Id24. Proton 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] ⁺ ; Hydrogen spectrum of Id24: 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 (6mL) were sequentially added into the double-neck flask, and after being dissolved and clarified, NEt ₃ (873μL, 6.28mmol, 2eq) was added under the ice bath, and after stirring for 10 minutes, continue Ethyl isocyanate (513μl, 6.28mmol, 2eq) diluted 10 times with DMF was added dropwise, and reacted in an ice bath for 1 hour. TLC showed that the reaction of the raw materials was basically complete and a major fluorescent spot was generated. Post-processing: the reaction solution was extracted with EA/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, the liquid was separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and successively washed with acetonitrile + Methanol, ethyl acetate beating and filtering to obtain 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 (5mL) were sequentially added into a double-neck flask, and after being dissolved and clarified, NEt ₃ (219μL, 1.57mmol, 1eq) was added under ice cooling, and after stirring for 10 minutes, Continue to add ethyl isocyanate (107 μl, 1.65 mmol, 1.05 eq) diluted 10 times with DMF dropwise, and react in ice bath for 1 h, TLC shows that the reaction of raw materials is basically complete. Post-processing: The reaction solution was extracted with EA/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, the liquid was separated, the organic phase was washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and column chromatography , concentrated to give 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=7.5,1.3Hz,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 (5mL) were sequentially added into a double-neck flask, and after being dissolved and clarified, NEt ₃ (656μL, 4.72mmol, 3eq) was added under an ice bath, and after stirring for 10 minutes, Continue to add carbamoyl chloride (294mg, 3.14mmol, 2 eq) dropwise. After 2 hours of reaction in ice bath, TLC shows that the raw materials are basically reacted completely, and two main fluorescent spots are produced. After continuing to react for 2 hours, TLC shows no obvious change . Post-processing: the reaction solution was extracted with EA/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, the liquid was separated, the organic phase was washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, the sample was mixed with silica gel, and the column Chromatography and concentration yielded compounds Id28 and Id29. Proton spectrum of Id28: 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] ⁺ . Proton spectrum of Id29: 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

Compound Ic17 (1.5g, 3.79mmol, 1eq) and DMF (13mL) were added to the double-neck flask successively, the undissolved clarification, nitrogen replacement, NEt ₃ (581μl, 4.17mmol, 1.1eq) was added dropwise under ice bath, and stirred After 10 minutes, ethyl isocyanatoacetate (492 μL, 4.17 mmol, 1.1 eq) diluted 10 times with DCM was added dropwise. After reacting in ice bath for about 1.5 h, TLC showed that the raw material was almost completely reacted. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M hydrochloric acid, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and mixed with silica gel , column chromatography (DCM:MeOH=600:1 product), concentrated to obtain the crude compound Id30, followed by PE:EA=1:1, acetonitrile + a small amount of methanol beating purification to obtain 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

Compound Ic14 (200mg, 0.5mmol, 1eq) and DMF (2mL) were sequentially added to the double-necked flask, and the undissolved clarified, NEt ₃ (77μL, 0.56mmol, 1.1eq) was added dropwise under ice bath, and after stirring for 10mins, continue Carbamoyl chloride (50mg, 0.53mmol, 1.05eq) diluted 10 times with DCM was added dropwise, and reacted in ice bath for about 6h. The reaction solution was extracted with EA/H ₂ O, the aqueous layer was adjusted to weakly acidic with 1M hydrochloric acid, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain Compound Id31: yellow solid, 76 mg. 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

Compound Ic14 (200mg, 0.5mmol, 1eq) and DMF (2mL) were sequentially added to the double-necked flask, and the undissolved clarified, NEt ₃ (77μL, 0.56mmol, 1.1eq) was added dropwise under ice bath, and after stirring for 10mins, continue Add N,N'-dimethylcarbamoyl chloride (49μl, 0.53mmol, 1.05eq) diluted 10 times with DCM dropwise, react in ice bath for about 1h, TLC shows that the raw material has no obvious change, add 2eqNEt ₃ to continue the reaction for 9h back. The reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M hydrochloric acid, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain Compound Id32: yellow-brown solid, 30 mg. 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

Add compound Ic5 (636mg, 2mmol, 1eq) and NMP (8mL) into the double-neck flask in sequence, after dissolving and clarifying, add powdered potassium carbonate (276mg, 2mmol), stir in ice bath for 15mins, then continue to add isocyanide dropwise Isopropyl acetoacetate (314mg, 2.2mmol, 1.1 eq) was reacted in ice bath for 3h, and TLC showed that the reaction of the starting material was almost complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 530 mg of the yellow compound Id33 was obtained. ¹ H NMR (500MHz, DMSO-d6) δ12.71(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

Add compound Ic5 (636mg, 2mmol, 1eq) and NMP (8mL) into the double-neck flask in sequence, after dissolving and clarifying, add powdered potassium carbonate (276mg, 2mmol), stir in ice bath for 15mins, then continue to add isocyanide dropwise Acyl 2-isopropylpentanoic acid ethyl ester (376mg, 2.2 mmol, 1.1eq), reacted in ice bath for 3h, TLC showed that the reaction of the raw material was basically complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 630 mg of yellow compound Id35 and a total of 100 mg of Id36 were obtained. 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=7.1 Hz,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

Add compound Ic5 (636mg, 2mmol, 1eq) and NMP (8mL) into the double-neck flask in sequence, after dissolving and clarifying, add powdered potassium carbonate (276mg, 2mmol), stir in ice bath for 15mins, then continue to add isocyanide dropwise Acyl 2-isopropylvaleric acid isopropyl ester (407mg, 2.2 mmol, 1.1eq), reacted in ice bath for 3h, TLC showed that the reaction of the raw material was basically complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 200 mg of the yellow compound Id37 was obtained. 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=6.7Hz,1H),1.22( dd, J = 6.3, 1.2 Hz, 6H), 1.00 - 0.90 (m, 6H). MS (ESI, ev): m/z = 504.3 [M+H] ⁺ .

Example 53: Synthesis of Compounds Id38 and Id39

Add compound Ic5 (636mg, 2mmol, 1eq) and NMP (8mL) into the double-neck flask in sequence, after dissolving and clarifying, add powdered potassium carbonate (276mg, 2mmol), stir in ice bath for 15mins, then continue to add isocyanide dropwise Isopropyl isopropionate (345mg, 2.2mmol, 1.1eq), reacted in ice bath for 3h, TLC showed that the reaction of the raw material was basically complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 450 mg of yellow compound Id38 and a total of 120 mg of Id39 were obtained. 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 HNMR (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

Add compound Ic5 (636mg, 2mmol, 1eq) and NMP (8mL) into the double-neck flask in sequence, after dissolving and clarifying, add powdered potassium carbonate (276mg, 2mmol), stir in ice bath for 15mins, then continue to add isocyanide dropwise Isopropyl acetoacetate (402 mg, 2.2 mmol, 1.1 eq) was reacted in ice bath for 3 h, TLC showed that the reaction of the raw material was basically complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 450 mg of the yellow compound Id40 was obtained. ¹ H NMR (500MHz, DMSO-d6) δ12.71(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

Add compound Ic5 (636mg, 2mmol, 1eq) and NMP (8mL) into the double-neck flask in sequence, after dissolving and clarifying, add powdered potassium carbonate (276mg, 2mmol), stir in ice bath for 15mins, then continue to add isocyanide dropwise Cyclopentyl acetate (372mg, 2.2mmol, 1.1 eq), reacted in ice bath for 3h, TLC showed that the reaction of the raw material was basically complete. Post-processing: the reaction solution was extracted with DCM/H ₂ O, the pH of the aqueous layer was adjusted to weak acidity with 1M HCl, separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, purified and concentrated by column chromatography A total of 450 mg of the yellow compound Id41 was obtained. ¹ H NMR (500MHz, DMSO-d6) δ12.71(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 (10mL) were sequentially added to an eggplant-shaped bottle to dissolve, then K ₂ CO ₃ (434mg, 3.14mmol, 1eq) was added, replaced with nitrogen, and 4-chloroform was added under ice bath Base-5-methyl-1,3-dioxol-2-one (171μL, 3.14mmol, 1 eq), after ice-bath reaction for 2h, TLC showed no significant change, added 0.5eq NaI, and transferred Reaction at room temperature for 2 hours, about 40% of the raw material remained in TLC, no significant change after heating up to 40°C for 2 hours, continue to add 1eq K ₂ CO ₃ , after 2 hours of reaction at 40°C, TLC showed two newly formed small polar spots fluorescence Lighter, stock thicker. Post-processing: the filtrate was extracted three times with DCM/H ₂ O (the pH of the aqueous layer was adjusted to weak acidity with 1M hydrochloric acid), separated, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, passed through the column Purified by chromatography to obtain the compounds respectively:

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 Example

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

main reagent

main instrument

Microplate reader (SYNERGY-H1);

Microanalytical balance (XP26, Mettler-Toledo Instruments Shanghai Co., Ltd.);

Vortexer vortex shaker (SI-A256, Scientific Industries, Inc.);

centrifuge (5417R, Eppendorf);

Pipette gun (Eppendorf);

LC (Waters), Mass (Applied Biosystems) in LC/MS-MS;

Biological safety cabinet (AC2-3S1, ESCO);

Carbon dioxide incubator (Thermo Scientific HERAcell 150i, Thermo Scientific);

Pure water instrument (seepage source SYS ultrapure water machine);

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

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

^T100TM Thermal Cycler (1861096, BIO-RAD);

Centrifuge (Micro21/21R Thermo Scientific, Thermo Scientific).

Enzymes, Cells and Viruses

3CL protease: self-made recombinant full-length coronavirus 3CL protease based on the coronavirus genome sequence, GenBank numbers of SARS-CoV-2, SARS-CoV, MERS-CoV, H229E-CoV, HKU1-CoV, NL63-CoV and OC43-CoV genomes used They are MN908947.3, AAP13442.1, MT387202.1, AF304460.1, AY597011.2, AY567487.2, and AY903459.1. The DNA sequences required for the expression of seven coronavirus 3CL protease proteins were provided by Nanjing KingScript Co., Ltd. Synthesis of companies;

Vero E6, HepG2, HEK293 and L02 cells were purchased from ATCC;

SARS-CoV-2 was obtained from the National Virus Resource Bank.

Pharmacological Example 1: Test of Baicalein Derivatives Ia1-7, Ib1-7 and Ic1-8 on SARS-CoV-2 3CLpro inhibitory activity

The inhibitory activity of baicalein derivatives Ia1-7, Ib1-7 and Ic1-8 on SARS-CoV-2 3CLpro enzyme activity was evaluated by fluorescence resonance energy transfer method. The volume of the entire enzymatic reaction system is 120 μL, the final concentration of protease is 30 nM, and the final concentration of substrate is 20 μM. The buffer of the reaction system includes 50mM Tris pH7.3, 1mM EDTA. Add SARS-CoV-2 3CLpro protease and different concentrations of compounds to the 96-well plate, incubate at 30°C for 10 minutes, add the substrate and quickly put it into a microplate reader for reading. Excitation light and emission light were 320nM and 405nM, respectively. The test time is 3.5min, and the fluorescence value is read every 35s. The final result takes the reading value of the first 2 minutes to fit the reaction rate, and compares it with the control group (DMSO) to calculate the inhibition rate. The calculation formula is: inhibition rate=[1–(test group reaction rate/control group reaction rate)]× 100%. The _IC50 value and the inhibition rate curve were obtained by fitting the software GraphPad Prism 8. Figures 1-7 are the inhibition curves of baicalein derivatives Ia7, Ib1-3, and Ic3-5 on SARS-CoV-2 3CLpro, respectively.

The _inhibition rates and IC values of baicalein derivatives Ia1-7, Ib1-7 and Ic1-8 against SARS-CoV-2 3CLpro inhibition are listed in Table 1 below.

Table 1: Inhibitory effects of baicalein derivatives Ia1-7, Ib1-7 and Ic1-8 on SARS-CoV-2 3CLpro

"-" means not measured.

Pharmacological Example 2: Evaluation of the Replication Inhibition Activity of Compound Ic5 on SARS-CoV-2 WIV04 Strain and South African Strain B.1.351

The test compound Ic5 was dissolved in DMSO into a 40mM mother solution. On the day of the test, the compound mother solution was serially diluted 10 times with DMEM, that is, 1 μL of the compound mother solution was added to 9 μL DMSO, and after two 10-fold dilutions, a 0.4 mM dilution was obtained. Afterwards, 20-fold dilution to 20 μM, and then continued three-fold serial dilution to obtain 6.7, 2.2, 0.74, 0.25, 0.08 μM in sequence.

Vero E6 cells were used in the test, and Vero E6 cells (50,000 cells/well) were added to a 48-well plate, 100 μL/well of a medium containing gradient concentrations of compounds was added, and SARS-CoV-2 WIV04 strain or South African strain B was added one hour later .1.351 with a multiplicity of infection (MOI) of 0.01. After co-incubating for 1 hour, the supernatant was aspirated, washed and re-added with 200 μL/well of medium containing gradient concentrations of compounds, and incubated at 37°C for 24 hours. After 24 hours, the cell supernatant was collected, viral RNA was extracted from the supernatant, and the virus copy number of the supernatant was detected by real-time fluorescent quantitative PCR method. The inhibition rate of the compound was calculated according to the virus copy number, and the EC ₅₀ of the compound was calculated.

Data analysis and processing used GraphPad Prism 6 and Excel software. The degree of inhibition of the replication of SARS-CoV-2 by different concentrations of compounds was calculated by the following formula:

Inhibition % = [1–(I/I ₀ )]×100%

Wherein, inhibition % represents the inhibitory percentage of the compound to SARS-CoV-2 replication, and I and _I represent respectively the copy number of SARS-CoV-2 viral RNA in the cell supernatant in the compound and the control well (DMSO group).

Compound EC ₅₀ was calculated by fitting the following equation using GraphPad Prism 6 software:

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

Wherein, X is the Log value of the detected concentration of the test substance, Y is the inhibition percentage at the corresponding concentration, Bottom and Top are the minimum and maximum inhibition percentages, respectively.

The test results showed that the half effective concentration EC ₅₀ of Ic5 inhibiting WIV04 strain in Vero E6 cells was 0.72±0.18 μM, as shown in FIG. 8 .

The test results show that Ic5 can significantly inhibit the replication of SARS-CoV-2 South African strain B.1.351 in Vero E6 cells, with an EC ₅₀ of 1.21±0.44, as shown in Figure 9.

Pharmacological Example 3: Detection of the effect of compound Ic5 on cell viability

The supernatant was removed from the cells in the 96-well plate, and the diluted compound Ic5 was added to each well. After incubation for 24 hours, the cell viability was detected with a CCK8 detection kit. The test results showed that the cytotoxicity of Ic5 was very weak, and the CC ₅₀ for Vero E6 cells was greater than 500 μM, as shown in FIG. 10 .

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

ICR mice (purchased from the Experimental Animal Management Department of Shanghai Family Planning Research Institute) intravenous injection dose 5mg/Kg, solvent: 5% dimethyl sulfoxide + 5% ethanol + 40% polyethylene glycol 300 + 50% Physiological saline; intragastric administration group dose 100mg/Kg, vehicle: 5% dimethyl sulfoxide + 95% 0.5% hydroxypropyl methylcellulose solution. Blood was collected through the submandibular vein, 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°C within 1 hour of collection and stored frozen at approximately -80°C. Precipitate an aliquot of 20 µL plasma sample containing 10 ng/mL IS with 400 µL methanol. The mixture was spun for 1 minute and centrifuged for 7 minutes. Transfer 400 µL of supernatant to a 96-well plate. Inject a 10 µL aliquot of the supernatant for LC-MS/MS analysis.

When the dosage of the intragastric administration group was 100mg/Kg, the test results were as shown in Table 2 below:

Table 2

Pharmacological Example 5: Test of the Inhibitory Activity of Baicalein Derivatives Ic10-21, Id5-6, Id8-9, Id14-16, Id18 and Id30-32 on SARS-CoV-2 3CLpro

The fluorescence resonance energy transfer method was used to evaluate and measure the inhibitory activity of some baicalein derivatives on the enzyme activity of SARS-CoV-2 3CLpro. The volume of the entire enzymatic reaction system is 120 μL, the final concentration of protease is 30 nM, and the final concentration of substrate is 20 μM. The buffer of the reaction system includes 50mM Tris pH7.3, 1mM EDTA. Add SARS-CoV-23CLpro protease and different concentrations of compounds to the 96-well plate, incubate at 30°C for 10 min, add the substrate and quickly put it into a microplate reader for reading. Excitation light and emission light were 320nM and 405nM, respectively. The test time is 3.5min, and the fluorescence value is read every 35s. For the final result, the reading value of the first 2 minutes was used to fit the reaction rate, and compared with the control group (DMSO), the inhibition rate was calculated.

Table 3: Inhibitory effects of baicalein derivatives Ic10-21, Id5-6, Id8-9, Id14-16, Id18 and Id30-32 on SARS-CoV-2 3CLpro

"-" means not measured.

Pharmacological Example 6: Evaluation of the inhibitory activity of some baicalein derivatives on the replication of SARS-CoV-2 WIV04 strain.

The test compound was dissolved in DMSO into a 40 mM stock solution. On the day of the test, the compound stock solution was serially diluted with DMEM to obtain 1 μM.

Vero E6 cells were used in the test, and Vero E6 cells (50,000 cells/well) were added to a 48-well plate, and 100 μL/well of medium containing gradient concentration compounds was added, and SARS-CoV-2 WIV04 strain was added one hour later or, the multiplicity of infection (MOI) is 0.01. After co-incubation for 1 hour, the supernatant was aspirated, washed, and 200 μL/well medium containing 1 μM compound was added again, and incubated at 37°C for 24 hours. After 24 hours, collect the cell supernatant, extract the supernatant virus RNA and use real-time fluorescent quantitative PCR method to detect the supernatant virus copy number, calculate the compound inhibition rate according to the virus copy number, and the results are represented by A, B or C respectively, where A :70～100％, B:30～69％, C:10～29％.

Table 4: Inhibitory activity of baicalein derivatives Ic13-18, Id8, Id16, Id18, Id21 and Id28-32 on the replication of SARS-CoV-2WIV04 strain

Pharmacological Example 7. ICR mouse PK property test experiment of compound Id16

Intravenous injection dosage of ICR mice is 10mg/kg, vehicle: 40% PEG400+10% Solutol HS 15+50% distilled water; dosage of ICR mice intragastric administration group is 100mg/kg, vehicle: 5% DMSO+ 5% Solutol HS 15+90% normal saline, sodium hydroxide to adjust the pH to 8. Blood was collected through the submandibular vein, 0.03 mL/time point. Samples were placed in tubes containing K2-EDTA and stored on ice until centrifugation. Blood samples were centrifuged at 6800 g for 6 min at 2–8°C within 1 hour of collection and stored frozen at approximately -80°C. A 20 μL plasma sample containing 10 ng/mL IS was precipitated with 400 μL methanol (1 mL methanol: 0.1 mL (1M HCl + 1% VC)). The mixture was spun for 1 minute and centrifuged for 7 minutes. Transfer 400 µL of supernatant to a 96-well plate. Inject a 10 μL aliquot of the supernatant for LC-MS/MS analysis to detect Id16. As shown in Table 5 of the test results, the compound has a high oral bioavailability of 57.5% in mice, and the blood drug exposure is relatively high.

Pharmacokinetic parameters when table 5.Id16 ICR mice were injected intravenously with 10mg/kg and administered orally with 100mg/kg

The dosage of the ICR mouse gavage administration group is 200 mg/kg, and the vehicle: 10% PEG400+90% (0.5% hydroxypropyl methylcellulose). According to the same administration and plasma processing method when the dose of Id16 intragastric administration was 100 mg/kg, LC-MS/MS detected Id16, the hydrolyzed metabolite Ic5 and the hydrolyzed glucuronidated metabolite Id8, respectively. The test results are shown in Table 6 below. The metabolites Ic5 and Id8 of compound Id16 in mice still have good anti-new coronavirus activity. Id16, Ic5 and Id8 of drug exposure were also detected.

Pharmacokinetic parameters when table 6.Id16 ICR mice were given 200mg/kg orally

Pharmacological Example 8. Cynomolgus monkey PK property test experiment of compound Id16

Cynomolgus monkeys (purchased from Guangzhou Xusheng Biotechnology Co., Ltd.) administered intravenously at a dose of 10 mg/kg, vehicle: 5% DMSO+5% Solutol HS 15+90% normal saline, adjusted pH to 8 with sodium hydroxide; The dose of the crab monkey gavage administration group was 100 mg/kg, and the vehicle: 10% PEG400+90% (0.5% hydroxypropyl methylcellulose). Blood will be collected via the femoral vein or other suitable vein, 1.0 mL/time point. 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°C within 1 hour of collection and stored frozen at approximately -80°C. A 40 μL plasma sample containing 10 ng/mL IS was precipitated with 400 μL methanol (1 mL methanol: 0.1 mL (1M HCl + 1% VC)). The mixture was spun for 1 min and centrifuged at 18000 g for 7 min. Transfer 400 µL of supernatant to a 96-well plate. Inject a 1 µL aliquot of the supernatant for LC-MS/MS analysis. Id16 and metabolites Ic5 and Id8 were detected, respectively. The test results are shown in Table 7 below. The compound Id16 and the metabolites Ic5 and Id8 have high blood drug exposure, and the PK properties in cynomolgus monkeys are good.

Pharmacokinetic parameters when table 7.Id16 cynomolgus monkeys were given intravenous injection of 10 mg/kg and intragastric administration of 100 mg/kg

Pharmacological Example 9. Compounds Ic5, Id8, Id16 Cell Level Liver and Kidney Cytotoxicity Test Experiments

The stock solutions of each compound (100 μM in dimethylsulfoxide) were diluted to 200, 100, 50, 25, 10, 5, 2.5, 1, 0.5 μM with cell culture medium. The negative control was 0.5% dimethyl sulfoxide in cell culture medium. Human liver cancer cells HepG2, human embryonic kidney cells HEK293 and human normal liver cells L02 were seeded in a 96-well plate at a density of 1×10 ⁴ cells per well and incubated for 48 hours. Then different concentrations of compounds were added to the cells and incubated for 48 hours (n=6). Cytotoxicity was detected with a CCK8 detection kit. Absorbance was measured at a wavelength of 450 nm by an automated microplate reader (Biotek, Winooski, VT, USA). The half inhibitory concentration (IC ₅₀ ) value of each compound was calculated using GraphPad Prism 6 software. The results are shown in Table 8. The IC ₅₀ of the three tested compounds against different liver and kidney cells were all greater than 25 μM.

Table 8. Inhibitory activity of compounds Ic5, Id8, and Id16 on liver and kidney-related cells.

Pharmacological Example 10. The anti-new coronavirus effect of compounds Ic5 and Id16 on the new coronavirus mouse model prepared by adenovirus vector transduction hACE2

H11-K18-hACE2 mice aged 7-8 weeks were transferred to the BSL-3 laboratory of the institute. Each mouse was infected with 1×10 ³ PFU of SARS-CoV-2 virus by intranasal drip, and this day was regarded as day 0. Two hours after the mice were infected with SARS-CoV-2, the drug was administered by intragastric administration. Mice were divided into 3 groups. Control group (BID, oral administration of vehicle), Ic5 administration of 200mg/kg group (BID, oral administration) of 10 rats, Id16 administration of 200mg/kg group (BID, oral administration). The administration was given once on the 0th day, twice on the 1st, 2nd, and 3rd days, respectively, with an interval of 9 hours, and the body weight of the mice was recorded every day.

On day 2, 5 mice in each group were sacrificed, and on day 4, 5 mice in each group were sacrificed. Then the lung tissue of the mouse was extracted, and the right lung was ground with DMEM, and part of the homogenate was used to extract RNA, and the rest was stored at -80°C.

PremixEx Taq^TMII(Tli RNaseH Plus)利用实时荧光定量PCR技术对组织中的病毒RNA拷贝数进行绝对定量检测。样品拷贝数通过标准质粒浓度进行计算。结果如图11所示，Ic5在第4天表现出一定的抗新冠病毒作用，Id16在第2天和第4天均表现出显著的抗新冠病毒作用。Determination of virus copy number: Use the kit TaKaRa MiniBEST Viral RNA/DNA Extraction KitVer.5.0 to extract the RNA in the ground lung tissue, follow the instructions of the kit TaKaRa PrimeScript ^TM RT reagentKit with gDNA Eraser to extract the RNA and perform reverse transcription, and then by TaKaRa

PremixEx Taq ^TM II (Tli RNaseH Plus) utilizes real-time fluorescent quantitative PCR technology for absolute quantitative detection of viral RNA copy number in tissues. Sample copy numbers were calculated from standard plasmid concentrations. The results are shown in Figure 11, Ic5 showed a certain anti-new coronavirus effect on the 4th day, and Id16 showed a significant anti-new coronavirus effect on the second day and the fourth day.

Pharmacological Example 11. Subacute Toxicity Test of Compound Id16 on Mice

Male ICR mice were set up in the control group, 200mg/kg group, 400mg/kg group, and 800 mg/kg group, with 10 mice in each group, administered once a day, and observed food intake, body weight and symptoms. The results showed that repeated administration for 7 days The mice in each dose group had no obvious symptoms, no significant difference in body weight, and no significant difference in food intake. Based on the above, no obvious toxicity was found in the subacute toxicity test of compound Id16 on mice. See Tables 9a, 9b and 9c for details.

Table 9a. Compound Id16 observed in mice when repeated administration in 7 days

Table 9b. Changes in body weight of mice when compound Id16 was administered repeatedly in 7 days

Table 9c. Changes in food intake of mice when compound Id16 was administered repeatedly in 7 days

Pharmacological Example 12. Test of Compound Ic5's Inhibitory Activity on Different 3CLpro Enzymes

The inhibitory activity of Ic5 to other sources of 3CLpro enzyme activity was evaluated by fluorescence resonance energy transfer method. The experimental method is the same as in Pharmacological Example 1, except that the 3CLpro enzyme is from other coronaviruses shown in Table 10 below. The experimental results 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, with IC ₅₀ all less than 0.5 μM.

The compound of the present invention has stronger SARS-CoV-2 3CLpro inhibitory activity, stronger cellular level anti-new coronavirus replication, and can be orally absorbed, and is expected to be used for the prevention and treatment of diseases caused by new coronavirus.

Claims (10)

1. 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,

-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;

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;

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;

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 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;

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 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;

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.

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;

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.

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;

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.

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:

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 in any one of claims 1 to 7;

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 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:

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 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,

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 in any one of claims 1 to 7, 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 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:

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,

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.

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