CN117865993A

CN117865993A - AAK1 inhibitor and preparation and application thereof

Info

Publication number: CN117865993A
Application number: CN202410031946.8A
Authority: CN
Inventors: 叶向阳; 毛念栋; 高园; 车昊; 张航; 徐月莹; 段吉隆; 袁滢惠; 谢恬
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2023-01-10
Filing date: 2024-01-09
Publication date: 2024-04-12

Abstract

The invention discloses an AAK1 inhibitor, and preparation and application thereof. The invention provides an AAK1 inhibitor with a novel structure shown in a formula (I), a pharmaceutical composition and a hydrate containing the compound shown in the formula (I), and isotope derivatives, chiral isomers, allosteric isomers, different salts, prodrugs, preparations and the like of the compound. The invention also provides a preparation method and application of the AAK1 inhibitor with novel structure.

Description

AAK1 inhibitor and preparation and application thereof

Technical Field

The invention belongs to the technical field of synthesis of anti-coronavirus drugs, and particularly relates to an AAK1 inhibitor, and preparation and application thereof.

Background

There are 6 links in infection replication of coronaviruses: (1) Attachment, i.e., attachment of the viral S glycoprotein to a specific receptor on the surface of a host cell, occurs with specific binding; (2) Invasion, i.e., into a cell of a host by endocytosis or membrane fusion; (3) Uncoating, i.e., the capsid of the virus is degraded and destroyed by the host cell or the virus' own enzymes, releasing the viral nucleic acid; (4) Synthesis, i.e., the replication of RNA and the synthesis of viral proteins within the host cell; (5) Assembling, namely assembling each part of the synthesized nucleic acid, protein capsids and the like into new virus particles; (6) The newly produced virions leave the host cell by blebbing. In each of the above links, coronavirus invasion is a crucial step. Successful intervention in this step, the virus can be rejected from the host cell. The known mode of endocytosis is mainly clathrin-dependent endocytosis.

AAK1 is a serine-threonine kinase, is a key kinase for threonine phosphorylation at 156 th subunit mu 2 of AP-2 protein, and the phosphorylated AP-2 protein mediates clathrin-dependent endocytosis, so that AAK1 can be effectively inhibited and blocked, thereby playing a role in preventing coronaviruses from entering host cells and achieving the effect of killing viruses.

Disclosure of Invention

It is a first object of the present invention to address the deficiencies of the prior art and to provide an AAK1 inhibitor compound.

An AAK1 inhibitor compound, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, having the structure shown in formula (I):

x in formula (I) is selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

wherein the method comprises the steps ofRepresents the site of attachment of X to 7-azaindole; />Represents the site of attachment of X to Y; />Represents the site of Y to Z linkage;

preferably, an AAK1 inhibitor compound, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, has a structural formula shown in formula (I);

x in formula (I) is selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

more preferably, an AAK1 inhibitor compound, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, has a structural formula shown in formula (I);

x in formula (I) is selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

most preferably, the compound has a structural formula as shown in any one of formulas 1 to 27:

a second object of the present invention is to provide a method for preparing an AAK1 inhibitor.

When (when)Or->When the method comprises the following steps:

(1) Carrying out substitution reaction on the compound a and the compound b to obtain a compound c;

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

(4) Carrying out Suzuki reaction on the compound e and the compound f to obtain a compound g;

(5) Halogenating the compound g and N-bromosuccinimide (NBS) or N-iodosuccinimide (NIS) to obtain a compound h;

(6) Carrying out a Sonogashira reaction on the compound h and the compound i to obtain a compound Ia;

the synthetic route is as follows:

when A in the compound h is Br, X in the formula (I) is selected fromIs the case for (a);

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

wherein the method comprises the steps ofRepresents the site of attachment of X to 7-azaindole; />Represents the site of attachment of X to Y; />Represents the site where Y is linked to Z.

When (when)Or->When the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

(7) The compound Ia is subjected to reduction reaction to obtain a compound I;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

When (when)When the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

(4) The compound e is subjected to Miyaurabelation reaction to obtain a compound f;

(5) Reacting the compound g with the compound h through decarboxylative Knoevenagel to obtain a compound i;

(6) Carrying out Suzuki reaction on the compound f and the compound I to obtain a compound I;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

When (when)When the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

(5) Carrying out amide coupling reaction on the compound g and the compound h to obtain a compound i;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3, y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

When (when)When the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) The compound d undergoes bromine substitution reaction to obtain a compound e;

(6) Carrying out Suzuki reaction on the compound h and the compound i to obtain a compound Ib;

the synthetic route is as follows:

wherein,selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

The compound represented by the formula (I) of the present invention can be produced by the above-mentioned method, however, the conditions of the method, such as reactants, solvents, amounts of the compounds used, reaction temperature, time required for the reaction, etc., are not limited to the above-mentioned explanation. The compounds of the present invention may also optionally be conveniently prepared by combining the various synthetic methods described in this specification or known in the art, such combinations being readily apparent to those skilled in the art to which the present invention pertains.

A third object of the present invention is to provide the use of the above AAK1 inhibitor compounds, or optical isomers, racemates, single enantiomers, possible diastereomers thereof, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, solvates thereof, for the preparation of an anti-coronavirus medicament.

A fourth object of the present invention is to provide an anti-coronavirus drug containing a safe and effective amount of the AAK1 inhibitor compound of novel structure, or an optical isomer, a racemate, a single enantiomer, a possible diastereomer, or a pharmaceutically acceptable salt, a prodrug, a deuterated derivative, a hydrate, or a solvate thereof.

Preferably, the anti-coronavirus drug may further comprise a pharmaceutically acceptable carrier.

Pharmaceutical compositions and methods of administration

Because the compounds of the present invention have activity in inhibiting invasion of various coronaviruses, the compounds of the present invention and various crystalline forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present invention as main active ingredients are useful for treating, preventing and alleviating various diseases caused by coronaviruses.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 5-1000mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or solubilisers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, such as glycerin; (d) Disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents such as paraffin; (f) absorption accelerators, such as quaternary amine compounds; (g) humectants, such as cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin; (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain location within the gut. Examples of embedding components that can be used are polymeric substances and waxy substances. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredients are mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 5000mg, preferably 5 to 2000mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Compared with the prior art, the invention has the main advantages that: provided are novel AAK1 inhibitors of the structure shown in formula (I), pharmaceutical compositions and hydrates of the compounds of formula (I), and optical isomers, racemates, single enantiomers, possible diastereomers of the compounds, or pharmaceutically acceptable salts, prodrugs, deuterated derivatives, hydrates, solvates thereof. The invention also provides a preparation method of the AAK1 inhibitor with novel structure, and activity of inhibiting various coronaviruses and application of the AAK1 inhibitor in resisting coronaviruses. The AAK1 inhibitor with novel structure is expected to become candidate anti-coronavirus drugs.

Drawings

FIG. 1 shows the results of green fluorescent protein expression under a fluorescent microscope in example 22.

FIG. 2 shows the results of luciferase assay in example 22.

Detailed Description

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1: preparation of Compound 1

To a solution of compound 1b (10.0 g,64.94 mmol) and potassium hydroxide (KOH, 85%,12.0g,216.45 mmol) in dimethyl sulfoxide (DMSO, 100.0 mL) was added compound 1a (8.0 mL,72.15 mmol). After the addition, the reaction mixture was stirred at room temperature for 3 hours, heated to 60℃and stirred for 24 hours. The reaction solution is cooled to room temperature, and is slowly dripped into ice water under stirring, and the precipitated solid product is collected by suction filtration. The solid was dissolved in Dichloromethane (DCM), washed twice with water and then once with saturated brine, and the organic phase was washed with anhydrous sodium sulfate (Na ₂ SO ₄ ) And (5) drying. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure, and the obtained crude product was recrystallized from Ethyl Acetate (EA) to give compound 1c (8.1 g, yield 52.9%) as a yellow solid.

To compound 1c (10.0 g,42.37 mmol) and ammonium chloride (NH) with stirring ₄ Cl,6.7g,127.12 mmol) ethanol (EtOH, 100.0 mL) and water (H ₂ To the mixed solution of O,25.0 mL) was added iron powder (Fe, 11.9g,211.87 mmol). After the addition, the reaction mixture was heated to 70℃and stirred overnight. The reaction was cooled to room temperature, the buchner funnel was filled with celite, filtered off with suction and the filter cake was washed with DCM. The filtrate was concentrated under reduced pressure. The solid was dissolved in DCM, washed twice with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (EA/pe+0.5% ammonia water system elution) to give compound 1d (6.3 g, 72.2%) as a pale yellow solid.

To H of Compound 1d (5.2 g,25.0 mmol) in an ice bath ₂ An aqueous solution (48%, 85.0 mL) of hydrogen bromide was added to a solution of O (50.0 mL), followed by dropwise addition of sodium nitrite (NaNO) ₂ ) H of (2) ₂ O solution (17.0 mL,1 g/mL). After the addition, the reaction was stirred in an ice bath for 40 minutes and then cuprous bromide (CuBr, 7.2g,50.0 mmol) was added. After the reaction mixture was heated to 60℃and stirred for 15 minutes, copper bromide (CuBr) was added thereto ₂ 11.2g,50.0 mmol). After the addition, the reaction mixture was stirred at 60℃overnight. The reaction solution was cooled to room temperature, and an appropriate amount of EA was added thereto, followed by stirring with saturated potassium carbonate (K) ₂ CO ₃ ) And (3) an aqueous solution until no bubbles are generated in the reaction solution. The buchner funnel was filled with celite, the solids were removed by suction filtration, and the filtrate was transferred to a separatory funnel. Separating the organic phase and the aqueous phase, extracting the aqueous phase with EA twice, combining the organic phases, washing the combined phases with saturated brine once, and washing the combined phases with anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure, and the obtained crude product was purified by silica gel column chromatography (eluting with EA/PE system) to give compound 1e (3.1 g, yield 46.2%) as a white solid.

To compound 1e (1.1 g,4.0 mmol), compound 1f (1.2 g,4.8 mmol) and K ₂ CO ₃ (1.7 g,12.0 mmol) dioxane (dioxane, 40.0 mL) and H ₂ A nitrogen stream was introduced into the mixed solution of O (10.0 mL) to purge the reaction mixture of air (15 min), and then XPhos Pd G3 (338 mg,0.4 mmol) as a catalyst was added. After the addition, the reaction mixture was heated to 90℃in a closed system and stirred overnight. The reaction solution was cooled to room temperature, the solution was concentrated under reduced pressure, the resulting solid was dissolved in DCM, washed twice with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to give 1g (1.0 g, 84.2% yield) of a pale yellow solid compound.

N-bromosuccinimide (NBS, 107mg,0.6 mmol) was added in portions to a solution of compound 1g (154 mg,0.5 mmol) in DCM (15.0 mL) under ice-bath. After the addition, the reaction solution was reacted in an ice bath for 2.5 hours. The reaction was diluted with the appropriate amount of DCM and concentrated in saturated sodium bicarbonate (NaHCO ₃ ) Washing with aqueous solution once, then washing with saturated saline, and washing the organic phase with anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to give compound 1 (123 mg, yield 63.8%) as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.09(s,1H),8.53(d,J＝2.1Hz,1H),7.91(d,J＝2.0Hz,1H),7.73(d,J＝2.6Hz,1H),7.20(dd,J＝8.4,2.2Hz,1H),7.11(d,J＝2.2Hz,1H),6.95(d,J＝8.6Hz,1H),4.26(dd,J＝10.6,2.5Hz,1H),4.03–3.95(m,1H),3.95–3.84(m,2H),3.63(td,J＝11.5,8.4Hz,2H),3.24–3.11(m,2H),2.73(td,J＝12.1,3.6Hz,1H).

Example 2: preparation of Compound 2

N-iodosuccinimide (NIS, 743mg,3.3 mmol) was added in portions to a solution of compound 1g (921 mg,3.0 mmol) in DCM (90.0 mL) under ice-bath. After the addition, the reaction solution was stirred in an ice bath for 2.5 hours. The precipitated solid product was collected by suction filtration to give compound 2a (800 mg, yield 61.6%) as a pale yellow solid.

A solution of compound 2a (173 mg,0.4 mmol), compound 2b (45 mg,0.44 mmol) and triethylamine (0.17 mL,1.2 mmol) in N, N-dimethylformamide (DMF, 4.0 mL) was purged with a stream of nitrogen for 15min, and then catalyst Pd (PPh) ₃ ) ₂ Cl ₂ (28 mg,0.04 mmol) and copper iodide (CuI, 15mg,0.08 mmol). After the addition, the reaction was stirred overnight at room temperature under nitrogen. The reaction solution was diluted with an appropriate amount of DCM, washed three times with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to give compound 2 (50 mg, 30.7% yield) as a white solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.19(d,J＝2.1Hz,1H),8.54(d,J＝1.8Hz,1H),8.17(d,J＝2.0Hz,1H),7.94(d,J＝2.5Hz,1H),7.64–7.56(m,2H),7.48–7.34(m,3H),7.24(dd,J＝8.4,2.1Hz,1H),7.14(d,J＝2.1Hz,1H),6.97(d,J＝8.5Hz,1H),4.27(dd,J＝10.6,2.3Hz,1H),4.04–3.85(m,3H),3.72–3.56(m,2H),3.25–3.10(m,2H),2.79–2.65(m,1H).

Example 3: preparation of Compound 3

Referring to the procedure for the synthesis of compound 2 in example 2, compound 3 (40 mg, 33.8%) was obtained as a yellow solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.31(s,1H),8.61(d,J＝24.9Hz,2H),8.17(s,1H),8.05(d,J＝2.6Hz,1H),7.84(t,J＝7.6Hz,1H),7.72(d,J＝7.0Hz,1H),7.38(s,1H),7.24(dd,J＝8.3,2.0Hz,1H),7.14(d,J＝2.1Hz,1H),6.97(d,J＝8.5Hz,1H),4.27(dd,J＝10.6,2.4Hz,1H),4.03–3.95(m,1H),3.95–3.87(m,2H),3.69–3.59(m,2H),3.23–3.13(m,2H),2.79–2.68(m,1H).

Example 4: preparation of Compound 4

Referring to the procedure for the synthesis of compound 2 in example 2, compound 4 (48 mg, 29.4%) was obtained as a pale yellow solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.27(d,J＝2.2Hz,1H),8.83(s,1H),8.56(d,J＝1.8Hz,2H),8.23(d,J＝2.0Hz,1H),8.06–7.95(m,2H),7.46(dd,J＝7.8,4.8Hz,1H),7.25(dd,J＝8.4,2.1Hz,1H),7.16(d,J＝2.1Hz,1H),6.98(d,J＝8.5Hz,1H),4.27(dd,J＝10.6,2.3Hz,1H),4.05–3.85(m,3H),3.72–3.57(m,2H),3.25–3.11(m,2H),2.80–2.66(m,1H).

Example 5: preparation of Compound 5

Referring to the procedure for the synthesis of compound 2 in example 2, compound 5 (48 mg, 29.4%) was obtained as a white solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.36(d,J＝2.3Hz,1H),8.70–8.58(m,2H),8.57(d,J＝2.1Hz,1H),8.24(d,J＝2.0Hz,1H),8.06(d,J＝2.7Hz,1H),7.59(d,J＝5.9Hz,2H),7.25(dd,J＝8.4,2.1Hz,1H),7.16(d,J＝2.1Hz,1H),6.98(d,J＝8.5Hz,1H),4.28(dd,J＝10.6,2.4Hz,1H),4.04–3.85(m,3H),3.73–3.58(m,2H),3.25–3.12(m,2H),2.79–2.65(m,1H).

Example 6: preparation of Compound 6

To Compound 4 (61 mg,0.15 mmol) in methanol (CH ₃ To the OH,5.0 mL) solution was added Lindlar catalyst (10 mg). After the addition was completed, the air was replaced with hydrogen three times, and the reaction solution was stirred at 60℃overnight in a hydrogen atmosphere. The reaction solution is prepared with a proper amount of CH ₃ OH was diluted, lindlar catalyst was removed by suction filtration, the filtrate was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (EA/PE system elution) to give compound 6 (20 mg, yield 32.5%) as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ11.93(d,J＝2.1Hz,1H),8.81(d,J＝1.8Hz,1H),8.56(d,J＝1.7Hz,1H),8.40(d,J＝2.0Hz,1H),8.06(d,J＝8.0Hz,1H),7.83(d,J＝2.5Hz,1H),7.45(d,J＝2.0Hz,1H),6.98(d,J＝8.5Hz,1H),6.94(d,J＝12.1Hz,1H),6.89(d,J＝8.5Hz,1H),6.86(dd,J＝8.4,2.0Hz,1H),6.78(d,J＝2.0Hz,1H),6.55(d,J＝12.1Hz,1H),4.26(ddd,J＝15.5,10.6,2.6Hz,2H),4.01–3.85(m,5H),3.63(ddd,J＝12.4,6.4,3.7Hz,2H).

Example 7: preparation of Compound 7

Referring to the procedure for the synthesis of compound 6 in example 6, compound 7 (20 mg, yield 19.4%) was obtained as an off-white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ11.34–11.26(m,1H),8.46(s,1H),8.39(d,J＝2.0Hz,2H),8.05(d,J＝2.0Hz,1H),7.72–7.65(m,1H),7.29(dd,J＝7.8,4.8Hz,1H),7.22(d,J＝2.0Hz,1H),7.16(dd,J＝8.4,2.2Hz,1H),7.08(d,J＝2.1Hz,1H),6.95(d,J＝8.6Hz,1H),4.26(dd,J＝10.6,2.6Hz,1H),3.98(dd,J＝10.3,3.0Hz,1H),3.94–3.86(m,2H),3.70–3.58(m,2H),3.14(ddd,J＝13.2,7.2,2.7Hz,1H),3.07–3.02(m,2H),3.02–2.96(m,2H).

Example 8: preparation of Compound 8

To compound 1 (193 mg,0.5 mmol), compound 8a (123 mg,0.6 mmol) and K ₂ CO ₃ (207 mg,1.5 mmol) of dioxane (5.0 mL) and H ₂ A mixed solution of O (1.5 mL) was purged with nitrogen (15 min), and then catalyst Pd (PPh) was added ₃ ) ₄ (58 mg,0.05 mmol). After the addition, the reaction mixture was stirred overnight in a closed system at 90 ℃. The reaction solution was cooled to room temperature, the solution was concentrated under reduced pressure, the resulting solid was dissolved in DCM, washed twice with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to give compound 8 (20 mg, yield 10.4%) as a pale yellow solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ12.23–12.00(m,1H),9.03(d,J＝2.0Hz,1H),8.52(d,J＝1.9Hz,1H),8.47(dd,J＝4.7,1.4Hz,1H),8.36(d,J＝1.9Hz,1H),8.22(dt,J＝7.9,1.8Hz,1H),8.05(d,J＝2.4Hz,1H),7.47(dd,J＝7.8,4.8Hz,1H),7.24(dd,J＝8.4,2.1Hz,1H),7.17(d,J＝2.1Hz,1H),6.97(d,J＝8.5Hz,1H),4.27(dd,J＝10.6,2.4Hz,1H),4.05–3.96(m,1H),3.95–3.85(m,2H),3.73–3.58(m,2H),3.25–3.09(m,2H),2.79–2.64(m,1H).

Example 9: preparation of Compound 9

Referring to the synthetic procedure for compound 2 in example 2, compound 9 (43 mg, 10.2%) was obtained as a pale yellow solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.06(s,1H),8.53(s,1H),8.26(s,1H),8.12–8.09(m,1H),7.83(d,J＝2.5Hz,1H),7.56(d,J＝8.6Hz,1H),7.22(dd,J＝8.4,2.1Hz,1H),7.12(d,J＝2.1Hz,1H),6.96(d,J＝8.5Hz,1H),6.51(s,1H),6.33(s,2H),4.26(dd,J＝10.6,2.5Hz,1H),3.98(dd,J＝10.7,3.0Hz,1H),3.93–3.86(m,2H),3.68–3.59(m,2H),3.23–3.11(m,2H),2.73(td,J＝11.1,10.4,3.2Hz,1H).

Example 10: preparation of Compound 10

Referring to the procedure for the synthesis of compound 2 in example 2, compound 10 (81 mg, 19.2%) was obtained as a brown solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.11–12.04(m,1H),8.53(s,1H),8.18(d,J＝1.7Hz,1H),7.91(d,J＝2.6Hz,1H),7.29(dd,J＝7.6,1.4Hz,1H),7.23(dd,J＝8.4,2.1Hz,1H),7.13(d,J＝2.1Hz,1H),7.09–7.03(m,1H),6.96(d,J＝8.5Hz,1H),6.77–6.73(m,1H),6.56(td,J＝7.5,1.0Hz,1H),5.43(s,2H),4.26(dd,J＝10.6,2.5Hz,1H),3.98(dd,J＝10.6,3.3Hz,1H),3.92–3.86(m,2H),3.72–3.57(m,3H),3.21–3.13(m,2H).

Example 11: preparation of Compound 11

Referring to the synthetic procedure for compound 2 in example 2, compound 11 (298 mg, 42.4%) was obtained as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.12(d,J＝2.2Hz,1H),8.53(d,J＝2.0Hz,1H),8.08(d,J＝2.1Hz,1H),7.89(d,J＝2.8Hz,1H),7.22(dd,J＝8.4,2.1Hz,1H),7.12(d,J＝2.1Hz,1H),7.05(t,J＝7.8Hz,1H),6.97(d,J＝8.6Hz,1H),6.80–6.76(m,1H),6.72(d,J＝7.6Hz,1H),6.57(ddd,J＝8.1,2.2,0.8Hz,1H),5.24(s,1H),4.27(dd,J＝10.6,2.5Hz,1H),3.99(dd,J＝10.7,3.1Hz,1H),3.95–3.87(m,2H),3.71–3.59(m,2H),3.24–3.12(m,2H),2.74(td,J＝11.3,10.7,3.4Hz,1H).

Example 12: preparation of Compound 12

Referring to the synthetic procedure of compound 2 in example 2, compound 12 (67 mg, 10.6%) was obtained as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.00(d,J＝2.2Hz,1H),8.51(d,J＝2.1Hz,1H),8.07(d,J＝2.1Hz,1H),7.79(d,J＝2.6Hz,1H),7.34–7.17(m,3H),7.11(d,J＝2.1Hz,1H),6.98–6.93(m,1H),6.65–6.50(m,2H),5.49(s,1H),4.26(dd,J＝10.6,2.6Hz,1H),3.98(dd,J＝10.3,2.8Hz,1H),3.94–3.85(m,2H),3.69–3.59(m,2H),3.23–3.11(m,2H),2.73(td,J＝13.3,12.6,3.6Hz,1H).

Example 13: preparation of Compound 13

Referring to the procedure for the synthesis of compound 2 in example 2, compound 13 (129 mg, 18.0%) was obtained as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ11.96(s,1H),8.50(d,J＝2.2Hz,1H),8.04(d,J＝2.2Hz,1H),7.72(s,1H),7.19(dd,J＝8.4,2.2Hz,1H),7.08(d,J＝2.2Hz,1H),6.96(d,J＝8.6Hz,1H),4.26(dd,J＝10.6,2.5Hz,1H),3.99(dd,J＝10.5,3.0Hz,1H),3.95–3.83(m,2H),3.71–3.59(m,2H),3.56(s,2H),3.22–3.12(m,2H),2.78–2.69(m,1H).

Example 14: preparation of Compound 14

Referring to the procedure for the synthesis of compound 2 in example 2, compound 14b (310 mg, 26.1%) was obtained as a white solid.

To CH of compound 14b (300 mg,0.63 mmol) with stirring ₃ To a solution of OH (1.0 mL) was added dropwise dioxane hydrochloride (4N, 3.5 mL). After the addition, the reaction was stirred at room temperature overnight. Concentrating the reaction solution under reduced pressure, and dissolving the obtained solid in H ₂ In O, DCM was washed once. Then under ice bath, go to H ₂ Slowly adding a proper amount of sodium hydroxide (NaOH) solid particles into the O solution, collecting the precipitated solid product by suction filtration, and using H to prepare a filter cake ₂ And (3) washing. The crude product obtained was purified by column chromatography on silica gel (DCM/CH ₃ OH system elution) to give compound 14 (105 mg, 44.4% yield) as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.07(s,1H),8.65(d,J＝2.3Hz,1H),8.50(d,J＝2.2Hz,1H),7.96(s,1H),7.14(dd,J＝8.4,2.1Hz,1H),7.03(d,J＝2.1Hz,1H),6.97(d,J＝8.5Hz,1H),4.26(dd,J＝10.6,2.5Hz,1H),4.03–3.85(m,5H),3.63(tt,J＝11.4,5.6Hz,2H),3.23–3.12(m,2H),2.94(t,J＝8.1Hz,2H),2.73(td,J＝12.1,3.5Hz,1H),1.90(p,J＝7.7Hz,2H).

Example 15: preparation of Compound 15

Referring to the procedure for the synthesis of compound 2 in example 2, compound 15b (177 mg, 18.1%) was obtained as a white solid.

Referring to the procedure for the synthesis of compound 14 in example 14, compound 15 (70 mg, 51.8%) was obtained as a white solid. ¹ HNMR(500MHz,DMSO-d ₆ )δ12.12(s,1H),10.09(s,1H),8.56(s,1H),8.13(s,1H),7.89(d,J＝2.6Hz,1H),7.63(d,J＝8.6Hz,2H),7.52(dd,J＝8.9,2.1Hz,2H),7.22(dd,J＝8.4,2.1Hz,1H),7.13(d,J＝2.1Hz,1H),6.96(d,J＝8.6Hz,1H),4.26(dd,J＝10.6,2.5Hz,1H),3.98(dd,J＝10.8,3.1Hz,1H),3.94–3.85(m,2H),3.69–3.59(m,2H),3.23–3.12(m,2H),2.73(td,J＝11.3,10.8,3.4Hz,1H),2.06(s,3H).

Example 16: preparation of Compound 16

To a solution of compound 10 (84 mg,0.2 mmol) and triethylamine (40 mg,0.4 mmol) in DCM (2.0 mL) under ice-bath was slowly added dropwise acetyl Chloride (CH) ₃ COCl,16mg,0.2 mmol). After the addition, the reaction solution was stirred in an ice bath until the starting material disappeared. The reaction solution was diluted with an appropriate amount of DCM, washed three times with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to give compound 16 (40 mg, 43.1% yield) as a pale yellow solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.37–12.03(m,1H),9.55(s,1H),8.56(s,1H),8.29(d,J＝1.7Hz,1H),7.95(d,J＝2.5Hz,1H),7.76(t,J＝7.1Hz,1H),7.57(dd,J＝7.7,1.2Hz,1H),7.38–7.31(m,1H),7.25(dd,J＝8.4,1.9Hz,1H),7.21–7.14(m,2H),6.97(d,J＝8.6Hz,1H),4.27(dd,J＝10.6,2.5Hz,1H),3.99(dd,J＝11.1,3.1Hz,1H),3.94–3.85(m,2H),3.72–3.57(m,2H),3.23–3.12(m,2H),2.74(td,J＝11.8,3.5Hz,1H),2.16(s,3H).

Example 17: preparation of Compound 17

Referring to the procedure for the synthesis of compound 16 in example 16, compound 17 (42 mg, 45.3%) was obtained as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.25–12.10(m,1H),10.03(s,1H),8.55(s,1H),8.12(d,J＝1.6Hz,1H),7.96(d,J＝2.7Hz,1H),7.85(s,1H),7.52(d,J＝8.0Hz,1H),7.33(t,J＝7.9Hz,1H),7.28–7.21(m,2H),7.13(d,J＝2.1Hz,1H),6.97(d,J＝8.6Hz,1H),4.27(dd,J＝10.6,2.5Hz,1H),4.01–3.87(m,3H),3.70–3.59(m,2H),3.23–3.12(m,2H),2.74(td,J＝11.4,11.0,3.4Hz,1H),2.07(s,3H).

Example 18: preparation of Compound 18

Referring to the procedure for the synthesis of compound 16 in example 16, compound 18 (39 mg, 42.0%) was obtained as a white solid. ¹ HNMR(500MHz,DMSO-d ₆ )δ12.13(s,1H),10.09(s,1H),8.56(s,1H),8.14(s,1H),7.89(d,J＝2.6Hz,1H),7.63(d,J＝8.6Hz,2H),7.52(dd,J＝8.9,2.1Hz,2H),7.23(dd,J＝8.4,2.1Hz,1H),7.13(d,J＝2.1Hz,1H),6.97(d,J＝8.6Hz,1H),4.27(dd,J＝10.6,2.5Hz,1H),4.02–3.85(m,3H),3.70–3.59(m,2H),3.23–3.12(m,2H),2.74(td,J＝11.3,10.8,3.4Hz,1H),2.07(s,3H).

Example 19: preparation of Compound 19

Referring to the procedure for the synthesis of compound 16 in example 16, compound 19 (50 mg, 62.2%) was obtained as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ12.03(d,J＝2.2Hz,1H),8.51(d,J＝2.1Hz,1H),8.38(t,J＝5.0Hz,1H),8.02(d,J＝2.0Hz,1H),7.78(d,J＝2.7Hz,1H),7.19(dd,J＝8.4,2.1Hz,1H),7.08(d,J＝2.2Hz,1H),6.97(d,J＝8.6Hz,1H),4.27(dd,J＝10.6,2.5Hz,1H),4.17(d,J＝5.3Hz,2H),3.99(dd,J＝10.7,3.1Hz,1H),3.95–3.82(m,2H),3.72–3.56(m,2H),3.23–3.12(m,2H),2.74(td,J＝11.2,10.6,3.4Hz,1H),1.86(s,3H).

Example 20: preparation of Compound 20

Referring to the procedure for the synthesis of compound 2 in example 2, compound 20 (22 mg, 11.3%) was obtained as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ11.99(s,1H),8.56(s,1H),7.99(s,1H),7.74(s,1H),7.18(dd,J＝8.4,2.1Hz,1H),7.07(d,J＝2.1Hz,1H),6.97(d,J＝8.5Hz,1H),5.41(s,1H),4.27(dd,J＝10.6,2.5Hz,1H),4.04–3.85(m,3H),3.72–3.58(m,2H),3.24–3.10(m,2H),2.73(td,J＝13.2,12.5,3.5Hz,1H),1.51(s,6H).

Example 21: preparation of Compound 21

To compound 1e (5.40 g,20.00 mmol), bis-pinacolato diboron (10.16 g,40.00 mmol) and KOA _C (5.88 g,60.00 mol) of dioxane (54.0 mL) was purged with a nitrogen stream to empty the reaction solution of air (15 min), and then catalyst Pd (PPh) ₃ ) ₄ (1.16 g,1.00 mmol). After the addition, the reaction mixture was heated to 95℃in a closed system and stirred overnight. The reaction solution was cooled to room temperature, the solution was concentrated under reduced pressure, the resulting solid was dissolved in DCM, washed twice with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ The OH system eluted) to give compound 21a as a white solid (2.74 g,yield 43.2%).

To compound 21b (411 mg,3.00 mmol) and compound 21c (450 mg,2.00 mmol) were added dioxane (dioxane, 5.0 mL) and piperidine (5.0 mL) TEA (768 mL,7.6 mmol). After the addition, the reaction mixture was heated to 100℃in a closed system and stirred overnight. The reaction solution was cooled to room temperature, the solution was concentrated under reduced pressure, the resulting solid was dissolved in DCM, washed twice with water and then once with saturated brine, and the organic phase was taken up in anhydrous Na ₂ SO ₄ And (5) drying. The drying agent was removed by filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to afford compound 21d (471 mg, 78.5% yield) as a pale yellow solid.

The procedure for the synthesis of 1g of compound of example 1 was followed to give compound 21 (66 mg, yield 32.2%) as a white solid. ¹ HNMR(500MHz,DMSO-d ₆ )δ11.92(d,J＝2.7Hz,1H),8.80(d,J＝2.3Hz,1H),8.55(d,J＝2.1Hz,1H),8.49(d,J＝2.1Hz,1H),8.39(dd,J＝4.7,1.6Hz,1H),8.04(dt,J＝8.0,2.0Hz,1H),7.82(d,J＝2.6Hz,1H),7.58(d,J＝16.7Hz,1H),7.37(dd,J＝8.0,4.7Hz,1H),7.26(dd,J＝8.4,2.2Hz,1H),7.23–7.16(m,2H),6.98(d,J＝8.5Hz,1H),4.28(dd,J＝10.6,2.6Hz,1H),3.99(dd,J＝11.1,3.4Hz,1H),3.95–3.87(m,2H),3.71–3.60(m,2H),3.21(t,J＝10.5Hz,1H),3.17–3.11(m,1H),2.74(td,J＝11.6,3.6Hz,1H).

Example 22: preparation of Compound 22

To a solution of compound 22b (427 mmol,2.40 mmol) in dichloromethane (4.0 mL) was added a solution of compound 22a (424 mg,2.00 mmol) in pyridine (4.0 mL). The reaction mixture was stirred at room temperature overnight. The precipitate was collected by filtration and the resulting crude product was purified by silica gel column chromatography (DCM/CH ₃ OH system elution) to afford compound 22c as a brown solid (311 mg, 40.8% yield).

The procedure for the synthesis of 1g of compound of example 1 was followed to give compound 22 (112 mg, yield 32.8%) as a white solid. ¹ H NMR(500MHz,DMSO-d ₆ )δ11.51(d,J＝2.6Hz,1H),10.51(s,1H),9.17(d,J＝1.5Hz,1H),8.77(dd,J＝4.7,1.7Hz,1H),8.55(d,J＝2.2Hz,1H),8.49(d,J＝2.2Hz,1H),8.35(dt,J＝7.9,2.0Hz,1H),7.95(d,J＝2.6Hz,1H),7.59(dd,J＝7.5,5.2Hz,1H),7.19(dd,J＝8.4,2.2Hz,1H),7.09(s,1H),6.97(d,J＝8.5Hz,1H),4.26(dd,J＝10.6,2.6Hz,1H),3.98(dd,J＝10.6,3.4Hz,1H),3.94–3.86(m,2H),3.69–3.59(m,2H),3.20(t,J＝10.5Hz,1H),3.14(tt,J＝10.7,2.5Hz,1H),2.73(td,J＝11.3,10.7,3.5Hz,1H).

Example 23: partial compound in vitro AAK1 inhibition activity assay

1. Experimental materials

AAK1(Innitrogen,Cat.No.A32880)；LP-922761(MCE,Cat.No.HY-120179)；ADP-Glo KinaseAssay(Promege,Cat.No.v9102/3)；HEPES,pH7.5(Gibco,Cat.No.11344-041)；Brij-35solution(Sigma,Cat.No.B4184)；EDTA(Gibco,Cat.No.15575-038)；MgCl ₂ (Sigma,Cat.No.M2670-500g)；DTT(Sigma,Cat.No.D0632-10G)；96well plate(Corning,Cat.No.3365)；384well Echo plate(Labcyte,Cat.No.PP-0200)；PROXIPLATE-384PLUS(PE,Cat.No.6008289)。

2. Experimental method

2.1 serial dilution of Compounds to prepare Source plate

1) The initial concentration of the compound tested was 0.5. Mu.M, formulated at a 100-fold concentration, i.e., 50. Mu.M. 995. Mu.L of 100% DMSO was added to the EP tube, followed by 5. Mu.L of 10mM compound solution, to prepare a 50. Mu.M compound solution. mu.L of the above compound solution was added to the second well of the 96-well plate, and 80. Mu.L of 100% DMSO was added to the other wells. Adding 20 mu L of compound solution from the second hole into the third hole, sequentially diluting downwards for 5 times, and diluting for 4 concentrations;

2) 100 μl of 100% dmso was added as a no compound control and no enzyme control, respectively, to the same 96 well plate, and the plate was labeled as a source plate;

2.2 preparation of intermediate plate

Transfer 40 μl of compound from the source plate onto a new 384 well plate as an intermediate plate;

2.3 preparation of analysis plate

Transfer 50nL of compound to assay plate by Echo in 100% dmso;

2.4 preparation of buffer solution

40mM Tris pH 7.5，20mM MgCl ₂ ，0.01mg/ml BSA，1mM DTT；

2.5 preparation of kinase solution

1) Preparing an AAK1 solution in a kinase buffer;

2) 2.5. Mu.L of kinase solution was added per well except for the control wells without enzyme (replaced with 2.5. Mu.L of kinase buffer);

3) A rocking plate;

2.6 preparation of substrate solution

1) Preparing a substrate solution of an AAK1 peptide substrate and ATP in a 1x kinase reaction buffer;

2) 2.5. Mu.L of substrate solution was added to each well of the assay plate to initiate the reaction;

3) A rocking plate;

2.7 kinase reaction

The assay plate was covered and incubated for 1 hour at room temperature;

2.8 kinase assay

1) Equilibrate ADP-Glo reagent to room temperature;

2) The reaction was stopped by adding 5. Mu.L of ADP-Glo reagent per well;

3) Stirring with a centrifuge, slowly shaking on a shaking table, and balancing for 120 min;

4) Adding 10 mu L of kinase detection reagent into each hole, oscillating for 1min, balancing for 30min, and reading and emitting light on a flat-plate reader;

2.9 reading of the plate

Collecting data;

2.10 Curve fitting

1) Copying the value of the RLU from Envision;

2) Converting the RLU value to a percent inhibition value;

a. inhibition ratio = (max-sample RLU)/(max-min) ×100;

"min" refers to RLU without enzyme, "max" refers to RLU with DMSO;

3) Fitting the data in XLFIT excel 5.4.0.8 version to obtain IC ₅₀ A value;

the formula is Y =Bottom+(Top-Bottom)/(1+(IC ₅₀ /X)*HillSlope)；

Top is the highest point of the curve, bottom is the lowest point of the curve, hillSlope represents the curvature of the curve.

3. Experimental results

Inhibition of AAK1 by a portion of the target compounds was determined by the above-described experimental methods. The results are shown in Table 1.

TABLE 1 half inhibition concentration of target compounds on AAK1

Example 24: compound 4, 9 inhibiting SARS-CoV-2 pseudovirus from infecting ACE2 high expression cell

1. Cells and viruses used in experiments

SARS-CoV-2 pseudovirus is purchased from Biotechnology Co., ltd. In Fubai, and has a product number of FNV215, and the surface of the envelope of the virus contains Spike glycoprotein (Spike Protein) of a novel coronavirus, and the virus is internally coated with RNA sequences of Green Fluorescent Protein (GFP) and Luciferase (Luciferase). The infection efficiency can be judged by observing the expression of green fluorescent protein and detecting the activity of luciferase after the virus infects target cells 48-72H.

The HEK-293 cell line hACE2-HEK293 overexpressing human ACE2 protein was also purchased from Biotechnology Co., ltd., fubai, st. No. FBC2591, and the cell culture broth was DMEM medium containing 10% FBS, 1% Streptomyces lividans mixed solution according to conventional subculture.

2. Experimental procedure and results:

2.1 drug treatment:

the drug to be tested was diluted to 10mM with DMSO, then the 10mM drug solution was diluted to 1mM with cell culture solution (dulbecco' smodified eagle medium, DMEM), sterilized and filtered through a 0.22 μm filter, and sub-packaged and stored at-20deg.C for use.

2.2 evaluation of the effect of drugs on invasion of SARS-CoV-2 into host cells:

hACE2-293 cells were cultured at a density of 5X 10 ⁵ Inoculating into 24-well plate at 37deg.C with 5% CO ₂ After the cells are completely adhered after being cultured for 6 hours in an incubator. hACE2-HEK293 cells were treated with 5. Mu.M of compounds 4 and 9 in DMEM, 5. Mu.M of LX9211 was added as a positive control group, 5. Mu.M of compound 28 (J.Med. Chem.2019,62, 5810-5831) was added as a reference group, cells treated without any drug were used as a negative control group, and after 1 hour of drug treatment, SARS-CoV-2 pseudovirus (MOI=5) was added and cells were continuously infected at 37℃for 48 hours. The green fluorescent protein expression was observed by photographing under a fluorescent microscope, and the result is shown in fig. 1. After photographing, cells were lysed and the luminescence value of luciferases was measured using the Luciferase Assay System kit (Promega Co.) and the results are shown in FIG. 2.

Wherein the structure of compound 28 is shown in the formula:

FIG. 1 shows that compounds 4, 9, 28 and LX9211 (BMS-986176) inhibit SARS-CoV-2 virus invasion into host cells as measured by 50 μm when green fluorescent protein expression is observed under a fluorescent microscope.

The luciferase assay of fig. 2 shows that compounds 4, 9, 28 and LX9211 (BMS-986176) inhibit invasion of SARS-CoV-2 virus into host cells with P <0.01.

Experimental results show that the AAK1 inhibitor obtained through structural modification, the compounds 4 and 9 can enhance the activity of corresponding SARS-CoV-2 virus invasion host cells, and can be used for preventing or treating related diseases caused by novel coronavirus infection, and are expected to provide new candidate drug molecules for clinically treating pneumonia caused by the novel coronavirus infection and upper respiratory tract infection caused by the novel coronavirus infection.

In summary, the present invention provides the preparation of compounds of formula I, and their inhibition of AAK1 and their use in the preparation of anti-coronavirus drugs.

Claims

1. An AAK1 inhibitor compound, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, characterized in that the structural formula of the compound is shown in formula (I):

x in formula (I) is selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:z=0 or 1;

wherein the method comprises the steps ofRepresents the site of attachment of X to 7-azaindole;/>represents the site of attachment of X to Y; />Represents the site where Y is linked to Z.

2. An AAK1 inhibitor compound according to claim 1, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, wherein X in formula (I) is selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

wherein the method comprises the steps ofRepresents the position of attachment of X to 7-azaindoleA dot; />Represents the site of attachment of X to Y; />Represents the site where Y is linked to Z.

3. An AAK1 inhibitor compound according to claim 2, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, wherein X in formula (I) is selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

wherein the method comprises the steps ofRepresents X linked to 7-azaindoleIs a site of (2); />Represents the site of attachment of X to Y; />Represents the site where Y is linked to Z.

4. An AAK1 inhibitor compound according to claim 1, wherein the compound has the structural formula of any one of formulae 1 to 27:

5. a process for preparing an AAK1 inhibitor compound, characterized in that when x=When the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3; y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

6. A process for preparing an AAK1 inhibitor compound, characterized in that when x=When the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

(7) The compound Ia is subjected to reduction reaction to obtain a compound I;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3, y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

wherein the method comprises the steps ofRepresents the site of attachment of X to 7-azaindole; />Represents the site of attachment of X to Y; />Representation ofThe site where Y is linked to Z.

7. A process for preparing an AAK1 inhibitor compound, characterized in that whenWhen the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3, y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

8. A process for preparing an AAK1 inhibitor compound, characterized in that whenWhen the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

(3) Carrying out Sandmeyer reaction on the compound d to obtain a compound e;

the synthetic route is as follows:

wherein Y is selected from one of the following structures:

wherein n is a natural number from 1 to 3, y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

9. A process for preparing an AAK1 inhibitor compound, characterized in that whenWhen the method comprises the following steps:

(2) The compound c undergoes a reduction reaction to obtain a compound d;

the synthetic route is as follows:

wherein,selected from one of the following structures:

y is selected from one of the following structures:

wherein n is a natural number from 1 to 3, y=0 or 1;

z is selected from one of the following structures:

z=0 or 1;

10. Use of an AAK1 inhibitor compound according to any one of claims 1-4, or an optical isomer, racemate, single enantiomer, possible diastereoisomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof, for the preparation of an anti-coronavirus drug.

11. An anti-coronavirus agent comprising a safe and effective amount of said AAK1 inhibitor compound, or an optical isomer, racemate, single enantiomer, possible diastereomer, or a pharmaceutically acceptable salt, prodrug, deuterated derivative, hydrate, solvate thereof.

12. An anti-coronavirus agent according to claim 9, further comprising a pharmaceutically acceptable carrier.