Dnj derivatives, pharmaceutical compositions and uses thereof

Abstract

The application discloses a DNJ derivative, a pharmaceutical composition and application thereof; the DNJ derivative has the following structural formula: the application provides a series of novel DNJ derivatives in structure, the DNJ derivative can pass through the misfolding of the S protein of the coronavirus cell membrane protein S protein, so that the infectivity of the coronavirus is reduced, and the purpose of inhibiting the coronavirus infection is achieved.The DNJ derivative is expected to be used for treating and / or preventing the coronavirus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a DNJ derivative, a pharmaceutical composition and applications thereof. Background Art

[0002] In recent years, the periodic outbreaks of highly pathogenic coronavirus pandemics, represented by SARS-CoV-2, have not only posed a significant threat to public health but also inflicted immeasurable losses and heavy burdens on economic and social development. This highlights the fact that emerging infectious diseases caused by coronaviruses have become a major challenge to the survival and development of all humanity. Currently known coronaviruses are just the tip of the iceberg, and new, more harmful zoonotic coronaviruses are likely to emerge in the future. Therefore, the development of highly effective, broad-spectrum anti-coronavirus drugs is crucial to defeating coronaviruses.

[0003] DNJ (1-Deoxynojirimycin, CAS No. 19130-96-2) is an aminosugar compound extracted from the traditional Chinese medicine mulberry leaves and white mulberry bark. Its structure is highly similar to glucose. Bayer has discovered that it is a potent α-glucosidase inhibitor, showing significant inhibition against various types of α-glucosidase, and is used to treat type 2 diabetes in adults. DNJ analogs, miglitol (Glyset™) and miglustat (Zavesca™), are also used clinically as α-glucosidase inhibitors and glycotransferase inhibitors, respectively, to treat type 2 diabetes, type 1 Gaucher disease, and Niemann-Pick disease type C.

[0004] Studies have shown that existing DNJ and its derivatives often have weak antiviral activity, and there are currently no reports of DNJ derivatives being used against coronaviruses.

[0005] Therefore, it is necessary to develop a DNJ derivative with novel structure and anti-coronavirus effect. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the first aspect of the present invention provides a DNJ derivative having anti-coronavirus characteristics.

[0007] The second aspect of the present invention also provides a pharmaceutical composition.

[0008] The third aspect of the present invention also provides a use of a DNJ derivative.

[0009] According to the first aspect of the present invention, the DNJ derivative provided has the following structural formula:

[0010]

[0011] Wherein, R is selected from C 5～10 Alkyl, methoxy substituted C 5～8 Alkyl, C substituted by alkynyl, hydroxyl or methoxyphenyl 1～5 The alkyl group, m≥1, n≥1, x≥1, y≥1, z≥1.

[0012] The DNJ derivative according to the embodiment of the present invention has at least the following beneficial effects:

[0013] The present invention provides a series of novel DNJ derivatives that can inhibit coronavirus infection by reducing the infectivity of the coronavirus by misfolding the coronavirus membrane protein S protein. They are expected to be used to treat and / or prevent coronavirus infection.

[0014] According to a preferred embodiment of the present invention, R is selected from C 8～10 Alkyl, methoxy substituted C 5～8 Alkyl, C substituted by alkynyl, hydroxyl or methoxyphenyl 1～5 The alkyl group, m is selected from 1 to 12, n is selected from 1 to 10, x is selected from 1 to 12, y is selected from 1 to 112, and z is selected from 1 to 12.

[0015] According to a preferred embodiment of the present invention, R is selected from

[0016] m is selected from 3 to 12, n is selected from 3 to 10, x is selected from 3 to 12, y is selected from 3 to 12, and z is selected from 3 to 12.

[0017] According to a preferred embodiment of the present invention, the DNJ derivative is selected from the following structures:

[0018]

[0019]

[0020] According to a preferred embodiment of the present invention, the DNJ derivative is prepared by the following method, and the reaction equation is as follows:

[0021]

[0022] S1, mixing compound 1, a reducing agent, and an organic solvent I to carry out reaction I, solid-liquid separation, and collecting the solid phase to obtain compound 2;

[0023] S2, mixing the compound 2, the acyl halide and the organic solvent II to carry out reaction II, and then adding an organic amine to react to obtain an intermediate solution; mixing the intermediate solution, a boron reducing agent, an ammonium salt and the organic solvent III to carry out reaction III, solid-liquid separation, and collecting the solid phase to obtain compound 3;

[0024] S3, mixing the compound 3, R-COH, palladium catalyst I, organic carboxylic acid and organic solvent IV to carry out reaction IV to obtain compound 4;

[0025] Alternatively, the compound 3, Br-R, potassium carbonate, potassium iodide and an organic solvent V are mixed to carry out reaction V to obtain compound 4;

[0026] S4. Mix the compound 4, an ether solvent, an inorganic acid and a palladium catalyst II to carry out reaction VI to obtain.

[0027] According to a preferred embodiment of the present invention, in step S1, the reducing agent includes at least one of lithium aluminum hydride, diisobutylaluminum hydride (DIBAL-H), dimethoxyethoxyaluminum hydride, potassium borohydride, lithium borohydride, sodium borohydride, and sodium cyanoborohydride.

[0028] According to a preferred embodiment of the present invention, in step S1, the organic solvent I includes at least one of tetrahydrofuran (THF), diethyl ether, methyl tert-butyl ether, toluene, benzene, and 1,4-dioxane.

[0029] According to a preferred embodiment of the present invention, in step S1, the molar ratio of the reducing agent to compound 1 is 1:3-4.

[0030] According to a preferred embodiment of the present invention, in step S1, the temperature of the reaction I is -5°C to 5°C.

[0031] According to a preferred embodiment of the present invention, in step S2, the acyl halide includes acyl chloride.

[0032] According to a preferred embodiment of the present invention, in step S2, the acyl chloride includes at least one of oxalyl chloride, acetic anhydride, acetyl chloride, trifluoroacetic anhydride, trichloroacetic anhydride, and trichloroacetyl chloride.

[0033] According to a preferred embodiment of the present invention, in step S2, the organic solvent II includes at least one of a halogenated hydrocarbon and a sulfone solvent.

[0034] According to a preferred embodiment of the present invention, the halogenated hydrocarbon includes at least one of dichloromethane (DCM), 1,2-dichloroethane, chloroform, and carbon tetrachloride.

[0035] According to a preferred embodiment of the present invention, the sulfone solvent includes at least one of dimethyl sulfoxide (DMSO) and diphenyl sulfoxide.

[0036] According to a preferred embodiment of the present invention, the organic amine includes at least one of triethylamine (ET3N), DBU, diisopropylethylamine and DABCO.

[0037] According to a preferred embodiment of the present invention, the molar ratio of the compound 2, the acyl halide and the organic amine is 1:1.5-2.5:2.5-3.5.

[0038] According to a preferred embodiment of the present invention, the boron reducing agent includes at least one of NaBH3CN, NaBH(OAc)3, NaBH4, and LiBHEt3.

[0039] According to a preferred embodiment of the present invention, the ammonium salt is at least one of ammonium acetate and ammonium formate.

[0040] According to a preferred embodiment of the present invention, the molar ratio of the ammonium salt to the boron reducing agent is 1:1.9-2.1.

[0041] According to a preferred embodiment of the present invention, the reaction temperature of Reaction II is -90°C to -70°C.

[0042] According to a preferred embodiment of the present invention, the reaction temperature of Reaction III is 20°C to 25°C.

[0043] According to a preferred embodiment of the present invention, the molar ratio of R-COH to compound 3 is 2:3-4.

[0044] According to a preferred embodiment of the present invention, the palladium catalyst I comprises at least one of palladium-carbon catalyst, palladium hydroxide, platinum oxide, platinum hydroxide, ruthenium-carbon, and rhodium-carbon.

[0045] According to a preferred embodiment of the present invention, the organic carboxylic acid includes at least one of formic acid, acetic acid and propionic acid.

[0046] According to a preferred embodiment of the present invention, the organic solvent III and the organic solvent IV are independently selected from at least one of methanol, ethanol and propanol.

[0047] According to a preferred embodiment of the present invention, the organic solvent V includes acetonitrile and N,N-dimethylformamide.

[0048] According to a preferred embodiment of the present invention, the reaction temperature of reaction V is 60-80°C.

[0049] According to a preferred embodiment of the present application, the ether solvent comprises at least one of dimethyl ether, diethyl ether and methyl tert-butyl ether (MTBE).

[0050] According to a preferred embodiment of the present application, the inorganic acid comprises at least one of sulfuric acid, hydrogen chloride and hydrogen bromide.

[0051] According to a preferred embodiment of the present application, the molar ratio of the inorganic acid to compound 4 is 1:5-10.

[0052] According to a preferred embodiment of the present application, the palladium catalyst II comprises at least one of palladium-carbon catalyst, palladium hydroxide, platinum oxide, platinum hydroxide, ruthenium-carbon and rhodium-carbon.

[0053] According to a second aspect of the present application, a pharmaceutical composition is provided, comprising the DNJ derivative or the pharmaceutically acceptable salt thereof according to the first aspect of the present application, and a pharmaceutically acceptable excipient.

[0054] According to a preferred embodiment of the present application, the DNJ derivative or the pharmaceutically acceptable salt thereof accounts for 0.1%-99% of the total mass, calculated based on the mass of the pharmaceutical composition.

[0055] According to a preferred embodiment of the present application, the DNJ derivative or the pharmaceutically acceptable salt thereof accounts for 0.5%-95% of the total mass, calculated based on the mass of the pharmaceutical composition.

[0056] According to a preferred embodiment of the present application, the dosage form of the pharmaceutical composition comprises a solid, a semi-solid or a solution. For example, it can be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a soft capsule, a granule, a pill, an oral solution, a dry suspension, a dripping pill, a dry extract, an injection or an infusion.

[0057] According to a preferred embodiment of the present application, the administration mode of the pharmaceutical can be a conventional administration mode in the art, including but not limited to injection administration or oral administration. The injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc.

[0058] According to a preferred embodiment of the present application, the administration mode of the pharmaceutical can be a conventional administration mode in the art, including but not limited to injection administration or oral administration. The injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection, etc.

[0059] According to a preferred embodiment of the present application, the coronavirus comprises SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and SARS-CoV.

[0060] Definitions and General Terms

[0061] “C 5-10 "alkyl" means an alkyl group having a total of 5 to 10 carbon atoms, including C 5-10 Straight chain alkyl, C 5-10 Branched alkyl and C 5-10 The cycloalkyl group may be, for example, a straight-chain alkyl group having 5, 6, 7, 8, 9, or 10 carbon atoms, a branched-chain alkyl group having 5, 6, 7, 8, 9, or 10 carbon atoms, or a cycloalkyl group having 5, 6, 7, 8, 9, or 10 carbon atoms, such as n-pentyl, isopentyl, n-hexyl, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, cyclopentyl, methylcyclopentyl, cyclohexyl, n-heptyl, isoheptyl, octyl, decyl, etc. 8-10 The "alkyl group" has a similar explanation, except that the number of carbon atoms is different.

[0062] "Methoxy-substituted C 5～8 Alkyl" and "C 5-10 The explanation of "alkyl" is basically similar, except that C 5～8 Any one of the hydrogen atoms in the alkyl group is substituted by a methoxy group.

[0063] "C substituted by alkynyl, hydroxyl or methoxyphenyl 1～5 Alkyl, " and "C 5-10 The explanation of "alkyl" is basically similar, except that C 1～5 One of the hydrogen atoms in the alkyl group is optionally substituted by an alkynyl group, a hydroxyl group or a methoxyphenyl group.

[0064] Pharmaceutically acceptable excipients of the present invention include any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, glidant or lubricant, etc., suitable for the specific target dosage form. As described in the following documents: In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, the contents of these documents are summarized, indicating that different excipients can be used in the preparation of pharmaceutically acceptable compositions and their known preparation methods. Except to the extent that any conventional excipients are incompatible with the compounds of the present invention, for example by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present invention.

[0065] Examples of pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffer substances, such as phosphates; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blocking polymers; lanolin; sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethyl cellulose. sodium cellulose, ethylcellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered solution; and other nontoxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; release agents; coatings; sweeteners; flavoring agents; fragrances; preservatives and antioxidants.

[0066] Pharmaceutically acceptable salts of the present application include salts with inorganic acids, organic acids, alkali metals, alkaline earth metals and basic amino acids. These include, but are not limited to: hydrochlorides, sulfates, bisulfates, nitrates, hydrobromides, hydroiodides, carbonates, bicarbonates, sulfites, bisulfites, pyrosulfates, monohydrogen phosphates, dihydrogen phosphates, perchlorates, persulfates, hemisulfates, bisulfates, thiocyanates, phosphates, pyrophosphates, metaphosphates; suitable organic acid salts include, but are not limited to: formates, acetates, propionates, butyrates, benzoates, malonates, succinates, pyruvates, mesylates, esylates, propylsulfonates, citrates, 4-nitrobenzoates, phenylsulfonates, p-toluenesulfonates, malates, propiolates, 2-butynoates, 2-hydroxy-ethanesulfonates, vinylacetates, tartrates, L-tartrates, fumarates, isethionates, maleates, lactates, lactobionates, pamoates, salicylates, galactarates, glucoheptanoates, mandelates, 1,2-ethanedisulfonates, 2-naphthalenesulfonates, oxalates, trifluoroacetates, trifluoromethanesulfonates, adipates, suberates, sebacates, butyn-1,4-dioates, hexyn-1,6-dioates, glycolates, alginates, ascorbates, isoascorbates, aspartates, L-aspartates, glutarates, L-glutarates, 2-phenoxybenzoates, 2-(4-hydroxybenzoyl)benzoates, acetoacetates, 2-hydroxyethanesulfonates, benzenesulfonates, borates, chlorobenzoates, camphorates, itaconates, camphorsulfonates, levocamphorsulfonates, methylbenzoates, dinitrobenzoates, sulfamates, lactobionates, galacturonates, cyclopentanepropionates, dodecylsulfates, acrylates, cyclopentanepropionates, glycerophosphates, methoxybenzoates, digluconates, gluconates, heptanoates, hexanoates, 2-hydroxy-ethanesulfonates, trimethylacetates, glucuronates, laurates, phthalates, phenylacetates, lauryl sulfates, 2-acetoxybenzoates, nicotinates, cinnamates, oleates, palmitates, pamoates, pectates, phthalates, glutarates, hydroxymaleates, hydroxybenzoates, phenylacetates, 3-hydroxy-2-naphthoates, 3-phenylpropionates, isobutyrates, neopentanoates, picrates, stearates, 2,2-dichloroacetates, acylated amino acid salts, alginates, 4-acetamidobenzoates, cucurbitates, cholate, octanoates, nonanoates, cyclamic acids, pthalates, hemisulfates, sorbates, pamoates, mucates, glycine hydrochlorides, naphthalene disulfonates, xylene sulfonates, cysteine hydrochlorides, undecanoates, polyvinylsulfonates, sulfosalicylates, phenylbutyrates, 4-hydroxybutyrates, polyvinylsulfates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and valerates.

[0067] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0069] Figure 1 This is a graph showing the experimental results of co-treatment of a pseudovirus model with DNJ derivatives prepared in an embodiment of the present invention;

[0070] Figure 2 This is a graph showing the experimental results of the pre-treatment of pseudoviruses by DNJ-25 derivatives in an embodiment of the present invention;

[0071] Figure 3 This is a graph showing the inhibition of pseudovirus co-treatment by DNJ-25 derivatives in an embodiment of the present invention;

[0072] Figure 4 This is a graph showing the inhibition of pseudovirus pretreatment by DNJ-25 derivatives in an embodiment of the present invention;

[0073] Figure 5 This is a graph showing a co-treatment time gradient experiment of DNJ-25 derivatives inhibiting a pseudovirus model according to an embodiment of the present invention;

[0074] Figure 6 This is a curve diagram of the DNJ-25 derivatives inhibiting the live virus model in the examples of the present invention and a diagram of the mRNA levels of NP and Spike proteins that inhibit the live virus.

[0075] Marking Description:

[0076] *: P<0.05; **: P<0.01; ***: P<0.001. DETAILED DESCRIPTION

[0077] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0078] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0079] Synthesis of raw material DNJ-1: The synthetic route and preparation method are as follows:

[0080]

[0081] First step: To a suspension of lithium aluminum hydride (2.28 g, 60 mmol) in 25 mL of tetrahydrofuran, compound 1 (10.82 g, 20 mmol) in 50 mL of tetrahydrofuran was added slowly dropwise at 0 °C under ice-bath condition. TLC was used to monitor the progress of the reaction. After the reaction was completed, sodium sulfate decahydrate was added to quench the lithium aluminum hydride under ice-bath condition. Then, diatomite was added to assist filtration. The filtrate was rotary evaporated and purified by flash column chromatography (DCM:MeOH = 100:1) to give compound 2 (10.4 g, 95%) as a white solid.

[0082] Second step: To a solution of oxalyl chloride (7.34 mL, 86 mmol) in 50 mL of anhydrous dichloromethane, a solution of dimethyl sulfoxide (7.52 mL, 106 mmol) in 10 mL of dichloromethane was added dropwise at -78 °C. After half an hour, a solution of compound 2 (10.86 g, 20 mmol) in 50 mL of dichloromethane was added dropwise. After 2 h, triethylamine (30 mL, 216 mmol) was added dropwise. The reaction was slowly warmed to room temperature. TLC was used to monitor the progress of the reaction. After the starting material was consumed, the reaction was slowly added to a pre-cooled solution of ammonium acetate (15.4 g, 200 mmol) and sodium cyanoborohydride (5.04 g, 80 mmol) in anhydrous methanol at 0 °C. The reaction was warmed to room temperature. TLC was used to monitor the progress of the reaction. After the reaction was completed, the solvent was rotary evaporated. Then, 50% aqueous sodium hydroxide solution (100 mL) was added. The organic phase was extracted with dichloromethane. The organic phase was washed with 37% sodium hypochlorite solution (100 mL). The organic phase was adjusted to acidic pH with 2 M hydrochloric acid solution. The product was obtained as a white solid by slurry with acetone. The white solid was filtered and dried to give compound 3 as a hydrochloride salt. The hydrochloride salt was dissolved in 50% aqueous sodium hydroxide solution. The solution was extracted with ethyl acetate. The organic phase was washed with saturated brine. The yellow oil of compound 3 was obtained by rotary evaporation (4.2 g, 42%).

[0083] Third step: Compound 3 (1.05 g, 2 mmol) was dissolved in 10 mL of anhydrous dichloromethane and stirred at -78 °C for 0.5 h. Then, boron tribromide (1.5 g, 6 mmol) was added dropwise slowly. The reaction was continued at room temperature for 0.5 h. TLC was used to monitor the progress of the reaction. After the reaction was completed, the reaction was quenched with triethylamine. The reaction was extracted with ionized water. The aqueous phase was rotary evaporated and dissolved in methanol. The product was obtained as a white solid by column chromatography on neutral alumina with MeOH→MeOH:H2O = 2:1 as the eluent (228 mg, 70%).

[0084] The data of DNJ-1 are as follows:

[0085] [α] D 25 = -175 (c = 1.3, MeOH).

[0086] 1 H NMR (400MHz, D2O) δ3.70(dd,J=11.6,3.0Hz,1H),3.49(dd,J=11.7,6.3Hz,1H),3.36(ddd,J=10.8,9.1,5.1Hz,1H),3.18(t,J=9. 1Hz, 1H), 3.10 (t, J = 9.4Hz, 1H), 2.98 (dd, J = 12.3, 5.2Hz, 1H), 2.41 (ddd, J = 9.5, 6.2, 2.9Hz, 1H), 2.32 (dd, J = 12.3, 10.8Hz, 1H).

[0087] Example 1

[0088] This example provides a DNJ-3 derivative and a DNJ-4 derivative, and the preparation method thereof is as follows:

[0089]

[0090] Synthesis of DNJ-3: DNJ-1 (49.2 mg, 0.3 mmol) was dissolved in a mixture of methanol and water (MeOH:H₂O = 2:1). Nonanal (51.2 mg, 0.36 mmol), 10% Pd / C (64 mg), and acetic acid (0.18 mg, 0.03 mmol) were added to the mixture. The hydrogen atmosphere was displaced and the reaction was allowed to proceed at room temperature. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the palladium-on-carbon was filtered off. The filtrate was dried and dissolved in methanol. DNJ-3 was purified by column chromatography using a neutral alumina column eluting with MeOH → MeOH:H₂O = 2:1 to afford DNJ-3 (64 mg, 74%) as a white solid.

[0091] The data of DNJ-3 is as follows:

[0092] [α] D 25 =-60 (c=1, MeOH);

[0093] 1H NMR(400MHz,MeOD)δ3.93–3.81(m,2H),3.49(ddd,J=10.5,9.1,4.9Hz,1H), 3.40–3.31(m,3H),3.14(t,J=9.1Hz,1H),3.00(dd,J=11.1,4.9Hz,1H),2.85 –2.74(m,1H),2.65–2.54(m,1H),2.18(t,J=10.9Hz,1H),2.12(dt,J=9.6,2 .8Hz,1H),1.51(p,J=7.3Hz,2H),1.33(p,J=4.5Hz,15H),0.96–0.89(m,3H).

[0094] Synthesis of DNJ-4: DNJ-1 (189 mg, 1.15 mmol) was dissolved in 3 mL of a mixture of methanol and water (MeOH:H₂O = 2:1). n-Decanal (216 mg, 1.15 mmol), 10% Pd / C (122 mg), and acetic acid (7 mg, 0.115 mmol) were added to the mixture. The hydrogen atmosphere was displaced and the reaction was allowed to proceed at room temperature. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, the palladium-on-carbon was filtered off. The filtrate was dried and dissolved in methanol. DNJ-4 was purified by column chromatography using a neutral alumina column with an elution ratio of MeOH → MeOH:H₂O = 2:1 to afford DNJ-4 (49 mg, 15%) as a white solid.

[0095] The data of DNJ-4 is as follows:

[0096] [α] D 25 =-36 (c=1, MeOH);

[0097] 1 H NMR(500MHz,D2O)δ3.99(d,J=11.3Hz,1H),3.91–3.81(m,1H),3.72–3.61(m,1H),3.54(t,J=9.2Hz,1H),3.34(t,J=9.0Hz,1H),3.2 2–3.12(m,1H),2.99–2.89(m,1H),2.86(d,J=18.9Hz,1H),2.56–2.41(m,2H),1.59(s,2H),1.48–1.22(m,14H),0.96–0.86(m,3H).

[0098] Example 2

[0099] This example provides a DNJ-5 derivative, the reaction equation and preparation method of which are as follows:

[0100]

[0101] First step: Sodium hydride (2.4 g, 60 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) in an ice bath at 0 °C, and a solution of compound 5 (8.01 g, 50 mmol) in anhydrous tetrahydrofuran was added dropwise to the resulting suspension. After stirring for 0.5 h, potassium iodide (8.52 g, 60 mmol) was added slowly dropwise. After the reaction was completed as monitored by thin layer chromatography, saturated ammonium chloride solution was added to quench the reaction, and the organic phase was extracted with ethyl acetate three times, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by flash column chromatography (petroleum ether:EtOAc = 4:1) to obtain compound 6 (3.5 g, 40%).

[0102] Second step: Compound 6 (870 mg, 5 mmol) was dissolved in dichloromethane (25 mL), and pyridine chlorochromate (1.3 g, 6 mmol) was added slowly. The reaction was carried out at room temperature, and after the reaction was completed as monitored by thin layer chromatography, the insoluble material was filtered off using celite, and the filtrate was evaporated. Compound 7 (620 mg, 72%) was obtained by flash column chromatography (petroleum ether:EtOAc = 30:1).

[0103] Third step: DNJ-1 (252 mg, 1.54 mmol) was dissolved in a mixture of methanol and water (MeOH:H2O = 2:1, 6 mL), and compound 7 (530 mg, 3 mmol), 10% Pd / C (100 mg), and acetic acid (9.6 mg, 0.16 mmol) were added. The reaction was carried out under a hydrogen atmosphere, and after the reaction was completed as monitored by thin layer chromatography, the palladium carbon was filtered off. DNJ-5 (69 mg, 15%) was obtained as a white solid by dissolving the filtrate in methanol and passing it through a neutral alumina column using MeOH→MeOH:H2O = 2:1 as the eluent.

[0104] The data for DNJ-5 are as follows:

[0105] [α] D 25 = -10 (c = 3, MeOH). 1H NMR(500MHz,D2O)δ3.84–3.70(m,2H),3.45(td,J=10.1,4.9Hz,1H),3.39(t,J=6.7 Hz,2H),3.29(t,J=9.5Hz,1H),3.25(s,3H),3.17(t,J=9.3Hz,1H),2.94(dd,J=11. 5, 5.0 Hz, 1H), 2.65 (td, J = 11.5, 9.9, 6.2 Hz, 1H), 2.54 (ddd, J = 13.4, 10.1, 6.0 Hz, 1H), 2.27–2.16 (m, 2H), 1.48 (t, J = 6.8 Hz, 2H), 1.39 (s, 2H), 1.28–1.12 (m, 10H). Example 3

[0106] This example provides a DNJ-6 derivative, the reaction equation and preparation method of which are as follows:

[0107]

[0108] Step 1: Compound 4-iodoanisole (420.6 mg, 5 mmol), bistriphenylphosphine palladium dichloride (176.25 mg, 0.25 mmol), and cuprous iodide (95 mg, 0.5 mmol) were weighed into an anhydrous and oxygen-free reaction flask, followed by the addition of anhydrous N,N-dimethylformamide (10 mL) and triethylamine (1.01 g, 10 mmol). The mixture was reacted at room temperature for 0.5 h, and then compound 4-pentyn-1-ol (1.29 g, 5.5 mmol) was added dropwise. The reaction progress was monitored by thin-layer chromatography. After completion of the reaction, saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by flash column chromatography (Petrol ether:EtOAc=5:1) to give compound 8 (675 mg, 71%).

[0109] Step 2: Compound 8 (190.24 mg, 1 mmol) was dissolved in a suspension of 10% Pd / C (45 mg) in methanol (10 mL), and the mixture was reacted in an oil bath at 50°C under a hydrogen atmosphere. After completion of the reaction, the palladium carbon was filtered off by thin-layer chromatography. The filtrate was dried and then purified by flash column chromatography (Petrol ether:EtOAc=4:1) to give compound 9 (133 mg, 68%).

[0110] Step 3: Compound 9 (110 mg, 0.56 mmol) was dissolved in dichloromethane (5 mL), and pyridinium chlorochromate (244.11 mg, 1.13 mmol) was added. The reaction was allowed to react at room temperature. After completion of the reaction, the insoluble matter was filtered off with celite, and the filtrate was dried by rotary evaporation and purified by flash column chromatography (Petrol ether:EtOAc=3:1) to give compound 10 (70 mg, 65%).

[0111] Step 4: DNJ-1 (34 mg, 0.2 mmol), compound 10 (60 mg, 0.31 mmol), and acetic acid (1.2 mg, 0.02 mmol) were added to 2 mL of a mixed solvent of methanol and water (MeOH:H2O=2:1) ​​containing 10% Pd / C (30 mg) and reacted under a hydrogen atmosphere. The reaction progress was monitored by thin layer chromatography. After completion, the palladium carbon was filtered off, the filtrate was dried and dissolved in methanol, and chromatographed on a neutral alumina column with an elution system of MeOH→MeOH:H2O=2:1 to obtain DNJ-6 (49 mg, 72%) as a white solid.

[0112] The data of DNJ-6 is as follows:

[0113] [α] D 25 =-18 (c=2, MeOH), mp: 117~118°C.

[0114] 1 H NMR(500MHz,D2O)δ6.84(d,J=8.1Hz,2H),6.59(d,J=8.1Hz,2H),3.75(d,J=11.8Hz,1H),3.66(d,J=12 .2Hz,1H),3.47(td,J=9.7,4.8Hz,1H),3.42(s,3H),3.34(t,J=9.4Hz,1H),3.16(t,J=9.2Hz,1H),2.8 5(q,J=5.0Hz,1H),2.57–2.46(m,1H),2.41(q,J=6.6,5.4Hz,1H),2.28(q,J=7.8,6.3Hz,2H),2.12(t, J=10.9Hz,1H),2.03(d,J=9.6Hz,1H),1.33(t,J=7.7Hz,2H),1.23(s,2H),1.00(q,J=7.3,6.5Hz,2H).

[0115] Example 4

[0116] This example provides a DNJ-7 derivative, the reaction equation and preparation method of which are as follows:

[0117]

[0118] Step 1: Compound 3 (2.1 g, 4 mmol) was dissolved in acetonitrile (20 mL), and potassium carbonate (1.13 g, 8.2 mmol) and propargyl bromide (975.8 mg, 8.2 mmol) were added. The mixture was refluxed in an oil bath at 80°C. After completion of the reaction, saturated ammonium chloride solution was added for quenching, and the mixture was extracted three times with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by flash column chromatography (Petrol ether:EtOAc = 5:1) to give compound 11 (1.73 g, 77%).

[0119] Step 2: Compound 11 (561.72 mg, 1 mmol) was dissolved in anhydrous dichloromethane (10 mL), stirred at -78 ° C for 0.5 h, and then boron tribromide (751.62 mg, 3 mmol) was slowly added dropwise. After reacting for 0.5 h, the mixture was moved to room temperature. After the reaction was completed by thin layer chromatography monitoring, triethylamine was added dropwise in an ice bath to quench the reaction system, and the mixture was extracted with deionized water. The aqueous phase was spin-dried and dissolved in methanol. The mixture was chromatographed on a neutral alumina column with an elution system of MeOH → MeOH:H2O = 2:1 to obtain a brown solid DNJ-7 (167 mg, 83%).

[0120] The data of DNJ-7 is as follows:

[0121] [α]D25=-50 (c=0.8, MeOH).

[0122] 1 H NMR (600MHz, D2O) δ3.83–3.77(m,2H),3.64(d,J=17.7Hz,1H),3.51(td,J=10.0,5.0Hz,1H),3.40(d,J=17.8Hz,1H),3.35( t,J=9.5Hz,1H),3.21(d,J=9.3Hz,1H),2.88(dd,J=11.3,5.1Hz,1H),2.46(t,J=11.0Hz,1H),2.27(dt,J=9.9,2.4Hz,1H).

[0123] Example 5

[0124] This example provides a DNJ-8 derivative, the reaction equation and preparation method of which are as follows:

[0125]

[0126] Step 1: Compound 6-heptynol (448.68 mg, 4 mmol) was dissolved in dichloromethane (20 mL), and pyridinium chlorochromate (1.3 g, 6 mmol) was added. The reaction was allowed to react at room temperature for 2 h. After completion of the reaction, the insoluble matter was filtered off with celite, and the filtrate was dried and flash column chromatography (Petrol ether:EtOAc = 40:1) was performed to obtain compound 12 (225 mg, 51%).

[0127] Step 2: DNJ-1 (32.8 mg, 0.2 mmol) was dissolved in 3 mL of a mixed solvent of methanol and water (MeOH:H2O=2:1), and compound 12 (33 mg, 0.3 mmol), sodium cyanoborohydride (25 mg, 0.4 mmol), and acetic acid (1.2 mg, 0.02 mmol) were added. The reaction was carried out at room temperature. After the reaction was completed, the solvent was dried and the mixture was dissolved in methanol and passed through a neutral alumina column. The elution system was MeOH→MeOH:H2O=2:1 to obtain DNJ-8 (40 mg, 78%) as a white solid.

[0128] The data of DNJ-8 is as follows:

[0129] [α] D 25 =-42 (c=3, MeOH).

[0130] 1 H NMR(400MHz,D2O)δ3.81(qd,J=12.8,2.6Hz,2H),3.49(ddd,J=10.7,9.2,4.9Hz,1H),3 .33(t,J=9.5Hz,1H),3.21(t,J=9.2Hz,1H),2.99(dd,J=11.5,5.0Hz,1H),2.71(ddd,J =13.6,9.8,6.6Hz,1H),2.60(ddd,J=13.5,9.7,6.2Hz,1H),2.30(dd,J=6.9,4.3Hz,1H ),2.27–2.22(m,1H),2.16(t,J=7.0Hz,2H),1.47(q,J=7.3Hz,3H),1.36–1.27(m,2H).

[0131] Example 6

[0132] This example provides a DNJ-9 derivative, the reaction equation and preparation method of which are as follows:

[0133]

[0134] Step 1: Compound 3 (1.05 g, 2 mmol) was dissolved in acetonitrile (10 mL), and then benzyl-2-bromoethyl ether (300 mg, 2.4 mmol) and potassium carbonate (331.2 mg, 2.4 mmol) were added. The mixture was reacted in an oil bath at 80°C. After the reaction was completed by thin-layer chromatography, saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by flash column chromatography (Petrol ether:EtOAc=6:1) to give compound 13 (640 mg, 50%).

[0135] Step 2: Compound 13 (640 mg, 1 mmol) was dissolved in anhydrous dichloromethane (10 mL), stirred at -78 ° C for 0.5 h, and then boron tribromide (1.5 g, 6 mmol) was slowly added dropwise. After reacting for 0.5 h, the mixture was moved to room temperature. After the reaction was completed by thin layer chromatography, triethylamine was added dropwise in an ice bath to quench the system, and then deionized water was added for extraction. The aqueous phase was spin-dried and dissolved in methanol. Neutral alumina column chromatography was used with an elution system of MeOH→MeOH:H2O=2:1 to obtain DNJ-9 (60 mg, 30%) as a white oily liquid.

[0136] The data of DNJ-9 is as follows:

[0137] [α] D 25 =-40 (c=1.6, MeOH).

[0138] 1 H NMR(400MHz,D2O)δ3.92(qd,J=13.1,2.7Hz,2H),3.80(td,J=5.7,2.5Hz,2H),3.63(ddd,J=10.8,9.2,4.9Hz,1H),3.46(t, J=9.6Hz,1H),3.38–3.25(m,2H),3.16(dt,J=12.8,6.1Hz,1H),2.92(dt,J=14.5,5.4Hz,1H),2.63(q,J=11.6,9.6Hz,2H).

[0139] Example 7

[0140] This example provides a DNJ-10 derivative, the reaction equation and preparation method of which are as follows:

[0141]

[0142] First step: Compound 2'-hydroxyacetophenone (1.36 g, 10 mmol) was dissolved in tetrahydrofuran (40 mL) and added to a round bottom flask containing sodium hydride (1 g, 25 mmol) under anhydrous and oxygen free conditions. After stirring for 30 min, a solution of compound p-methoxybenzaldehyde (1.36 g, 10 mmol) in tetrahydrofuran (10 mL) was added dropwise. The reaction was monitored by thin layer chromatography after 2 h and quenched by the addition of saturated ammonium chloride solution. The reaction mixture was extracted with ethyl acetate three times and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and purified by flash column chromatography (petroleum ether: DCM = 2: 1) to give compound 14 (1.64 g, 65%).

[0143] Second step: Compound 14 (508.56 mg, 2 mmol) was dissolved in dimethyl sulfoxide (10 mL) and iodine (1.35 g, 5 mmol) was added. The reaction was refluxed in an oil bath at 170 °C. After 1.5 h, the reaction was quenched by the addition of 50 mL of water and extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and purified by flash column chromatography using dichloromethane to give compound 15 (492 mg, 97%). ( 5 mg, 0.02 mmol) was added dropwise. The reaction was refluxed in an oil bath at 170 °C. After 1.5 h, the reaction was quenched by the addition of 50 mL of water and extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and purified by flash column chromatography using dichloromethane to give compound 15 (492 mg, 97%).

[0144] Third step: Compound 15 (252.27 mg, 1 mmol) was dissolved in dichloromethane (5 mL) under anhydrous and oxygen free conditions and stirred in a -78 °C cold bath for 30 min. Boron tribromide (375.81 mg, 1.5 mmol) was added dropwise and the reaction was allowed to warm to room temperature. After 3 h, the reaction was quenched using saturated sodium bicarbonate solution and filtered to give compound 16 (226 mg, 95%).

[0145] Fourth step: Compound 16 (119.12 mg, 0.5 mmol) was dissolved in N,N- dimethylformamide (5 mL) and potassium carbonate (345.5 mg, 2.5 mmol), compound 2-20 (574.85 mg, 2.5 mmol) were added. The reaction was carried out in an oil bath at 80 °C. After 3 h, the reaction was monitored by thin layer chromatography and quenched by the addition of saturated ammonium chloride solution. The reaction mixture was extracted with ethyl acetate three times and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and purified by flash column chromatography using dichloromethane to give compound 17 (84 mg, 30%). ( 345.5 mg, 2.5 mmol), compound 2-20 (574.85 mg, 2.5 mmol), in an oil bath at 80 °C. After 3 h, the reaction was monitored by thin layer chromatography and quenched by the addition of saturated ammonium chloride solution. The reaction mixture was extracted with ethyl acetate three times and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and purified by flash column chromatography using dichloromethane to give compound 17 (84 mg, 30%).

[0146] Step 5: DNJ-1 (32.8 mg, 0.2 mmol) and potassium carbonate (55.28 mg, 0.4 mmol) were added to 2 mL of N,N-dimethylformamide, stirred for 0.5 h, and then compound 17 (85 mg, 0.22 mmol) was added. The reaction was carried out at 80 ° C for 12 h. After the reaction was completed, the insoluble matter was filtered out after thin layer chromatography monitoring, and the filtrate was dried and flash column chromatography (DCM: MeOH = 5:1) was used to obtain DNJ-10 (37 mg, 40%).

[0147] The data of DNJ-10 is as follows:

[0148] [α] D 25 =-250 (c=1, MeOH), mp: 174~175°C.

[0149] 1 H NMR (400MHz, DMSO-d6) δ8.07–8.01(m,3H),7.86–7.74(m,2H),7.50(ddd,J=8.0,6.9,1.3Hz,1H),7.13– 7.07(m,2H),6.93(s,1H),4.22(t,J=5.2Hz,1H),4.05(t,J=6.5Hz,2H),3.77(d,J=11.4Hz,1H),3.60(dd ,J=9.8,5.7Hz,1H),3.31–3.21(m,1H),3.08(td,J=9.1,3.8Hz,1H),2.96(t,J=8.9Hz,1H),2.90–2.76( m,2H),2.42(ddd,J=13.5,8.3,5.1Hz,1H),2.02–1.93(m,2H),1.76(p,J=7.4Hz,2H),1.53–1.32(m,4H).

[0150] Example 8

[0151] This example provides a series of DNJ-11 to DNJ-13 derivatives, and their reaction equations and preparation methods are as follows:

[0152]

[0153] Step 1: Dissolve 6-hydroxyflavone (238.24 mg, 1 mmol) in N,N-dimethylformamide (5 mL) in three round-bottom flasks, add potassium carbonate (After stirring in an oil bath at 60°C for 0.5h, compound 1,6-dibromohexane (487.94mg, 2mmol), compound 1,8-dibromooctane (544.04mg, 2mmol), and compound 1,10-dibromodecane (600mg, 2mmol) were added respectively. After the reaction was completed by thin layer chromatography, saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and flash column chromatography (Petrol ether:EtOAc=16:1) gave compound 18-1 (261mg, 65%), compound 18-2 (200mg, 47%), and compound 18-3 (221mg, 49%), respectively.

[0154] Step 2: DNJ-1 (16.4 mg, 0.1 mmol) and potassium carbonate (17 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (0.5 mL) in three reaction tubes respectively. After stirring in an oil bath at 80°C for 0.5 h, compound 18-1 (48.16 mg, 0.12 mmol), compound 18-2 (51.5 mg, 0.12 mmol), and compound 18-3 (55 mg, 0.12 mmol) were dissolved in N,N-dimethylformamide (0.5 mL) and added dropwise to the three reaction tubes. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, the insoluble matter was filtered off, the filtrate was dried, and flash column chromatography (DCM:MeOH=5:1) was performed to give DNJ-11 (21 mg, 39%), DNJ-12 (24 mg, 42%), and DNJ-13 (30 mg, 31%).

[0155] The data of DNJ-11 is as follows:

[0156] [α] D 25 =-113 (c=1.3, MeOH).

[0157] 1H NMR(400MHz, DMSO-d6)δ8.10(dd,J=7.8,1.9Hz,2H),7.77–7.71(m,1H),7.63–7.57(m,3H),7.41(d,J=8.3Hz,2 H),7.02(s,1H),4.17(t,J=5.2Hz,1H),4.05(t,J=6.5Hz,2H),3.74(dt,J=11.4,3.0Hz,1H),3.57(ddd,J=11.5 ,5.9,3.5Hz,1H),3.23(tt,J=9.3,4.4Hz,1H),3.06(td,J=9.0,4.9Hz,1H),2.93(td,J=8.9,3.3Hz,1H),2.87– 2.72(m,2H),2.41(ddd,J=13.6,8.7,5.1Hz,1H),2.01–1.91(m,2H),1.75(p,J=6.8Hz,2H),1.50–1.23(m,6H).

[0158] The data of DNJ-12 is as follows:

[0159] [α] D 25 =-120 (c=1, MeOH), mp: 166~168°C.

[0160] 1 H NMR(500MHz,DMSO-d6)δ8.12–8.08(m,2H),7.76–7.72(m,1H),7.63–7.56(m,3H),7.42(d,J=8.3Hz,2H),7.02 (s,1H),4.15(s,1H),4.05(t,J=6.5Hz,2H),3.73(d,J=11.4Hz,1H),3.56(d,J=11.0Hz,1H),3.21(d,J=12.2Hz ,1H),3.05(td,J=9.1,3.8Hz,1H),2.93(t,J=8.9Hz,1H),2.81(dd,J=11.0,4.8Hz,1H),2.74(ddd,J=13.2,9.3 ,6.5Hz,1H),2.38(ddd,J=13.6,9.0,5.1Hz,1H),1.99–1.90(m,2H),1.74(p,J=6.7Hz,2H),1.46–1.17(m,8H).

[0161] The data for DNJ-13 is as follows:

[0162] [α] D 25=-65 (c=1, MeOH), mp: 168~169°C.

[0163] 1 H NMR (500MHz, DMSO-d6) δ8.11–8.08(m,2H),7.76–7.71(m,1H),7.63–7.55(m,3H),7.41(d,J=7.9Hz,2H),7. 01(s,1H),4.14(s,1H),4.04(t,J=6.5Hz,2H),3.71(d,J=11.4Hz,1H),3.55(d,J=11.6Hz,1H),3.21(td,J=9 .7,4.1Hz,1H),3.05(td,J=9.1,3.2Hz,1H),2.92(t,J=8.9Hz,1H),2.80(dd,J=11.0,4.8Hz,1H),2.75–2.6 8(m,1H),2.36(ddd,J=13.7,9.1,5.2Hz,1H),1.98–1.89(m,2H),1.73(p,J=6.8Hz,2H),1.45–1.13(m,12H).

[0164] Example 9

[0165] This example provides a series of DNJ-14 to DNJ-16 derivatives, the reaction equations and preparation methods of which are as follows:

[0166]

[0167] Step 1: Dissolve 3-hydroxyflavone (714.72 mg, 3 mmol) in N,N-dimethylformamide (15 mL) in three reaction bottles, and add potassium carbonate. ( After stirring in an oil bath at 60°C for 0.5h, compound 1,6-dibromohexane (1.01g, 4.5mmol), compound 1,8-dibromooctane (1.23g, 4.5mmol), and compound 1,10-dibromodecane (1.35g, 4.5mmol) were added respectively. After the reaction was completed by thin layer chromatography, saturated ammonium chloride solution was added to quench the reaction. The reaction was extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and flash column chromatography (Petrol ether:EtOAc=12:1) gave compound 19-1 (453mg, 38%), compound 19-2 (507mg, 39%), and compound 19-3 (602mg, 44%), respectively.

[0168] Step 2: DNJ-1 (49.2 mg, 0.13 mmol), potassium carbonate and (The mixture was stirred in an oil bath at 80 ° C for 0.5 h, and then compound 19-1 (180.6 mg, 0.45 mmol), compound 19-2 (186 mg, 0.45 mmol), and compound 19-3 (205.8 mg, 0.45 mmol) were dissolved in N, N-dimethylformamide (2 mL) and added dropwise to three reaction tubes. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, the insoluble matter was filtered off, the filtrate was dried, and flash column chromatography (DCM: MeOH = 5: 1) was used to give DNJ-14 (92 mg, 63%), DNJ-15 (101 mg, 66%), and DNJ-16 (122 mg, 75%).

[0169] The data of DNJ-14 is as follows:

[0170] [α] D 25 =-143 (c=1.3, MeOH).

[0171] 1 H NMR(500MHz,CD3OD)δ8.05(dd,J=8.1,1.6Hz,1H),7.98–7.94(m,2H),7.67(ddd,J=8.7,7.0,1.7Hz,1H),7.52(d,J=8.3Hz,1H),7.4 9–7.42(m,3H),7.38–7.34(m,1H),3.85(t,J=6.4Hz,2H),3.79(d,J=2.8Hz,2H),3.45(ddd,J=10.5,9.1,4.8Hz,1H),3.33(t,J=9.3 Hz,1H),3.11(t,J=9.1Hz,1H),2.93(dd,J=11.2,4.9Hz,1H),2.68(dt,J=13.3,7.7Hz,1H),2.51–2.45(m,1H),2.13(t,J=10.9Hz,1 H),2.07(dt,J=9.6,2.7Hz,1H),1.59–1.52(m,2H),1.34(p,J=7.7Hz,2H),1.26(p,J=7.3Hz,2H),1.12(qd,J=7.7,7.3,3.9Hz,2H).

[0172] The data of DNJ-15 is as follows:

[0173] [α] D 25 =-72 (c=2, MeOH).

[0174] 1H NMR(500MHz,CD3OD)δ8.07(dd,J=8.1,1.6Hz,1H),8.01–7.97(m,2H),7.69(ddd,J=8.7,7.1,1.7Hz,1H),7.55(dd,J=8.6,0.9Hz,1H),7.49–7.43(m,3H),7.37(ddd,J=8.1,7.0,1.1Hz,1H),3.86(t,J=6.4Hz,2H),3.80(d,J=2.7Hz,2H),3.45(ddd,J=10.5,9.0,4.8Hz,1H),3.35–3.30(m,1H),3.11(t,J=9.1Hz,1H),2.94(dd,J=11.2,4.9Hz,1H),2.76–2.68(m,1H),2.55–2.48(m,1H),2.14(t,J=10.9Hz,1H),2.08(dt,J=9.6,2.8Hz,1H),1.59–1.52(m,2H),1.39(p,J=7.6,7.2Hz,2H),1.28–1.09(m,9H).

[0175] DNJ-16的数据如下:

[0176] [α] D 25 =-19(c=3,MeOH)。

[0177] 1 H NMR(500MHz,CD3OD)δ8.07(dd,J=8.0,1.6Hz,1H),8.01–7.97(m,2H),7.69(ddd,J=8.6,7.0,1.7Hz,1H),7.55(d,J=8.5Hz,1H),7.45(qd,J=4.9,1.7Hz,3H),7.37(t,J=7.6Hz,1H),3.86(t,J=6.5Hz,2H),3.81(d,J=2.7Hz,2H),3.48–3.42(m,1H),3.34(t,J=9.3Hz,1H),3.12(t,J=9.1Hz,1H),2.96(dd,J=11.2,4.9Hz,1H),2.79–2.70(m,1H),2.58–2.50(m,1H),2.17(t,J=10.9Hz,1H),2.11(dt,J=9.8,2.8Hz,1H),1.58–1.51(m,2H),1.42(p,J=7.6Hz,2H),1.26–1.09(m,15H).

[0178] 实施例10

[0179] This example provides a series of DNJ-17 to DNJ-19 derivatives, the reaction equations and preparation methods of which are as follows:

[0180]

[0181] Step 1: Dissolve chrysin (254.24 mg, 1 mmol) in N,N-dimethylformamide (5 mL) in three reaction bottles, add potassium carbonate ( After stirring in an oil bath at 60°C for 0.5h, compound 1,6-dibromohexane (367mg, 1.5mmol), compound 1,8-dibromooctane (408mg, 1.5mmol), and compound 1,10-dibromodecane (450mg, 1.5mmol) were added respectively. After the reaction was completed by thin layer chromatography, saturated ammonium chloride solution was added to quench the reaction. The reaction was extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and flash column chromatography (Petrol ether:EtOAc=16:1) gave compound 20-1 (254mg, 41%), compound 20-2 (277mg, 41%), and compound 20-3 (296mg, 42%), respectively.

[0182] Step 2: DNJ-1 (33 mg, 0.2 mmol), potassium carbonate and ( The mixture was dissolved in N,N-dimethylformamide (2 mL) and stirred in an oil bath at 80 ° C for 0.5 h. Then, compound 20-1 (100.15 mg, 0.24 mmol), compound 2--2 (107 mg, 0.24 mmol), and compound 20-3 (114 mg, 0.24 mmol) were dissolved in N,N-dimethylformamide (0.5 mL) and added dropwise to three reaction tubes. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, the insoluble matter was filtered off, the filtrate was dried, and flash column chromatography (DCM: MeOH = 5: 1) was used to obtain DNJ-17 (34 mg, 34%), DNJ-18 (36 mg, 34%), and DNJ-19 (36 mg, 33%).

[0183] The data of DNJ-17 is as follows:

[0184] [α] D 25 =-58 (c=0.8, MeOH).

[0185] 1H NMR (400MHz, DMSO-d6) δ12.85(s,1H),8.20–8.12(m,2H),7.70–7.62(m,3H),7.10(s,1H),6.88(d,J=2. 3Hz,1H),6.44(d,J=2.2Hz,1H),4.21(d,J=5.3Hz,1H),4.15(t,J=6.5Hz,2H),3.82–3.76(m,1H),3.65–3 .57(m,1H),3.27(tt,J=8.9,3.8Hz,1H),3.10(td,J=9.3,4.5Hz,1H),2.97(t,J=8.5Hz,1H),2.90–2.76( m,2H),2.45(ddd,J=13.6,8.8,5.1Hz,1H),2.05–1.96(m,2H),1.80(p,J=6.8Hz,2H),1.55–1.25(m,7H).

[0186] The data for DNJ-18 is as follows:

[0187] [α] D 25 =-53 (c=1.8, MeOH), mp: 120~124°C.

[0188] 1 H NMR(500MHz,DMSO-d6)δ12.83(s,1H),8.16–8.11(m,2H),7.70–7.60(m,3H),7.06(s,1H),6.82(d,J=2.2Hz,1H ),6.40(d,J=2.2Hz,1H),4.20(s,1H),4.11(t,J=6.5Hz,2H),3.77(dd,J=11.6,2.4Hz,1H),3.61(d,J=11.5Hz,1 H),3.26(td,J=9.7,4.9Hz,1H),3.13–3.06(m,1H),2.97(t,J=8.8Hz,1H),2.86(dd,J=11.0,4.8Hz,1H),2.84– 2.75(m,1H),2.42(ddd,J=13.7,9.1,5.1Hz,1H),2.05–1.93(m,2H),1.77(p,J=6.9Hz,2H),1.51–1.18(m,13H).

[0189] The data for DNJ-19 is as follows:

[0190] [α] D 25= -29 (c = 1.3, MeOH), m.p.: 125-129 °C.

[0191] 1 H NMR (500 MHz, DMSO-d6) δ 12.83 (s, 1H), 8.13 (dd, J = 7.0, 1.9 Hz, 2H), 7.68 - 7.61 (m, 3H), 7.05 (s, 1H), 6.81 (d, J = 2.2 Hz, 1H), 6.39 (d, J = 2.2 Hz, 1H), 4.19 (d, J = 15.0 Hz, 1H), 4.11 (t, J = 6.6 Hz, 2H), 3.77 (d, J = 11.4 Hz, 1H), 3.61 (d, J = 11.6 Hz, 1H), 3.30 - 3.24 (m, 1H), 3.11 (td, J = 9.2, 3.5 Hz, 1H), 2.98 (t, J = 8.9 Hz, 1H), 2.86 (dd, J = 11.0, 4.8 Hz, 1H), 2.81 - 2.74 (m, 1H), 2.42 (ddd, J = 13.8, 9.0, 5.2 Hz, 1H), 2.03 - 1.96 (m, 2H), 1.76 (p, J = 6.9 Hz, 2H), 1.48 - 1.19 (m, 14H).

[0192] Example 11

[0193] This example provides a series of DNJ-20 to DNJ-22 derivatives, the reaction equation and preparation method as follows:

[0194]

[0195] First step: compound Kaempferol (1.71 g, 6 mmol) was dissolved in N, N- dimethylformamide (40 mL), then potassium carbonate (2.74 g, 19.8 mmol) was added, stirred in 60 °C oil bath for 0.5 h, then benzyl bromide (3.34 g, 19.8 mmol) was added, the reaction progress was monitored by thin layer chromatography. After 3 h, saturated ammonium chloride solution was added to quench the reaction system, extracted with ethyl acetate for 3 times, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and rotary evaporated, recrystallized to obtain yellow solid 21 (2.18 g, 50%).

[0196] Step 2: Compound 21 (556.61 mg, 1 mmol) was dissolved in N,N-dimethylformamide (10 mL) in three round-bottom flasks, and cesium carbonate (488.73 mg, 1.5 mmol) was added. The mixture was stirred in an oil bath at 60°C for 0.5 h, and then 1,6-dibromohexane (367 mg, 1.5 mmol), 1,8-dibromooctane (408 mg, 1.5 mmol), and 1,10-dibromodecane (450 mg, 1.5 mmol) were added. After the reaction was completed, saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by flash column chromatography (Petrol ether:EtOAc=5:1) to give compounds 22-1 (688 mg, 88%), 22-2 (493 mg, 66%), and 22-3 (444 mg, 57%), respectively.

[0197] Step 3: Dissolve DNJ-1 (49.2 mg, 0.3 mmol) and potassium carbonate (62 mg, 0.45 mmol) in N,N-dimethylformamide (1 mL) in three reaction tubes, stir in an oil bath at 80°C for 0.5 h, and then dissolve compound 22-1 (323.85 mg, 0.45 mmol), compound 22-2 (336.5 mg, 0.45 mmol), and compound 22-3 (342.8 mg, 0.45 mmol) in N,N-dimethylformamide (1 mL) and add dropwise to the three reaction tubes. Monitor the reaction progress by thin layer chromatography. After the reaction is completed, filter out the insoluble matter, spin dry the filtrate, and perform flash column chromatography (DCM:MeOH=7:1) to give compound 23-1 (215 mg, 72%), 23-2 (188 mg, 76%), and 23-3 (176 mg, 68%).

[0198] Step 4: Compounds 23-1 (115 mg, 0.14 mmol), 23-2 (188 mg, 0.23 mmol), and 23-3 (107 mg, 0.13 mmol) were dissolved in 2 mL of a tetrahydrofuran / MeOH mixture (tetrahydrofuran:methanol = 1:1). 42 mg of Pd(OH)2 / C was added to each of the solutions, and the mixture was reacted at 45°C under a hydrogen atmosphere for 3 h. After the reaction, the insoluble matter was filtered off, and the filtrate was dried. The solid was slurried with diethyl ether and filtered to give yellow solids DNJ-20 (58 mg, 78%), DNJ-21 (87 mg, 67%), and DNJ-22 (57 mg, 69%).

[0199] The data of DNJ-20 is as follows:

[0200] [α] D 25=-190 (c=1.4, MeOH), mp: 212~216°C.

[0201] 1 H NMR (500MHz, CD3OD) δ8.04(d,J=8.4Hz,2H),6.89(d,J=8.5Hz,2H),6.30(s,1H),4.03(q, J=6.6,4.7Hz,2H),3.87(d,J=2.8Hz,2H),3.51(td,J=9.8,4.8Hz,1H),3.40(d,J=9.3Hz,1 H),3.32–3.27(m,1H),3.17(t,J=9.1Hz,1H),3.03(td,J=9.2,7.2,4.9Hz,1H),2.89–2.8 1(m,1H),2.73–2.60(m,1H),2.31–2.16(m,2H),1.54(h,J=7.6Hz,5H),1.41–1.21(m,6H).

[0202] The data of DNJ-21 is as follows:

[0203] [α] D 25 =-100 (c=1.4, MeOH), mp: 212~216°C.

[0204] 1 H NMR(400MHz,CD3OD)δ8.01(d,J=8.5Hz,2H),6.85(d,J=8.5Hz,2H),6.42(s,1H),6.2 8(s,1H),3.45(ddd,J=10.7,9.1,4.8Hz,1H),3.34(t,J=9.3Hz,1H),3.26(p,J=1.7Hz ,1H),3.11(t,J=9.1Hz,1H),3.02–2.94(m,1H),2.77(dtd,J=15.9,8.6,7.5,4.4Hz,1 H),2.63–2.49(m,1H),2.25–2.11(m,2H),1.81(p,J=6.7Hz,2H),1.51–1.16(m,14H).

[0205] The DNJ-22's data is as follows:

[0206] [α] D 25 =-120 (c=1.5, MeOH), mp: 228~234°C.

[0207] 1H NMR(400MHz,CD3OD)δ8.05(d,J=8.4Hz,2H),6.89(d,J=8.4Hz,2H),6.45(s,1H), 6.31(s,1H),3.49(td,J=10.0,4.8Hz,1H),3.37(t,J=9.3Hz,1H),3.30(dt,J=3.3 ,1.6Hz,1H),3.15(t,J=9.1Hz,1H),2.99(dd,J=11.2,4.9Hz,1H),2.82–2.70(m,1 H),2.63–2.50(m,1H),2.25–2.11(m,2H),1.88–1.77(m,2H),1.52–1.16(m,20H).

[0208] Example 12

[0209] This example provides a series of DNJ-23 to DNJ-25 derivatives, the reaction equations and preparation methods of which are as follows:

[0210]

[0211] Step 1: Luteolin (1.15 g, 4 mmol) was dissolved in N,N-dimethylformamide (10 mL). Potassium carbonate (1.78 g, 12.8 mmol) was added and stirred in a 60°C oil bath for 0.5 h. Benzyl bromide (2.19 g, 12.8 mmol) was then added and the reaction progress monitored by thin-layer chromatography. After 3 h, saturated ammonium chloride solution was added to quench the reaction. A large amount of yellow solid precipitated, which was filtered, washed with ethyl acetate, and dried to obtain crude product 24 (1.5 g, 67%).

[0212] Step 2: Compound 24 (445.21 mg, 0.8 mmol) was dissolved in N,N-dimethylformamide (3 mL) in three reaction flasks, and cesium carbonate (391 mg, 1.2 mmol) was added. The mixture was stirred in an oil bath at 60°C for 0.5 h, and then 1,6-dibromohexane (293 mg, 1.2 mmol), 1,8-dibromooctane (326.4 mg, 1.2 mmol), and 1,10-dibromodecane (360 mg, 1.2 mmol) were added. After the reaction was completed, saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with ethyl acetate three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and purified by flash column chromatography (Petrol ether:EtOAc=5:1) to give compounds 25-1 (286 mg, 50%), 25-2 (299 mg, 50%), and 25-3 (357 mg, 58%), respectively.

[0213] Step 3: Dissolve DNJ-1 (41 mg, 0.25 mmol) and potassium carbonate (52 mg, 0.375 mmol) in N,N-dimethylformamide (1 mL) in three reaction tubes, stir in an oil bath at 80°C for 0.5 h, and then dissolve compound 25-1 (270 mg, 0.375 mmol), compound 25-2 (280.4 mg, 0.375 mmol), and compound 25-3 (291 mg, 0.375 mmol) in N,N-dimethylformamide (1 mL) and add dropwise to the three reaction tubes. Monitor the reaction progress by thin layer chromatography. After the reaction is completed, filter out the insoluble matter, spin dry the filtrate, and perform flash column chromatography (DCM:MeOH=10:1) to give 26-1 (134 mg, 67%), 26-2 (157 mg, 76%), and 26-3 (134 mg, 63%).

[0214] Step 4: Compounds 26-1 (134 mg, 0.17 mmol), 26-2 (157 mg, 0.19 mmol), and 26-3 (134 mg, 0.17 mmol) were dissolved in 2 mL of a tetrahydrofuran / methanol mixture (tetrahydrofuran:methanol = 1:1). 56 mg of Pd(OH)2 / C was added to each of the solutions, and the mixture was reacted at 45°C under a hydrogen atmosphere for 3 h. After the reaction, the insoluble matter was filtered off, and the filtrate was dried. The solid was slurried with diethyl ether and filtered to give yellow solids DNJ-23 (90 mg, 99%), DNJ-24 (97 mg, 91%), and DNJ-25 (85 mg, 85%).

[0215] The data of DNJ-23 is as follows:

[0216] [α] D 25 =-74 (c=1.2, MeOH), mp: 220~230°C.

[0217] 1H NMR (500MHz, CD3OD) δ7.36–7.29(m,2H),6.90(d,J=8.3Hz,1H),6.49(d,J=2.1Hz,1H),6.45(s,1H),6.34 (d,J=2.1Hz,1H),3.96–3.88(m,2H),3.57(ddd,J=10.6,9.0,4.9Hz,1H),3.45(t,J=9.4Hz,1H),3.33(p,J =1.6Hz,1H),3.23(t,J=9.1Hz,1H),3.13(dd,J=11.4,4.9Hz,1H),3.01–2.91(m,1H),2.76(ddd,J=13.3, 9.2,6.1Hz,1H),2.43–2.33(m,2H),1.87(p,J=6.7Hz,2H),1.65–1.54(m,4H),1.40(q,J=7.6,6.9Hz,2H).

[0218] The data of DNJ-24 is as follows:

[0219] [α] D 25 =-29 (c=1.6, MeOH), mp: 259~260°C.

[0220] 1 H NMR (500MHz, CD3OD) δ7.34–7.28(m,2H),6.87(d,J=8.2Hz,1H),6.48(d,J=2.1Hz,1H),6.45(s,1H),6 .34(d,J=2.1Hz,1H),3.90–3.82(m,2H),3.51(ddd,J=10.6,9.1,4.9Hz,1H),3.40(t,J=9.4Hz,1H),3. 30(p,J=1.7Hz,1H),3.17(t,J=9.1Hz,1H),3.06(dd,J=11.4,4.9Hz,1H),2.87(ddd,J=13.3,9.7,6.6H z,1H),2.66(ddd,J=13.4,9.6,6.1Hz,1H),2.34–2.24(m,2H),1.87–1.78(m,2H),1.56–1.23(m,12H).

[0221] The data of DNJ-25 is as follows:

[0222] [α] D 25 =-114 (c=1.2, MeOH), mp: 236~238°C.

[0223] 1 H NMR (500MHz, CD3OD) δ7.3–7.3(m,2H),6.8(d,J=8.2Hz,1H),6.4(d,J=2.1Hz,1H),6.4(s,1H) ,6.3(d,J=2.1Hz,1H),4.0(t,J=6.6Hz,2H),3.8(d,J=2.7Hz,2H),3.5–3.4(m,1H),3.3(t,J= 9.4Hz,1H),3.1(t,J=9.1Hz,1H),3.0(dd,J=11.2,4.9Hz,1H),2.8(qd,J=9.1,5.4Hz,1H),2. 6–2.5(m,1H),2.2(t,J=10.9Hz,1H),2.1(dt,J=9.6,2.7Hz,1H),1.8(p,J=6.8Hz,2H),1.5–1. 4(m,5H),1.4–1.2(m,13H).δ7.4–7.3(m,2H),6.9(d,J=8.2Hz,1H),6.5(d,J=2.1Hz,1H),6.5 (s,1H),6.4(d,J=2.1Hz,1H),4.0(t,J=6.6Hz,2H),3.9(d,J=2.7Hz,2H),3.5–3.4(m,1H),3.4 (t,J=9.4Hz,1H),3.2(t,J=9.1Hz,1H),3.0(dd,J=11.2,4.9Hz,1H),2.8(qd,J=9.1,5.4Hz,1 H),2.6–2.5(m,1H),2.3–2.1(m,2H),1.8(p,J=6.8Hz,2H),1.5–1.4(m,4H),1.4–1.2(m,12H).

[0224] Performance Testing

[0225] 1. The MTT method was used to determine the cytotoxicity of the DNJ derivatives prepared in the present invention to HELA-ACE2

[0226] Target cells HELA-ACE2 were seeded in 96-well culture plates one night in advance, about 3000-5000 cells per well, and the final volume of DMEM medium was 100 μL. The next day, different drugs were added to the target cells, with concentration gradients of: 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM. After 48 hours, the DMEM medium was removed, and MTT solution diluted with blank DMEM medium was added to each well, and incubation was continued for 4 hours. The MTT solution was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the blue-purple formazan crystals, and the mixture was shaken well on a shaker. After 10 minutes, the Biotek Cytation5 cell imaging microplate multifunctional enzyme label instrument was used to measure the absorbance at 490 nm; the results are shown in Table 1.

[0227] Table 1

[0228]

[0229] From the data in Table 1, DNJ 11-DNJ 13 derivatives have strong cytotoxicity, CC 50 <10 μM; DNJ 17-DNJ 19 derivatives have certain cytotoxicity, but CC50> 10 μM, and have little effect on the target cells themselves; other DNJ derivatives have no obvious cytotoxicity.

[0230] 2. Evaluation of DNJ derivative pseudovirus model co-treatment inhibition activity

[0231] S1, well-grown 293T cells were seeded in a 10 cm culture dish one night in advance, at a density of 30%-40% of the transfection density. After overnight culture, when the 293T cells grew to 70%-80%, the DMEM complete medium was replaced with blank DMEM medium. The packaging plasmid psPAX2, lentivirus plasmid pLenti-CMV-luc2-GFP-Puro and SARS-Cov-2 Spike protein plasmid were used in a ratio of 1:1:1 for transfection with transfection reagent Lipo 8000. After 6-8 hours of transfection, fresh DMEM complete medium was added, and incubation was continued for 48 hours. The 293T supernatant was collected, filtered through a 0.45 μm microporous filter, and the filtrate was centrifuged at 1000 rpm for 10 minutes to obtain the pseudovirus solution. The pseudovirus solution needs to be taken fresh, and freezing and thawing will reduce its infection effect.

[0232] S2, HELA-ACE2 cells were seeded in 96-well culture plates one night in advance at 1 × 10 4The density of the holes was seeded in a colorless transparent 96-well plate. Before the infection experiment, the HELA-ACE2 cells were pretreated with 10 μM of drug solution 1 hour in advance. Then, the pseudovirus solution, drug co-treatment HELA-ACE2 cells (ensure the final concentration of drug is 10 μM), while the setting of adding pseudovirus solution without drug treatment as control group, and the setting of not adding pseudovirus solution and not adding drug treatment as blank group. After 12 hours, replace with fresh DMEM complete medium and continue to culture. After 48 hours, the HELA-ACE2 cells were treated with cell lysis solution in the luciferase kit for 10 minutes, and the cell lysate was centrifuged at 12000 rpm for 10 minutes. Then, the supernatant was added with luciferase reaction solution in a black opaque 96-well plate, and the fluorescence value was immediately detected by Biotek Cytation 5 cell multifunctional imaging enzyme label instrument. The results are shown in Figure 1 As can be seen from the figure, DNJ-15 derivative, DNJ-16 derivative, DNJ-25 derivative can significantly inhibit the entry stage of SARS-CoV-2 pseudovirus infection of target cells HELA-ACE2, and the inhibition rate is 80.69%, 95.53%, 91.96% respectively.

[0233] 3. Pretreatment of DNJ derivative pseudovirus model inhibitory activity evaluation

[0234] S1, the 293T cells in good growth condition were seeded in a 10 cm culture dish one night in advance, and the density was 30%-40% of the transfection density. After overnight culture, when the 293T cells grew to 70%-80%, the DMEM complete medium was replaced with blank DMEM medium in advance. The packaging plasmid psPAX2, lentivirus plasmid pLenti-CMV-luc2-GFP-Puro and SARS-Cov-2 Spike protein plasmid were used in a ratio of 1:1:1 for transfection with transfection reagent Lipo 8000. After 12 hours of transfection, 10 μM of drug solution was used for subsequent treatment for 12 hours. Replace with fresh DMEM complete medium and continue to culture for 48 hours. Collect the 293T supernatant, filter it through a 0.45 μm microporous filter, and centrifuge the filtrate at 1000 rpm for 10 minutes to obtain the pseudovirus solution. The pseudovirus solution needs to be taken fresh, and freezing and thawing will reduce its infection effect.

[0235] S2, the HELA-ACE2 cells were seeded in a 96-well plate at 1×10 4The cells were seeded at a density of 100 μg / well in a colorless, transparent 96-well plate. Pseudovirus solutions treated with different drugs were added to the target cells. A control group was set up with a pseudovirus solution without drug treatment, and a blank group was set up with neither pseudovirus solution nor drug treatment. After 12 hours, the medium was replaced with fresh complete DMEM and culture continued. After 48 hours, HELA-ACE2 cells were treated with the cell lysis buffer in the luciferase kit for 10 minutes. The cell lysate was centrifuged at 12,000 rpm for 10 minutes, and the supernatant and luciferase reaction solution were added to a black, opaque 96-well plate. Fluorescence was immediately measured using a Biotek Cytation 5 multi-functional cell imaging microplate reader.

[0236] The results are as follows Figure 2 As shown in the figure, it can be seen that the DNJ derivatives provided by the present invention can reduce the infectivity of SARS-CoV-2 pseudovirus on target HELA-ACE2 cells by drug treatment during pseudovirus production in 293T cells. In particular, the DNJ-25 derivative can still significantly reduce the infectivity of SARS-CoV-2 pseudovirus on target HELA-ACE2 cells, with an inhibition rate of 71.15%. The present invention continues the following experiments using the DNJ-25 derivative as an example.

[0237] 4. DNJ-25 derivative co-treatment inhibition concentration curve determination experiment

[0238] The experimental steps for determining the inhibitory concentration curve of compound DNJ-25 were the same as those for evaluating the entry inhibitory activity of pseudovirus co-treatment, except that different concentrations of compound DNJ-25 were added to the 96-well plate, namely 20μM, 15μM, 10μM, 5μM, 2.5μM, 1.25μM, and 0.625μM. The results are shown in the figure. Figure 3 As shown in the figure, it can be seen that when the target cell HELA-ACE2 is treated with the pre-prepared SARS-CoV-2 pseudovirus and compound 25, it is found that the DNJ-25 derivative can inhibit the invasion of the pseudovirus in a concentration-dependent manner, and its EC 50 =5.192μM.

[0239] 5. DNJ-25 derivative pretreatment inhibition concentration curve determination experiment

[0240] The experimental steps for determining the inhibitory concentration curve of DNJ-25 derivatives are the same as those for evaluating the pseudovirus pretreatment entry inhibitory activity. The difference is that different concentrations of compound DNJ-25 are added to the 96-well plate, namely 20μM, 15μM, 10μM, 5μM, 2.5μM, 1.25μM, and 0.625μM. The results are shown in the figure. Figure 4As shown in the figure, the drug treatment during the production of pseudovirus in 293T cells showed that DNJ-25 derivatives could reduce the infectivity of SARS-CoV-2 pseudovirus to target cell HELA-ACE2 in a concentration-dependent manner, and its EC 50 =7.519μM.

[0241] 5. Experiment on the time of adding DNJ-25 derivatives

[0242] In order to determine the affected stage of the viral life cycle, compounds were added to the target cells HELA-ACE2 at different time points before, during and after pseudotyped novel coronavirus infection. Specifically, the freshly produced and collected pseudovirus solution was co-cultured with the target cells HELA-ACE2, and the compound solution with a final concentration of 20μM was added at -2 hours (ie, 2 hours of drug pretreatment), -1 hour (ie, 1 hour of drug pretreatment), 0 hours, 1 hour, 2 hours, 4 hours, and 6 hours of co-culture. After 12 hours of treatment (starting from the beginning of co-culture of the two), it was replaced with fresh DMEM complete medium, and after continuing to culture for 48 hours, the fluorescence value was detected using a Biotek Cytation 5 cell multifunctional imaging microplate reader. The results are as follows Figure 5 As shown, there was no significant difference in inhibitory effect between the addition of DNJ-25 derivatives before SARS-CoV-2 pseudovirus infection of target HELA-ACE2 cells and the co-treatment of pseudovirus, target cells, and the compound. However, the inhibitory effect gradually decreased with the passage of time after the addition of DNJ-25 derivatives. This suggests that DNJ-25 derivatives can reduce the infectivity of SARS-CoV-2 pseudoviruses to HELA-ACE2 cells in a concentration- and time-dependent manner.

[0243] 6. DNJ-25 derivative live virus verification experiment

[0244] The antiviral activity of DNJ-25 derivatives against SARS-CoV-2 was evaluated in HeLa-hACE2 cells. The cells were seeded into 24-well plates, pretreated with the compound (10 μM) for 2 hours, and then infected with wild-type SARS-CoV-2 at an infection multiplier (MOI) of 0.3 for 1 hour. Culture medium containing the compound (10 μM) was added. After culturing for 24 hours at 37°C and 5% CO2, the cells were collected, and the viral RNA was extracted using Trizol, followed by quantitative PCR reverse transcription (RT-qPCR) analysis. The results are shown in Figure 2. Figure 6 As shown, Figure 6 A is the EC of DNJ-25 derivatives inhibiting the live virus model 50 curve chart; Figure 6B is a graph showing the mRNA levels of NP and Spike proteins that inhibit live viruses. It can be seen from the figure that compared with the control group, DNJ-25 derivatives can significantly reduce the gene expression and protein levels of viral S protein and nucleocapsid protein in HeLa-hACE2 24 hours after infection, among which the EC for nucleocapsid protein is 50 The EC value for S protein is 1.667 μM. 50 Compared with compound DNJ-1 and luteolin (Lut), the inhibition rate of DNJ-25 derivative at 10 μM reached 91.44%.

[0245] In summary, the DNJ derivatives provided by the present invention exert their anti-coronavirus effects by reducing the infectivity of virus particles and inhibiting the invasion stage of the virus.

[0246] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the scope of the present invention.

Claims

1. A DNJ derivative or a pharmaceutically acceptable salt thereof, characterized in that: The DNJ derivative has the following structural formula: 。 2. A pharmaceutical composition, characterized in that The invention comprises the DNJ derivative or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, characterized in that Calculated based on the weight of the pharmaceutical composition, the DNJ derivative or a pharmaceutically acceptable salt thereof accounts for 0.1% to 99% of the total weight.

4. The pharmaceutical composition according to claim 3, characterized in that Calculated based on the weight of the pharmaceutical composition, the DNJ derivative or a pharmaceutically acceptable salt thereof accounts for 0.5% to 95% of the total weight.

5. The pharmaceutical composition according to claim 2, characterized in that The dosage form of the pharmaceutical composition includes solid dosage form, semisolid dosage form or solution dosage form.

6. Use of the DNJ derivative according to claim 1; or the pharmaceutical composition according to any one of claims 2 to 5 in the preparation of a drug for treating and / or preventing coronavirus; the coronavirus is SARS-CoV-2.

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