DNJ derivative, pharmaceutical composition and application thereof

Abstract

The invention discloses a DNJ derivative, a pharmaceutical composition and application of the DNJ derivative. The DNJ derivative provided by the invention has the following structural formula: # imgabs0 #, a series of DNJ derivatives with novel structures are provided, and the DNJ derivatives can reduce the infectivity of the coronavirus through misfolding of the S protein of the coronavirus cell membrane protein, so that the aim of inhibiting the coronavirus infection is fulfilled. The compound is expected to be used for treating and / or preventing coronavirus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a DNJ derivative, a pharmaceutical composition and application 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 huge threat to public health, but also brought immeasurable losses and heavy burdens to economic and social development, highlighting that new and emerging infectious diseases caused by coronaviruses have become one of the major challenges to survival and development faced by all mankind. The currently known coronaviruses are just the tip of the iceberg, and new and more harmful zoonotic coronaviruses may appear in the future. Therefore, the development of highly effective and broad-spectrum anti-coronavirus drugs is the key to defeating coronaviruses.

[0003] DNJ (1-Deoxynojirimycin; 1-deoxynojirimycin, CAS No. 19130-96-2) is an amino sugar compound extracted from traditional Chinese medicine mulberry leaves and mulberry bark. Its structure is highly similar to glucose. Bayer has found that it is a potent α-glycosidase inhibitor, which has a significant inhibitory effect on various types of α-glycosidases and is used to treat adult type II diabetes. DNJ analogs miglitol (GlysetTM) and miglustat (ZavescaTM) are also used as α-glycosidase inhibitors and glycotransferase inhibitors, respectively, and are clinically used to treat type II diabetes, type I 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, which has the characteristics of anti-coronavirus.

[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] The DNJ derivative provided according to the first aspect of the present invention has the following structural formula:

[0010]

[0011] Where 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, which can reduce the infectivity of coronavirus through the misfolding of the coronavirus membrane protein S protein, thereby inhibiting the infection of coronavirus. It is expected to be used for the treatment and / or prevention of 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, separating the solid and the liquid, 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 an organic solvent III to carry out reaction III, separating the solid and the liquid, and collecting the solid phase to obtain a 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 a compound 4;

[0026] S4, mixing 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 comprises acetonitrile or 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 invention, the ether solvent includes at least one of methyl ether, ethyl ether and methyl tert-butyl ether (MTBE).

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

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

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

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

[0054] According to a preferred embodiment of the present invention, 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.

[0055] According to a preferred embodiment of the present invention, 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.

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

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

[0058] The third aspect of the present invention provides a use of the DNJ derivative described in the first aspect of the present invention; or the pharmaceutical composition described in the second aspect of the present invention in the preparation of drugs for treating and / or preventing coronavirus.

[0059] According to a preferred embodiment of the present invention, the coronavirus includes 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 The 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 The alkyl group and the 5-10 The explanation of "alkyl" is basically similar, except that 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 1～5 Any one of the hydrogen atoms in the alkyl group is substituted by an alkynyl group, a hydroxy group or a methoxyphenyl group.

[0064] The pharmaceutically acceptable adjuvants of the present invention include any solvent, solid excipient, diluent, adhesive, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, 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. DB Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, the contents of the documents herein are combined to show that different adjuvants can be applied to the preparation of pharmaceutically acceptable compositions and their known preparation methods. Except insofar as any conventional excipients are incompatible with the compounds of the present invention, for example by producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present invention.

[0065] Substances that can be used as 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; polyvinyl pyrrolidone; 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 buffer solution; and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; coatings; sweeteners; flavorings; fragrances; preservatives and antioxidants.

[0066] The pharmaceutically acceptable salts of the present invention include salts formed with inorganic acids, organic acids, alkali metals, alkaline earth metals and basic amino acids. Including, but not limited to: hydrochloride, sulfate, bisulfate, nitrate, hydrobromide, hydroiodide, carbonate, bicarbonate, sulfite, bisulfite, pyrosulfate, monohydrogen phosphate, dihydrogen phosphate, perchlorate, persulfate, hemisulfate, bisulfate, thiocyanate, phosphate, pyrophosphate, metaphosphate; suitable organic acid salts include, but not limited to: formate, acetate, propionate, butyrate, benzoate, malonate, succinate, pyruvate, methanesulfonate, ethanesulfonate, propanesulfonate, citrate, 4-nitrobenzoate, benzenesulfonate, p-toluenesulfonate, malate, propiolate, 2-butyrate, 2-hydroxy-ethanesulfonate, Vinyl acetate, tartrate, L-tartrate, fumarate, isethionate, maleate, lactate, lactobionate, pamoate, salicylate, galactarate, glucoheptonate, mandelate, 1,2-ethanedisulfonate, 2-naphthalenesulfonate, oxalate, trifluoroacetate, trifluoromethanesulfonate, adipate, suberate, sebacate, butyne-1,4-dioate, hexyne-1,6-dioate, glycolate, alginate, ascorbate, isoascorbate, aspartate, L-aspartate, glutamate, L-glutamate, 2-phenoxybenzoate, 2-(4-hydroxybenzoyl)benzoate, acetoacetate, 2-hydroxy Ethylsulfonate, benzenesulfonate, borate, chlorobenzoate, camphorate, itaconate, camphorsulfonate, L-camphorsulfonate, methylbenzoate, dinitrobenzoate, sulfamate, lactobionate, galacturonate, cyclopentylpropionate, dodecyl sulfate, acrylate, cyclopentanepropionate, glycerophosphate, methoxybenzoate, digluconate, gluconate, heptanoate, hexanoate, 2-hydroxy-ethanesulfonate, trimethylacetate, glucuronate, laurate, phthalate, phenylacetate, lauryl sulfate, 2-acetoxybenzoate, nicotinate, cinnamate, oleate, palmitate, pamoate, pectinate, phthalate, Glutarate, hydroxymaleate, hydroxybenzoate, phenylacetate, 3-hydroxy-2-naphthoate, 3-phenylpropionate, isobutyrate, pivalate, picrate, stearate, 2,2-dichloroacetate, acylated amino acid salts, alginate, 4-acetamidobenzenesulfonate, decanoate, cholate, octanoate, nonanoate, cyclamate, phthalate, cysteine ​​hydrochloride, sorbate, pamoate, mucate, glycinate hydrochloride, naphthalene disulfonate, xylene sulfonate, cystine dihydrochloride, undecanoate, polyvinyl sulfonate, sulfosalicylate, phenylbutyrate, 4-hydroxybutyrate, polyvinyl sulfate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and valerate.

[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 easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0069] Figure 1 This is a graph showing the experimental results of co-treatment of a pseudovirus model with a DNJ derivative 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 the examples of the present invention;

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

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

[0073] Figure 5 This is a graph showing the co-treatment time gradient experiment of the pseudovirus model inhibited by DNJ-25 derivatives in the embodiment of the present invention;

[0074] Figure 6 It 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 inhibiting 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] Step 1: To a suspension of lithium aluminum hydride (2.28 g, 60 mmol) in 25 mL of tetrahydrofuran was slowly added a solution of compound 1 (10.82 g, 20 mmol) in 50 mL of tetrahydrofuran under ice bath conditions at 0°C. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, sodium sulfate decahydrate was added to quench the lithium aluminum hydride under ice bath conditions, and then diatomaceous earth was added to assist filtration. The filtrate was spin-dried and purified by flash column chromatography (DCM: MeOH = 100: 1) to obtain a milky white solid 2 (10.4 g, 95%).

[0082] Step 2: At -78°C, to a solution of oxalyl chloride (7.34 mL, 86 mmol) in anhydrous dichloromethane (50 mL) was added a solution of dimethyl sulfoxide (7.52 mL, 106 mmol) in dichloromethane (10 mL). After the addition was complete, the mixture was stirred for half an hour. Then, a solution of compound 2 (10.86 g, 20 mmol) in dichloromethane (50 mL) was added dropwise. After 2 hours, triethylamine (30 mL, 216 mmol) was added dropwise. After the addition was complete, the temperature was slowly raised to room temperature. The reaction progress was monitored by thin layer chromatography. After the raw materials are consumed, the above reaction solution is slowly added dropwise to an anhydrous methanol solution containing ammonium acetate (15.4 g, 200 mmol) and sodium cyanoborohydride (5.04 g, 80 mmol) precooled to 0°C, and the reaction system is heated to room temperature. After the reaction is completed by thin layer chromatography monitoring, the solvent is dried, and then 50% sodium hydroxide aqueous solution (100 mL) is added, extracted with dichloromethane, and the organic phase is washed with 37% sodium hypochlorite solution (100 mL). The organic layer is adjusted to acidic pH with 2M hydrochloric acid solution, and the product obtained by drying the organic phase is slurried with acetone, and the precipitated solid is filtered out. The white solid obtained by drying is the hydrochloride of compound 3. 50% sodium hydroxide solution is added to the obtained hydrochloride, extracted with ethyl acetate, washed with saturated brine, and dried to obtain yellow oily compound 3 (4.2 g, 42%).

[0083] Step 3: Dissolve compound 3 (1.05 g, 2 mmol) in anhydrous dichloromethane (10 mL) and stir at -78 °C for 0.5 h, then slowly drop boron tribromide (1.5 g, 6 mmol), react for 0.5 h, move to room temperature to continue the reaction, monitor the reaction progress with thin layer chromatography, after the reaction is completed, use triethylamine reaction system, use ionized water for extraction, spin dry the aqueous phase and dissolve it in methanol, use neutral alumina column chromatography, the elution system is MeOH→MeOH:H2O=2:1, and a white solid DNJ-1 (228 mg, 70%) is obtained.

[0084] The data of DNJ-1 is 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 mixed solvent of methanol and water (MeOH:H2O=2:1), and 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 system, and hydrogen was replaced and reacted at room temperature. The reaction progress was monitored by thin layer chromatography, and the palladium carbon was filtered off after the reaction was completed. The filtrate was dried and dissolved in methanol, and a neutral alumina column was used for chromatography, and the elution system was MeOH→MeOH:H2O=2:1 to obtain a white solid DNJ-3 (64 mg, 74%).

[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 mixed solvent of methanol and water (MeOH:H2O=2:1), and n-decyl aldehyde (216 mg, 1.15 mmol), 10% Pd / C (122 mg), and acetic acid (7 mg, 0.115 mmol) were added to the system to replace hydrogen and react at room temperature. The reaction progress was monitored by thin layer chromatography, and the palladium carbon was filtered off after the reaction was completed. The filtrate was dried and dissolved in methanol, and a neutral alumina column was used for chromatography, and the elution system was MeOH→MeOH:H2O=2:1 to obtain a white solid DNJ-4 (49 mg, 15%).

[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, and its reaction equation and preparation method are as follows:

[0100]

[0101] Step 1: Sodium hydride (2.4 g, 60 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) at 0°C in an ice bath. 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 slowly added dropwise. After the reaction was completed by TLC monitoring, 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=4:1) was performed to obtain compound 6 (3.5 g, 40%).

[0102] Step 2: Compound 6 (870 mg, 5 mmol) was dissolved in dichloromethane (25 mL), and then pyridinium chlorochromate (1.3 g, 6 mmol) was slowly added to react at room temperature. After the reaction was completed by monitoring with thin layer chromatography, the insoluble matter was filtered off with diatomaceous earth, the filtrate was dried by rotary evaporation, and compound 7 (620 mg, 72%) was obtained by flash column chromatography (Petrol ether: EtOAc = 30:1).

[0103] Step 3: DNJ-1 (252 mg, 1.54 mmol) was dissolved in 6 mL of a mixed solvent of methanol and water (MeOH:H2O=2:1), and then compound 7 (530 mg, 3 mmol), 10% Pd / C (100 mg), and acetic acid (9.6 mg, 0.16 mmol) were added to react under a hydrogen atmosphere. After the reaction was completed by TLC monitoring, the palladium carbon was filtered out, the filtrate was dried and dissolved in methanol and passed through a neutral alumina column. The elution system was MeOH→MeOH:H2O=2:1 to obtain a white solid DNJ-5 (69 mg, 15%).

[0104] The data of DNJ-5 is 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. 5,5.0Hz,1H),2.65(td,J=11.5,9.9,6.2Hz,1H),2.54(ddd,J=13.4,10.1,6.0Hz,1H),2.27–2.16(m,2H),1.48(t,J=6.8Hz,2H),1.39(s,2H),1.28–1.12(m,10H). Example 3

[0106] This example provides a DNJ-6 derivative, and its reaction equation and preparation method 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 bottle, and then anhydrous N,N-dimethylformamide (10 mL) and triethylamine (1.01 g, 10 mmol) were added. After reacting at room temperature for 0.5 h, compound 4-pentyn-1-ol (1.29 g, 5.5 mmol) was added dropwise. The reaction progress was monitored by thin layer chromatography. After the reaction was completed, saturated ammonium chloride solution was added to quench, and the mixture was extracted with ethyl acetate three times. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and flash column chromatography (Petrol ether:EtOAc=5:1) was performed to obtain 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 the reaction was completed by TLC monitoring, the palladium carbon was filtered off, and the filtrate was dried and subjected to flash column chromatography (Petrol ether:EtOAc=4:1) to obtain 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, and the reaction was carried out at room temperature. After the reaction was completed by monitoring with thin layer chromatography, the insoluble matter was filtered off with diatomaceous earth, the filtrate was dried by rotary evaporation, and compound 10 (70 mg, 65%) was obtained by flash column chromatography (Petrol ether: EtOAc = 3:1).

[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 the reaction was carried out under a hydrogen atmosphere. The reaction progress was monitored by thin layer chromatography. After the reaction, the palladium carbon was filtered off, the filtrate was dried and dissolved in methanol, and a neutral alumina column was used for chromatography. The elution system was MeOH→MeOH:H2O=2:1 to obtain a white solid DNJ-6 (49 mg, 72%).

[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, and its reaction equation and preparation method 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, and the mixture was refluxed 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, 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 flash column chromatography (Petrol ether:EtOAc=5:1) was performed to obtain 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 TLC monitoring, triethylamine was added dropwise in an ice bath to quench the reaction system. The mixture was extracted with deionized water, the aqueous phase was dried and dissolved in methanol, and a neutral alumina column was used for chromatography. The elution system was 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, and its reaction equation and preparation method are as follows:

[0125]

[0126] Step 1: Dissolve compound 6-heptynol (448.68 mg, 4 mmol) in dichloromethane (20 mL), add pyridinium chlorochromate (1.3 g, 6 mmol), and react at room temperature for 2 h. After the reaction is completed, filter out the insoluble matter with diatomaceous earth, spin dry the filtrate, and perform flash column chromatography (Petrol ether: EtOAc = 40: 1) to obtain compound 12 (225 mg, 51%).

[0127] Step 2: Dissolve DNJ-1 (32.8 mg, 0.2 mmol) in 3 mL of a mixed solvent of methanol and water (MeOH:H2O=2:1), and then add compound 12 (33 mg, 0.3 mmol), sodium cyanoborohydride (25 mg, 0.4 mmol), and acetic acid (1.2 mg, 0.02 mmol). React at room temperature. After monitoring the reaction by thin layer chromatography, spin dry the solvent and dissolve it with methanol through a neutral alumina column. The elution system is MeOH→MeOH:H2O=2:1 to obtain a white solid DNJ-8 (40 mg, 78%).

[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, and its reaction equation and preparation method 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, and the mixture was reacted in an oil bath at 80 °C. After the reaction was completed by TLC monitoring, 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 flash column chromatography (Petrol ether: EtOAc = 6: 1) was performed to obtain 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 TLC monitoring, 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. A neutral alumina column was used for chromatography, and the elution system was MeOH→MeOH:H2O=2:1 to obtain a white oily liquid DNJ-9 (60 mg, 30%).

[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, and its reaction equation and preparation method are as follows:

[0141]

[0142] Step 1: Dissolve the compound 2'-hydroxyacetophenone (1.36 g, 10 mmol) in tetrahydrofuran (40 mL), add it to an anhydrous oxygen-free round-bottom flask containing sodium hydride (1 g, 25 mmol), stir for 30 min, and then dropwise add a solution of the compound p-methoxybenzaldehyde (1.36 g, 10 mmol) in tetrahydrofuran (10 mL). After 2 h, the reaction was completed after monitoring by thin layer chromatography, and saturated ammonium chloride solution was added to quench, extracted with ethyl acetate 3 times, and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and flash column chromatography (Petrol ether:DCM=2:1) to obtain compound 14 (1.64 g, 65%).

[0143] Step 2: Dissolve compound 14 (508.56 mg, 2 mmol) in dimethyl sulfoxide (10 mL) and add iodine ( The reaction mixture was stirred for 1 h at 370 °C for 2 h. The mixture was refluxed in an oil bath at 170 °C for 1 h. After 1.5 h, the reaction was completed. 50 mL of water was added to the system to quench the reaction. The mixture was extracted three times with ethyl acetate. The organic phase was washed with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. Compound 15 (492 mg, 97%) was obtained by flash column chromatography using dichloromethane.

[0144] Step 3: Compound 15 (252.27 mg, 1 mmol) was dissolved in dichloromethane (5 mL) under anhydrous and oxygen-free conditions, and boron tribromide (375.81 mg, 1.5 mmol) was added dropwise after stirring in a -78°C cold trap for 30 min. The mixture was then warmed to room temperature. The reaction was completed after 3 h, and the reaction was quenched with saturated sodium bicarbonate solution. Compound 16 (226 mg, 95%) was obtained by filtration.

[0145] Step 4: Dissolve compound 16 (119.12 mg, 0.5 mmol) in N,N-dimethylformamide (5 mL) and add potassium carbonate ( 345.5 mg, 2.5 mmol) and compound 2-20 (574.85 mg, 2.5 mmol) were reacted in an oil bath at 80°C. After 3 h, the reaction was monitored 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 with dichloromethane was performed to obtain 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 mixture was reacted at 80 °C for 12 h. After the reaction was completed, the insoluble matter was filtered out after monitoring by thin layer chromatography. The filtrate was dried by rotary evaporation, and DNJ-10 (37 mg, 40%) was obtained by flash column chromatography (DCM: MeOH = 5:1).

[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 the 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 and add potassium carbonate. (The mixture was stirred in an oil bath at 60°C for 0.5 h, and then 1,6-dibromohexane (487.94 mg, 2 mmol), 1,8-dibromooctane (544.04 mg, 2 mmol), and 1,10-dibromodecane (600 mg, 2 mmol) were added respectively. 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 flash column chromatography (Petrol ether: EtOAc = 16: 1) was performed to obtain compound 18-1 (261 mg, 65%), compound 18-2 (200 mg, 47%), and compound 18-3 (221 mg, 49%).

[0154] Step 2: Dissolve DNJ-1 (16.4 mg, 0.1 mmol) and potassium carbonate (17 mg, 0.12 mmol) in N, N-dimethylformamide (0.5 mL) in three reaction tubes respectively, stir in an oil bath at 80 ° C for 0.5 h, then dissolve 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) in N, N-dimethylformamide (0.5 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 = 5: 1) to obtain 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 of 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, whose reaction equations and preparation methods 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. ( 622mg, 4.5mmol) was stirred in an oil bath at 60℃ for 0.5h, then 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, saturated ammonium chloride solution was added to quench, and 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) was performed to obtain compound 19-1 (453mg, 38%), compound 19-2 (507mg, 39%), and compound 19-3 (602mg, 44%).

[0168] Step 2: DNJ-1 (49.2 mg, 0.13 mmol), potassium carbonate and (62mg, 0.45mmol) was dissolved in N,N-dimethylformamide (0.5mL), and stirred in an oil bath at 80°C for 0.5h. Compound 19-1 (180.6mg, 0.45mmol), compound 19-2 (186mg, 0.45mmol), and compound 19-3 (205.8mg, 0.45mmol) were dissolved in N,N-dimethylformamide (2mL), respectively, 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-14 (92mg, 63%), DNJ-15 (101mg, 66%), and DNJ-16 (122mg, 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] 11H NMR (500 MHz, CD3OD) δ 8.07 (dd, J = 8.1, 1.6 Hz, 1H), 8.01–7.97 (m, 2H), 7.69 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.55 (dd, J = 8.6, 0.9 Hz, 1H), 7.49–7.43 (m, 3H), 7.37 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 3.86 (t, J = 6.4 Hz, 2H), 3.80 (d, J = 2.7 Hz, 2H), 3.45 (ddd, J = 10.5, 9.0, 4.8 Hz, 1H), 3.35–3.30 (m, 1H), 3.11 (t, J = 9.1 Hz, 1H), 2.94 (dd, J = 11.2, 4.9 Hz, 1H), 2.76–2.68 (m, 1H), 2.55–2.48 (m, 1H), 2.14 (t, J = 10.9 Hz, 1H), 2.08 (dt, J = 9.6, 2.8 Hz, 1H), 1.59–1.52 (m, 2H), 1.39 (p, J = 7.6, 7.2 Hz, 2H), 1.28–1.09 (m, 9H).

[0175] The data of DNJ-16 are as follows:

[0176] [α] D 25 = -19 (c = 3, MeOH).

[0177] 1 1H NMR (500 MHz, CD3OD) δ 8.07 (dd, J = 8.0, 1.6 Hz, 1H), 8.01–7.97 (m, 2H), 7.69 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.45 (qd, J = 4.9, 1.7 Hz, 3H), 7.37 (t, J = 7.6 Hz, 1H), 3.86 (t, J = 6.5 Hz, 2H), 3.81 (d, J = 2.7 Hz, 2H), 3.48–3.42 (m, 1H), 3.34 (t, J = 9.3 Hz, 1H), 3.12 (t, J = 9.1 Hz, 1H), 2.96 (dd, J = 11.2, 4.9 Hz, 1H), 2.79–2.70 (m, 1H), 2.58–2.50 (m, 1H), 2.17 (t, J = 10.9 Hz, 1H), 2.11 (dt, J = 9.8, 2.8 Hz, 1H), 1.58–1.51 (m, 2H), 1.42 (p, J = 7.6 Hz, 2H), 1.26–1.09 (m, 15H).

[0178] Example 10

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

[0180]

[0181] Step 1: Dissolve chrysin (254.24 mg, 1 mmol) in N,N-dimethylformamide (5 mL) in three reaction bottles and add potassium carbonate. ( The reaction 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 respectively. After the reaction was completed, saturated ammonium chloride solution was added to quench the reaction, and 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) was performed to obtain compound 20-1 (254 mg, 41%), compound 20-2 (277 mg, 41%), and compound 20-3 (296 mg, 42%).

[0182] Step 2: DNJ-1 (33 mg, 0.2 mmol), potassium carbonate and ( 33.2 mg, 1.2 mmol) was dissolved in N, N-dimethylformamide (2 mL), and stirred in an oil bath at 80 ° C for 0.5 h. 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), respectively, 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 of 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), mp: 125~129°C.

[0191] 1 H NMR(500MHz,DMSO-d6)δ12.83(s,1H),8.13(dd,J=7.0,1.9Hz,2H),7.68–7.61(m,3H),7.05(s,1H),6.81(d,J=2.2 Hz,1H),6.39(d,J=2.2Hz,1H),4.19(d,J=15.0Hz,1H),4.11(t,J=6.6Hz,2H),3.77(d,J=11.4Hz,1H),3.61(d,J=11 .6Hz,1H),3.30–3.24(m,1H),3.11(td,J=9.2,3.5Hz,1H),2.98(t,J=8.9Hz,1H),2.86(dd,J=11.0,4.8Hz,1H),2. 81–2.74(m,1H),2.42(ddd,J=13.8,9.0,5.2Hz,1H),2.03–1.96(m,2H),1.76(p,J=6.9Hz,2H),1.48–1.19(m,14H).

[0192] Embodiment 11

[0193] This example provides a series of DNJ-20 to DNJ-22 derivatives, and the reaction equations and preparation methods thereof are as follows:

[0194]

[0195] Step 1: Dissolve the compound kaempferol (1.71 g, 6 mmol) in N,N-dimethylformamide (40 mL), add potassium carbonate (2.74 g, 19.8 mmol), stir in a 60°C oil bath for 0.5 h, then add benzyl bromide (3.34 g, 19.8 mmol), and monitor the reaction progress with thin layer chromatography. After 3 h, add saturated ammonium chloride solution to quench the reaction system, extract 3 times with ethyl acetate, wash the organic phase with saturated sodium chloride solution, dry over anhydrous sodium sulfate, and spin dry, and recrystallize to obtain a 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, cesium carbonate (488.73 mg, 1.5 mmol) was added, and the mixture was stirred in an oil bath at 60°C for 0.5 h. 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 respectively. After the reaction was completed by TLC monitoring, saturated ammonium chloride solution was added to quench, 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 flash column chromatography (Petrol ether: EtOAc = 5: 1) was performed to obtain compounds 22-1 (688 mg, 88%), 22-2 (493 mg, 66%), and 22-3 (444 mg, 57%).

[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 respectively, stir in an oil bath at 80 °C for 0.5 h, 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 obtain 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 mixed solvent of tetrahydrofuran / MeOH (tetrahydrofuran: methanol = 1:1), and 42 mg of Pd(OH)2 / C were added, and the mixture was placed in a hydrogen atmosphere at 45°C for 3 h. After the reaction, the insoluble matter was filtered off, and the solid after the filtrate was dried was slurried with ether, and yellow solids DNJ-20 (58 mg, 78%), DNJ-21 (87 mg, 67%), and DNJ-22 (57 mg, 69%) were obtained by suction filtration.

[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 data of DNJ-22 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, and the reaction equations and preparation methods thereof are as follows:

[0210]

[0211] Step 1: Dissolve the compound luteolin (1.15 g, 4 mmol) in N,N-dimethylformamide (10 mL), add potassium carbonate (1.78 g, 12.8 mmol), stir in a 60°C oil bath for 0.5 h, then add benzyl bromide (2.19 g, 12.8 mmol), and monitor the reaction progress with thin layer chromatography. After 3 h of reaction, add saturated ammonium chloride solution to quench the reaction, and a large amount of yellow solid precipitates. Filter, wash the solid with ethyl acetate, and dry to obtain the 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 bottles, cesium carbonate (391 mg, 1.2 mmol) was added, and the mixture was stirred in an oil bath at 60°C for 0.5 h. 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 respectively. After the reaction was completed by TLC monitoring, saturated ammonium chloride solution was added to quench, 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 flash column chromatography (Petrol ether:EtOAc=5:1) was performed to obtain compounds 25-1 (286 mg, 50%), 25-2 (299 mg, 50%), and 25-3 (357 mg, 58%).

[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 respectively, stir in an oil bath at 80 °C for 0.5 h, 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 obtain 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 mixed solvent of tetrahydrofuran / methanol (tetrahydrofuran: methanol = 1:1), and 56 mg of Pd(OH)2 / C were added, and the mixture was placed in a hydrogen atmosphere at 45°C for 3 h. After the reaction, the insoluble matter was filtered off, and the solid after the filtrate was dried was slurried with ether, and yellow solids DNJ-23 (90 mg, 99%), DNJ-24 (97 mg, 91%), and DNJ-25 (85 mg, 85%) were obtained by suction filtration.

[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] The target cells HELA-ACE2 were seeded in a 96-well culture dish the night before, with about 3000-5000 cells per well, and the final volume of DMEM medium was 100μL. Different drugs were added to the target cells the next day, with a concentration gradient of 40μM, 20μM, 10μM, 5μM, and 2.5μM. After 48 hours, the DMEM medium was removed, and MTT solution diluted with blank DMEM medium was added to each well, and culture was continued for 4 hours. The MTT solution was removed, and 100μL dimethyl sulfoxide (DMSO) was added to dissolve the blue-purple crystalline formazan, and shaken on a shaker. After 10 minutes, the absorbance was measured at 490nm using the Biotek Cytation5 cell imaging microplate detection multifunctional microplate reader; the results are shown in Table 1.

[0227] Table 1

[0228]

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

[0230] 2. Evaluation of the inhibitory activity of co-treatment with DNJ derivative pseudovirus model

[0231] S1. Plant 293T cells with good growth conditions in a 10cm culture dish one night in advance, with a density of 30%-40% of the transfection density being the best. Culture overnight, and when 293T cells grow to 70%-80%, replace the DMEM complete medium with blank DMEM medium in advance. The packaging plasmid psPAX2, the lentiviral plasmid pLenti-CMV-luc2-GFP-Puro and the SARS-Cov-2Spike protein plasmid are transfected with the transfection reagent Lipo 8000 in a ratio of 1:1:1. After 6-8 hours of transfection, replace with fresh DMEM complete medium and continue to culture for 48 hours. Collect the 293T supernatant, filter through a 0.45μm microporous membrane, and centrifuge the filtrate at 1000rpm for 10 minutes to obtain a pseudovirus solution. The pseudovirus solution needs to be taken and used immediately, and freezing and thawing will reduce its infection effect.

[0232] S2 and HELA-ACE2 cells were cultured overnight at 1×10 4 / well were seeded in a colorless and transparent 96-well plate. Before the infection experiment, HELA-ACE2 cells were pretreated with a 10μM drug solution 1 hour in advance. Subsequently, the pseudovirus solution and the drug were co-treated with HELA-ACE2 cells (ensuring that the final drug concentration was 10μM). At the same time, the control group was set up with the pseudovirus solution but no drug treatment, and the blank group was set up without the pseudovirus solution or drug treatment. After 12 hours, the medium was replaced with fresh DMEM complete medium and culture continued. After 48 hours, the HELA-ACE2 cells were treated with the cell lysate in the luciferase kit for 10 minutes. After the cell lysate was centrifuged at 12000rpm for 10 minutes, the supernatant and the luciferase reaction solution were added to the black opaque 96-well plate, and the fluorescence value was immediately detected using the Biotek Cytation 5 cell multifunctional imaging microplate reader. The results are as follows Figure 1 As shown in the figure, it can be seen that DNJ-15 derivatives, DNJ-16 derivatives, and DNJ-25 derivatives can significantly inhibit the entry stage of SARS-CoV-2 pseudovirus-infected target cell HELA-ACE2, with inhibition rates reaching 80.69%, 95.53%, and 91.96%, respectively.

[0233] 3. Evaluation of inhibitory activity of DNJ derivatives in pseudovirus model pretreatment

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

[0235] S2 and HELA-ACE2 cells were cultured overnight at 1×10 4 / well were seeded in a colorless and transparent 96-well plate. The pseudovirus solution with different drug treatments was added to the target cells, and the pseudovirus solution without drug treatment was set as the control group, and the blank group was set without pseudovirus solution or drug treatment. After 12 hours, it was replaced with fresh DMEM complete medium and cultured. After 48 hours, HELA-ACE2 cells were treated with the cell lysate in the luciferase kit for 10 minutes. After the cell lysate was centrifuged at 12000rpm for 10 minutes, the supernatant and luciferase reaction solution were added to the black opaque 96-well plate, and the fluorescence value was immediately detected using the Biotek Cytation 5 cell multifunctional imaging microplate reader.

[0236] The results are as follows Figure 2 As shown in the figure, it can be seen that the drug treatment is carried out during the process of 293T cells producing pseudoviruses, and the DNJ derivatives provided by the present invention can reduce the infectivity of SARS-CoV-2 pseudoviruses to target cell HELA-ACE2; in particular, the DNJ-25 derivatives can still significantly reduce the infectivity of SARS-CoV-2 pseudoviruses to target cell HELA-ACE2, and the inhibition rate reaches 71.15%. Taking the DNJ-25 derivative as an example, the present invention continues the following experiment.

[0237] 4. Experiment on the inhibition concentration curve of DNJ-25 derivatives co-treatment

[0238] The experimental steps for determining the inhibitory concentration curve of compound DNJ-25 are consistent with the evaluation of pseudovirus co-treatment entry inhibitory activity, except 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 Figure 3 As shown in the figure, when the target cell HELA-ACE2 was treated with the pre-prepared SARS-CoV-2 pseudovirus and compound 25, it was found that the DNJ-25 derivative could inhibit the invasion of the pseudovirus in a concentration-dependent manner, and its EC 50 =5.192μM.

[0239] 5. Experiment on the determination of inhibition concentration curve of DNJ-25 derivative pretreatment

[0240] The experimental steps for determining the inhibitory concentration curve of DNJ-25 derivatives are consistent with the evaluation of pseudovirus pretreatment entry inhibitory activity, except 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 Figure 4As shown in the figure, the drug treatment during the production of pseudovirus in 293T cells can reduce the infectivity of SARS-CoV-2 pseudovirus to target cell HELA-ACE2 in a concentration-dependent manner. 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 HELA-ACE2 cells 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 HELA-ACE2 cells, and the compound solution with a final concentration of 20 μM was added at -2 hours (i.e., 2 hours of drug pretreatment), -1 hour (i.e., 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. After continuing to culture for 48 hours, the fluorescence value was detected using the Biotek Cytation 5 cell multi-function imaging microplate reader. The results are as follows Figure 5 As shown in the figure, there is no significant difference in the inhibitory effect between adding DNJ-25 derivatives before SARS-CoV-2 pseudovirus infects the target cell HELA-ACE2 and co-treatment of pseudovirus, target cells, and compounds. However, as the time of adding DNJ-25 derivatives goes by, its inhibitory effect gradually decreases. This shows that DNJ-25 derivatives can reduce the infectivity of SARS-CoV-2 pseudovirus 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 multiplicity (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 Graphs; Figure 6B is a graph of 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 of nucleocapsid protein 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 play an anti-coronavirus role 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 scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A DNJ derivative or a pharmaceutically acceptable salt thereof, characterized in that: It has the following structural formula: Where 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.

2. The DNJ derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: 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 12, and z is selected from 1 to 12.

3. The DNJ derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: R is selected from 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.

4. The DNJ derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that: Select from the following structures:

5. A pharmaceutical composition, characterized in that The invention comprises the DNJ derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, 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.

7. The pharmaceutical composition according to claim 6, 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.

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

9. Use of the DNJ derivative according to any one of claims 1 to 4; or the pharmaceutical composition according to any one of claims 5 to 8 in the preparation of drugs for treating and / or preventing coronavirus.

10. The use according to claim 9, characterized in that: The coronaviruses include SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV.

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