Flavonoid compound, and preparation method therefor, pharmaceutical composition comprising same, and use thereof

Abstract

Disclosed in the present invention are a flavonoid compound, and a preparation method therefor, a pharmaceutical composition comprising same, and a use thereof. The present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof. The compound of the present invention has broad-spectrum activity against diseases caused by infection with α coronaviruses (e.g., PEDV) and δ coronaviruses (e.g., PDCoV), and / or the compound of the present invention has biological activity in treating tumors.

Description

Flavonoids, their preparation methods, pharmaceutical compositions and uses

[0001] This application claims priority to Chinese patent application 2024110154936, filed on July 26, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention relates to flavonoids, their preparation methods, pharmaceutical compositions, and uses. Background Technology

[0003] Porcine epidemic diarrhea virus (PEDV), belonging to the genus *Alphacoronavirus* of the family Coronaviridae, causes massive mortality in piglets, resulting in huge losses to the global pig industry. Porcine deltacoronavirus (PDCoV), belonging to the genus *Deltacoronavirus*, also causes significant losses to the global pig industry; furthermore, PDCoV poses a risk of infecting humans. Vaccination is currently the primary means of controlling these coronaviruses, and there are no specific antiviral drugs. However, existing vaccines do not yet meet the needs for prevention and control in terms of protective efficacy and safety, especially lacking broad-spectrum activity. Therefore, the development of broad-spectrum and highly effective therapeutic drugs has become an urgent priority.

[0004] Viral infection triggers an integrated stress response (ISR) in cells, leading to phosphorylation of eIF2α (the α subunit of eukaryotic translation initiation factor 2). eIF2α phosphorylation downregulates overall protein synthesis and selectively upregulates genes such as transcription factor ATF4 (activating transcription factor 4) to defend against viral invasion. eIF2α kinases are responsible for completing eIF2α phosphorylation and play a pivotal regulatory role in the integrated antiviral stress response of eukaryotic cells. eIF2α kinases include four serine / threonine kinases: HRI (heme-regulated inhibitor kinase, EIF2AK1), PKR (double-stranded RNA-activated protein kinase / protein kinase R, EIF2AK2), PERK (pancreatic eIF2α kinase / PKR-like endoplasmic reticulum kinase, EIF2AK3), and GCN2 (general control nonderepressible-2, EIF2AK4). Summary of the Invention

[0005] The technical problem this invention aims to solve is the lack of drugs in the prior art with novel mechanisms of action against alpha and delta coronaviruses and / or with antitumor activity. This invention provides a flavonoid compound, its preparation method, pharmaceutical composition, and uses. The compounds of this invention possess broad-spectrum activity against diseases caused by alpha coronavirus (e.g., PEDV) and delta coronavirus infections (e.g., PDCoV), and / or, the compounds of this invention possess biological activity for treating tumors.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0008] Among them, R 1 Independently for C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl or C 1-6 Halogenated alkoxy groups;

[0009] Or, two Rs 1 Connected together, they form -O(CH2). p O-;

[0010] p is 1 or 2;

[0011] m is 1, 2, 3, 4 or 5;

[0012] Q is or R 3 ;

[0013] R 2 Independently cyano, halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl or C 1-6 Halogenated alkoxy groups;

[0014] n is 1, 2, 3, 4 or 5;

[0015] R 3 C 1-6 Alkyl, C 3-6 cycloalkyl, C 1-6 Haloalkyl, C 3-6 Halogenated cycloalkyl, -NH-C 1-6 Alkyl, -NH-C 3-6 cycloalkyl, -NH-C 1-6 Halogenated alkyl or -NH-C 3-6 Halogenated cycloalkyl groups;

[0016] It can be a single bond or a double bond.

[0017] In one embodiment, such as in a compound of formula I or a pharmaceutically acceptable salt thereof, certain groups may be defined as follows, and other groups may be defined as in any embodiment of the invention (hereinafter referred to as "in one embodiment"): R 1 Independently for C 1-6 Alkyl groups, such as methoxy groups.

[0018] In one particular scheme, m is 2.

[0019] In one particular scheme, R 2 Independently cyano or C 1-6 Halogenated alkyl; for example, cyano or trifluoromethyl.

[0020] In one particular scheme, R 3 C 3-6 cycloalkyl or -NH-C 3-6 Cycloalkyl; for example, cyclopropyl or -NH-cyclopropyl.

[0021] In one particular scheme, n is 2.

[0022] In one scheme, the C 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, for example, methyl.

[0023] In one embodiment, the halogen is independently fluorine, chlorine, bromine, or iodine, for example, fluorine.

[0024] In one scheme, the C 1-6 The alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy, for example, methoxy.

[0025] In one scheme, the C 1-6 C in haloalkyl 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, for example, methyl.

[0026] In one scheme, the C 1-6 The halogen in the haloalkyl group is independently fluorine, chlorine, bromine or iodine, for example fluorine.

[0027] In one scheme, the C 1-6 C in haloalkoxy 1-6 The alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy, for example, methoxy.

[0028] In one scheme, the C1-6 The halogen in a haloalkoxy group is independently fluorine, chlorine, bromine, or iodine, for example, fluorine.

[0029] In one scheme, the C 3-6 The cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, for example, cyclopropyl.

[0030] In one scheme, the C 3-6 C in halocycloalkyl 3-6 The cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, for example, cyclopropyl.

[0031] In one scheme, the C 3-6 The halogen in a halocycloalkyl group is fluorine, chlorine, bromine, or iodine, for example, fluorine.

[0032] In one embodiment, the compound of formula I is selected from:

[0033] For example, selected from:

[0034] Among them, R 1 The definitions of m and Q are as described in the previous scheme.

[0035] In one of the solutions, for For example,

[0036] In one of the solutions, for

[0037] In one of the solutions, for For example,

[0038] In one embodiment, the compound of formula I is selected from any of the following compounds:

[0039] This invention provides a method for preparing the compound of formula I, which is either method one or method two:

[0040] When Q is -NH-C 1-6 Alkyl, -NH-C 3-6 cycloalkyl, -NH-C 1-6 Halogenated alkyl or -NH-C 3-6 When the alkyl haloide is used, the preparation method is Method 1;

[0041] When Q is C 1-6 Alkyl, C 3-6 cycloalkyl, C1-6 Halogenated alkyl or C 3-6 When the alkyl haloide is used, the preparation method is Method Two:

[0042] Scheme 1 includes the following steps: In an organic solvent, in the absence of a base or in the presence of a base, compound S1 and compound S2 undergo a nucleophilic addition reaction to obtain compound I-(1);

[0043] Method 2 includes the following steps: In an organic solvent, in the presence of a base, compound S3 and compound S2 undergo an amidation reaction to obtain compound I-(2);

[0044] In compounds S1, S2, and S3, R 1 R 2 R 3 , m, n and Independently as defined in any of the preceding schemes; X is a halogen.

[0045] In the nucleophilic addition reaction, the organic solvent may be a haloalkane, such as dichloromethane.

[0046] In the nucleophilic addition reaction, the organic solvent may be an ester, such as ethyl acetate.

[0047] In the nucleophilic addition reaction, the base may be an organic base, such as triethylamine and / or pyridine.

[0048] The nucleophilic addition reaction can be carried out under the protection of an inert gas, such as nitrogen.

[0049] The nucleophilic addition reaction can be carried out under solvent reflux conditions.

[0050] In the amidation reaction, X can be chlorine.

[0051] In the amidation reaction, the organic solvent may be a haloalkane, such as dichloromethane.

[0052] In the amidation reaction, the base may be an organic base, such as triethylamine and / or pyridine.

[0053] The amidation reaction can be carried out under the protection of an inert gas, such as nitrogen.

[0054] The amidation reaction can be carried out under solvent reflux conditions.

[0055] This invention provides a compound of formula A, formula B, formula D, formula G, formula K, formula L, formula M, or formula N:

[0056] In the above compounds, R1 Independently as defined by any of the aforementioned schemes.

[0057] In a particular scheme, compound A, compound B, compound D, compound G, compound K, compound L, compound M, or compound N is selected from the following compounds:

[0058] The present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0059] This invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating eIF2α kinase-related diseases, wherein the eIF2α kinase-related diseases are preferably diseases caused by infection with α-coronaviruses or δ-coronaviruses, or malignant tumors.

[0060] In one embodiment, the alpha coronavirus is porcine epidemic diarrhea virus (PEDV).

[0061] In one embodiment, the δ-coronavirus is a porcine deltacoronavirus (PDCoV).

[0062] In one embodiment, the malignant tumor is a malignant solid tumor or a malignant hematological disease; the malignant solid tumor is, for example, breast cancer, and the malignant hematological disease is, for example, multiple myeloma.

[0063] The present invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament, preferably for treating diseases caused by infection with alpha coronaviruses or delta coronaviruses, or malignant tumors.

[0064] In one embodiment, the alpha coronavirus is porcine epidemic diarrhea virus (PEDV).

[0065] In one embodiment, the δ-coronavirus is a porcine deltacoronavirus (PDCoV).

[0066] In one embodiment, the malignant tumor is a malignant solid tumor or a malignant hematological disease; the malignant solid tumor is, for example, breast cancer, and the malignant hematological disease is, for example, multiple myeloma.

[0067] This invention provides the use of the compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of an eIF2α kinase modulator; the eIF2α kinase modulator is preferably an eIF2α kinase activator or an eIF2α kinase inhibitor, such as an eIF2α kinase activator.

[0068] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0069] The term "alkyl" refers to a saturated aliphatic hydrocarbon group; for example, an alkyl group with 1 to 6 carbon atoms, preferably an alkyl group with 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and their various branched isomers.

[0070] The term "alkoxy" refers to -O- (alkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, and butoxy.

[0071] "Haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above. Non-limiting examples include: trifluoromethyl.

[0072] "Haloalkoxy" refers to an alkoxy group that has been substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0073] The term "cycloalkyl" refers to a cyclic saturated aliphatic hydrocarbon group; for example, a cycloalkyl group with 3 to 6 carbon atoms. Non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0074] The term “pharmaceutically acceptable” as used herein refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0075] The term "pharmaceuticalally acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. See Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0076] The term “treatment” refers to a therapeutic approach. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0077] The term "prevention" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0078] The term "therapeutic effective amount" refers to an amount of compound sufficient to effectively treat the disease or condition described herein when administered to a patient. The "therapeutic effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but may be adjusted as needed by those skilled in the art.

[0079] The term "pharmaceuticalally acceptable excipients" refers to excipients and additives used in the manufacture and dispensing of pharmaceutical products. These are all substances included in pharmaceutical preparations, excluding the active ingredient. See Volume IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0080] The term "patient" refers to any animal, preferably a mammal, that is about to receive or has already received administration of the compound according to embodiments of the invention, with humans being the most preferred. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0081] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0082] The reagents and raw materials used in this invention are all commercially available.

[0083] The significant advantage of this invention lies in the fact that the compounds of this invention possess broad-spectrum activity against diseases caused by alpha coronaviruses (e.g., PEDV) and delta coronaviruses (e.g., PDCoV). As a control, the classic antiviral drug ribavirin has no activity against PDCoV.

[0084] Currently, the vast majority of clinically approved antiviral drugs are direct-acting antivirals (DAAs). However, DAAs have inherent drawbacks, including causing toxic side effects, lacking broad-spectrum activity, and inducing viral resistance. This invention focuses on antiviral host factors, providing target compounds that target host eIF2α kinase, eIF2α phosphorylation, and protein synthesis. By precisely regulating host-virus interactions, these compounds exert antiviral effects, which is beneficial for overcoming viral resistance and developing broad-spectrum antiviral drugs.

[0085] In addition, the compounds of this invention also have antitumor activity. Attached Figure Description

[0086] Figure 1 shows the expression of PEDV N protein in host Vero cells and PDCoV N protein in LLC-PK1 cells after treatment with different concentrations of I-1 and II-1, as determined by Western blot.

[0087] Figure 2 shows the changes in Vero cell protein synthesis and eIF2α phosphorylation as determined by Western blot after treatment with 30 μM concentrations I-1 and II-1 at different time points. Time refers to the time after treatment with the test compound. β-actin was used as a control. The intensity of the Western blot was quantified by ImageJ.

[0088] Figure 3 shows the changes in LLC-PK1 cell protein synthesis and eIF2α phosphorylation as determined by Western blot after treatment with 30 μM concentrations I-1 and II-1 at different time points. Time refers to the time after treatment with the test compound. β-actin was used as a control. The intensity of the Western blot was quantified using ImageJ. Detailed Implementation

[0089] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0090] Column chromatography separation and purification were performed using silica gel H₂. Nuclear magnetic resonance (NMR) measurements were performed on a Bruker Avance III 400 MHz or Bruker Avance Neo 600 MHz instrument. High-resolution mass spectrometry (HMS) measurements were performed on a ThermoScientific Q Exactive Plus instrument, using electrospray ionization (ESI) in positive ionization mode, with external calibrators.

[0091] Series A compounds (general formula IA) and their synthetic routes:

[0092] R 1 , m and Q are defined as in any of the aforementioned schemes.

[0093] Series B compounds (general formula IB) and their synthetic routes:

[0094] R 1 , m and Q are defined as in any of the aforementioned schemes.

[0095] Series of compounds (general formula IC) and their synthetic routes:

[0096] R 1 , m and Q are defined as in any of the aforementioned schemes.

[0097] Series of butyridine compounds (general formula ID) and their synthetic routes:

[0098] R 1 , m and Q are defined as in any of the aforementioned schemes.

[0099] Series of pentyl compounds (general formula IE) and their synthetic routes:

[0100] R 1 , m and Q are defined as in any of the aforementioned schemes.

[0101] Example 1: Preparation of Compound I-1

[0102] Preparation of N-(2-(3,4-dimethoxyphenyl)-4-oxo-4H-chromen-6-yl)acetamide (B-1)

[0103] At room temperature, a solution of sodium hydroxide (1.76 g, 44 mmol) in water (20 mL) was added to a solution of Z-1 (1.7 g, 8.8 mmol) in ethanol (20 mL), and the mixture was stirred at room temperature for 0.5 h. Z-2 (1.75 g, 10.5 mmol) was then added, and the mixture was stirred at room temperature for 24 h. The pH was adjusted from 11 to 3 with 10% hydrochloric acid, and the mixture was extracted with ethyl acetate (3 x 15 mL). The organic phases were combined, washed with saturated brine (20 mL), and rotary evaporated. The resulting liquid residue was subjected to silica gel column chromatography (petroleum ether (60-90 °C): ethyl acetate, 5:1 to 1:2), and dried under vacuum (80 °C, 2 h) to give a yellow solid A-1 (1.3 g, 43%).

[0104] 1 H NMR (600MHz, DMSO-d6) δ11.97(s,1H),9.89(s,1H),8.12(d,J=2.6Hz,1H),7.77(d,J=15.5Hz,1H),7.70(dd,J=8.9,2.6Hz,1H),7.67(d,J=15.5H z,1H),7.46(d,J=2.0Hz,1H),7.39(dd,J=8.4,2.0Hz,1H),7.05(d,J=8.4Hz,1H),6.95(d,J=8.9Hz,1H),3.85(s,3H),3.83(s,3H),2.03(s,3H).

[0105] At room temperature, iodine (79 mg, 0.3 mmol) was added to a solution of A-1 (0.5 g, 1.46 mmol) in dimethyl sulfoxide (15 mL), and the mixture was stirred at 180 °C for 12 h. The reaction solution was washed with saturated sodium thiosulfate aqueous solution (20 mL), and the aqueous phase was extracted with dichloromethane (3 x 15 mL). The organic phases were combined, washed with saturated brine (20 mL), and rotary evaporated. The residue was subjected to silica gel column chromatography (dichloromethane:methanol, 40:1) and dried under vacuum (70 °C, 2 h) to give a white solid B-1 (0.21 g, 47%).

[0106] 1H NMR (400MHz, DMSO-d6) δ10.26(s,1H),8.31(d,J=2.6Hz,1H),7.95(dd,J=9.1,2.7Hz,1H),7.75(d,J=9.0Hz,1H),7.71(d d,J=8.5,2.2Hz,1H),7.60(d,J=2.2Hz,1H),7.14(d,J=8.6Hz,1H),7.01(s,1H),3.89(s,3H),3.86(s,3H),2.09(s,3H).

[0107] Preparation of 6-amino-2-(3,4-dimethoxyphenyl)-4H-chromene-4-one hydrochloride (D-1)

[0108] B-1 (0.64 g, 1.89 mmol) was added to a mixed solution of concentrated hydrochloric acid (5 mL) and water (10 mL), and stirred under reflux for 3 h. After cooling to room temperature, a large amount of white solid precipitated in the reaction solution. The mixture was filtered, the filter cake was washed with water until neutral, and dried under vacuum (80 °C, 3 h) to obtain a nearly white powder D-1 (0.5 g, 79%).

[0109] 1 H NMR (400MHz, DMSO-d6) δ7.80(d,J=8.9Hz,1H),7.71(dt,J=5.3,2.3Hz,2H),7.60(d,J=2.2Hz,1 H),7.55(dd,J=8.9,2.8Hz,1H),7.14(d,J=8.6Hz,1H),7.04(s,1H),3.89(s,3H),3.86(s,3H).

[0110] Preparation of 1-(3-cyano-5-trifluoromethylphenyl)-3-(2-(3,4-dimethoxyphenyl)-4-oxo-4H-chromene-6-yl)urea (I-1)

[0111] Triphosgene (0.2 g, 0.68 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) at room temperature. Z-4 (0.2 g, 1 mmol) was added under ice-water bath conditions. The mixture was stirred at room temperature for 0.5 h, and then refluxed and stirred for 4 h. The reaction mixture was rotary evaporated to obtain a yellow solid residue Z-5 (0.23 g, 108%, 0.21 g pure).

[0112] Under nitrogen protection, D-1 (0.2 g, 0.6 mmol) was dissolved in anhydrous dichloromethane (10 mL), and triethylamine (0.2 g, 2 mmol) was added. A solution of Z-5 (0.21 g, 1 mmol) prepared in the first step in anhydrous dichloromethane (5 mL) was slowly added dropwise under reflux. After addition, the mixture was stirred under reflux for 3 h. The reaction was quenched with water (10 mL), and the organic phase was separated and washed successively with water (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The residue was subjected to silica gel column chromatography (petroleum ether (60-90 °C): ethyl acetate, 1:1) and recrystallized from ethanol, and dried under vacuum (100 °C, 5 h) to give a yellow solid I-1 (82 mg, 27%).

[0113] 1 H NMR (600MHz, DMSO-d6) δ9.47(s,1H),9.42(s,1H),8.27–8.25(m,1H),8.19(d,J=2.7Hz,1H),8.14–8.12(m,1H),7.91(br s,1H),7.88(dd,J=9.0,2.7Hz,1H),7.78(d,J=9.0Hz,1H),7.72(dd,J=8.5,2.2Hz,1H) ,7.61(d,J=2.2Hz,1H),7.15(d,J=8.6Hz,1H),7.02(s,1H),3.90(s,3H),3.86(s,3H).

[0114] ESI-HRMS: calcd for [M+H] + ,m / z:510.12713; found,m / z:510.12701;calcd for[M+Na] + ,m / z:532.10908; found,m / z:532.10866.

[0115] Example 2 Preparation of compound II-1

[0116] Preparation of 1-(3-cyano-5-trifluoromethylphenyl)-3-(2-(3,4-dimethoxyphenyl)-4-oxobenzodihydropyran-6-yl)urea (II-1)

[0117] Under nitrogen protection, E-1 (0.1 g, 0.33 mmol) and triethylamine (0.1 g, 1 mmol) were dissolved in anhydrous dichloromethane (15 mL), and a solution of Z-5 (0.11 g, 0.5 mmol) in anhydrous dichloromethane (5 mL) was slowly added dropwise under reflux. After the addition was complete, the mixture was stirred and refluxed for 2 h. The reaction was quenched with water (15 mL), the organic phase was separated, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The resulting solid residue was subjected to silica gel column chromatography (petroleum ether (60-90 °C): ethyl acetate, 4:1) and recrystallized from ethyl acetate, and dried under vacuum (80 °C, 5 h) to give a pale yellow solid II-2 (62 mg, 36%).

[0118] 1 H NMR(400MHz,DMSO-d6)δ9.38(s,1H),9.11(s,1H),8.23–8.20(m,1H),8.12–8.09(m ,1H),7.97(d,J=2.8Hz,1H),7.90–7.86(m,1H),7.61(dd,J=8.9,2.8Hz,1H),7.17( d,J=2.0Hz,1H),7.07(d,J=8.9Hz,1H),7.06(dd,J=8.4,1.9Hz,1H),6.98(d,J=8.3 Hz,1H),5.54(dd,J=13.1,2.8Hz,1H),3.78(s,3H),3.77(s,3H),3.34–3.25(m,2H).

[0119] ESI-HRMS: calcd for [M+H] + ,m / z:512.14278; found,m / z:512.14175;calcd for[M+NH4] + ,m / z:529.16933; found,m / z:529.16837;calcd for[M+Na] + ,m / z:534.12473; found,m / z:534.12362.

[0120] Example 3 Preparation of Compound III-1

[0121] Preparation of N-(2-(3,4-dimethoxyphenyl)-4-oxo-4H-chromen-7-yl)acetamide (G-1)

[0122] At room temperature, iodine (2.23 g, 8.8 mmol) was added to a solution of F-1 (3 g, 8.8 mmol) in dimethyl sulfoxide (15 mL), and the mixture was stirred at 180 °C for 12 h. The reaction solution was washed with saturated sodium thiosulfate aqueous solution (20 mL), and the aqueous phase was extracted with dichloromethane (3 x 15 mL). The organic phases were combined, washed with saturated brine (20 mL), and rotary evaporated. The residue was subjected to silica gel column chromatography (dichloromethane:methanol, 40:1 elution), and dried under vacuum (70 °C, 2 h) to give a white solid G-1 (1.24 g, 41%).

[0123] 1 H NMR (600MHz, DMSO-d6) δ10.49(s,1H),8.20(d,J=1.8Hz,1H),7.94(d,J=8.6Hz,1H),7.67(dd,J=8.6,2.2Hz,1H),7.5 5(d,J=2.1Hz,1H),7.41(d,J=8.6Hz,1H),7.12(d,J=8.5Hz,1H),6.95(s,1H),3.89(s,3H),3.85(s,3H),2.13(s,3H).

[0124] Preparation of 7-amino-2-(3,4-dimethoxyphenyl)-4H-chromen-4-one (H-1)

[0125] G-1 (1.4 g, 4.13 mmol) was added to a mixed solution of concentrated hydrochloric acid (7 mL) and ethanol (15 mL), and the mixture was refluxed and stirred for 3 h. The resulting reactants were cooled to room temperature and then rotary evaporated. The resulting red solid residue was recrystallized from ethanol to give a red solid. The solid was dissolved in dichloromethane (40 mL), and a saturated sodium carbonate aqueous solution (30 mL) was added and stirred for 0.5 h. The organic phase was separated, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The resulting red solid residue was subjected to silica gel column chromatography (dichloromethane:methanol, 50:0 to 50:1), and dried under vacuum (100 °C, 2 h) to give an orange solid H-1 (0.825 g, 67%).

[0126] 1 H NMR(600MHz,DMSO-d6)δ7.68(d,J=8.5Hz,1H),7.60(dd,J=8.5,2.2Hz,1H),7.50(d,J=2.2Hz,1H) ,7.11(d,J=8.5Hz,1H),6.76(s,1H),6.66(dd,J=8.6,2.0Hz,1H),6.64(d,J=2.0Hz,1H),6.27(br s,2H),3.87(s,3H),3.84(s,3H).

[0127] Preparation of 1-(3-cyano-5-trifluoromethylphenyl)-3-(2-(3,4-dimethoxyphenyl)-4-oxo-4H-chromene-7-yl)urea (III-1)

[0128] Under nitrogen protection, H-1 (0.2 g, 0.67 mmol) and triethylamine (0.2 g, 2 mmol) were dissolved in anhydrous dichloromethane (10 mL), and a solution of Z-5 (0.21 g, 1 mmol) in anhydrous dichloromethane (5 mL) was slowly added dropwise under reflux. After the addition was complete, the mixture was stirred under reflux for 1 h. The reaction was quenched with water (10 mL), and the mixture was extracted with dichloromethane (3 x 15 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The resulting solid residue was subjected to silica gel column chromatography (dichloromethane:methanol, 1:0–30:1), and dried under vacuum (100 °C, 10 h) to give a pale yellow solid III-1 (0.17 g, 50%).

[0129] 1 H NMR (400MHz, DMSO-d6) δ9.69(s,1H),9.56(s,1H),8.26(t,J=1.9Hz,1H),8.13(t,J=1.8Hz,1H),8.11(d,J=2.0Hz,1H),7.96(d,J=6.2Hz,1H),7.95(s ,1H),7.71(dd,J=8.5,2.1Hz,1H),7.58(d,J=2.3Hz,1H),7.35(dd,J=8.7, 2.1Hz,1H),7.13(d,J=8.6Hz,1H),6.97(s,1H),3.90(s,3H),3.86(s,3H).

[0130] ESI-HRMS: calcd for [M+H] + ,m / z:510.12713; found,m / z:510.12701;calcd for[M+Na] + ,m / z:532.10908; found,m / z:532.10786.

[0131] Example 4 Preparation of compound IV-1

[0132] Preparation of N-(4-(2-(3,4-dimethoxyphenyl)acetyl)-3-hydroxyphenyl)acetamide (K-1)

[0133] Z-6 (10 g, 60.54 mmol) and J-1 (15.6 g, 72.68 mmol) were dissolved in anhydrous dichloroethane (50 mL), and anhydrous aluminum trichloride (20.2 g, 151.5 mmol) was added in three portions under an ice-water bath. The resulting reaction mixture was stirred in an ice-water bath for 45 min, then stirred at room temperature for 4 h, and the reaction was quenched with water (20 mL). 4 mol / L hydrochloric acid (100 mL) was added to the resulting viscous mixture, and after 4 h, the suspension was filtered. The filter cake was washed with dichloromethane (20 mL). The filtrates were combined and extracted with dichloromethane (3 x 40 mL). The organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The resulting oily residue was subjected to silica gel column chromatography (petroleum ether (60-90℃): ethyl acetate, 5:1 to 2:1) to obtain oily substance K-1 (9.8g, 49%).

[0134] 1 H NMR (600MHz, DMSO-d6) δ12.25(s,1H),10.27(s,1H),8.01(d,J=8.8Hz,1H),7.35(d,J=2.1Hz,1H),7.06(dd,J=8.8,2.1Hz,1H), 6.91(d,J=2.0Hz,1H),6.88(d,J=8.2Hz,1H),6.79(dd,J=8.2,2.1Hz,1H),4.24(s,2H),3.72(s,3H),3.71(s,3H),2.08(s,3H).

[0135] Preparation of N-(3-(3,4-dimethoxyphenyl)-4-oxo-4H-chromen-7-yl)acetamide (L-1)

[0136] K-1 (9.3 g, 28.2 mmol) was dissolved in anhydrous DMF (20 mL). Boron trifluoride diethyl ether (23.0 g, 162.1 mmol) was added under ice-water bath conditions, followed by the addition of phosphorus pentachloride (5.1 g, 24.5 mmol) in portions under ice-water bath conditions. The mixture was stirred for 45 min under ice-water bath conditions, then at room temperature for 5 h. 40 mL of hot water at 90 °C was added, and the mixture was stirred at 85-95 °C for 1 h. Water (200 mL) was added to the resulting reaction mixture, and the mixture was stirred at room temperature, resulting in the precipitation of a solid. The mixture was filtered, and the filter cake was washed with water. The filter cake was recrystallized in ethanol (10 mL) and dried under vacuum (80 °C, 5 h) to obtain a yellow solid L-1 (4.3 g, 45%).

[0137] 1H NMR (600MHz, DMSO-d6) δ10.56(s,1H),8.44(s,1H),8.08(d,J=1.9Hz,1H),8.06(d,J=8.7Hz,1H),7.49(dd,J=8.7,2.0Hz ,1H),7.20(d,J=2.0Hz,1H),7.14(dd,J=8.2,2.0Hz,1H),7.01(d,J=8.4Hz,1H),3.78(s,3H),3.78(s,3H),2.13(s,3H).

[0138] 7-Amino-3-(3,4-dimethoxyphenyl)-4H-chromene-4-one (M-1)

[0139] L-1 (4.3 g, 12.7 mmol) was added to a mixed solution of concentrated hydrochloric acid (6 mL) and water (6 mL), and stirred under reflux for 3 h. The reaction solution was rotary evaporated, and water (15 mL) was added to the residue. The pH was then adjusted from 1 to 10 with ammonia, resulting in the precipitation of a large amount of solid. The solid was filtered, the filter cake was washed with water, recrystallized from ethanol, and dried under vacuum (80 °C, 3 h) to obtain a grayish-white powder M-1 (2.3 g, 61%).

[0140] 1 H NMR (600MHz, DMSO-d6) δ8.21(s,1H),7.77(d,J=8.7Hz,1H),7.16(d,J=2.0Hz,1H),7.09(dd,J=8.3,2.0Hz,1H),6 .98(d,J=8.3Hz,1H),6.69(dd,J=8.7,2.0Hz,1H),6.51(d,J=2.0Hz,1H),6.31(s,2H),3.77(s,3H),3.77(s,3H).

[0141] Preparation of 1-(3-cyano-5-trifluoromethylphenyl)-3-(3-(3,4-dimethoxyphenyl)-4-oxo-4H-chromene-7-yl)urea (IV-1)

[0142] Under nitrogen protection and reflux, a solution of Z-5 (154 mg, 0.72 mmol) in anhydrous dichloromethane (4 mL) was slowly added dropwise to a solution of M-1 (54 mg, 0.18 mmol) and triethylamine (73 mg, 0.72 mmol) in 3.5 mL of anhydrous dichloromethane. After addition, the mixture was refluxed and stirred for 4 h. The reaction was quenched with water, and the mixture was extracted with dichloromethane (3 x 10 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The residue was subjected to silica gel column chromatography (petroleum ether (60-90 °C): ethyl acetate, 1:1) to give a crude product. The crude product was decolorized in ethyl acetate with activated carbon, and the filtrate was rotary evaporated. The residue was slurried with diethyl ether to give a yellow solid IV-1 (27 mg, 29%).

[0143] 1 H NMR(600MHz,DMSO-d6)δ9.72(s,1H),9.57(s,1H),8.46(s,1H),8.25–8.23(m,1H),8.14–8.13(m,1H),8.07(d,J=8.7Hz,1H),7.96 –7.94(m,2H),7.42(dd,J=8.8,2.1Hz,1H),7.21(d,J=2.0Hz,1H),7.16(dd,J=8.3,2.1Hz,1H),7.01(d,J=8.4Hz,1H),3.79(s,6H).

[0144] ESI-HRMS: calcd for [M+H] + ,m / z:510.12713; found,m / z:510.12611;calcd for[M+Na] + ,m / z:532.10908; found,m / z:532.10799.

[0145] Example 5 Preparation of compound IV-2

[0146] Preparation of 1-cyclopropyl-3-(3-(3,4-dimethoxyphenyl)-4-oxo-4H-chromene-7-yl)urea (IV-2)

[0147] Under nitrogen protection and reflux, an anhydrous ethyl acetate solution of Z-7 (100 mg, 1.2 mmol) in 5 mL was added dropwise to a solution of M-1 (240 mg, 0.8 mmol) and triethylamine (240 mg, 2.4 mmol) in 15 mL of 15 mL of ethyl acetate. After addition, the mixture was refluxed and stirred for 4 h. The reaction was quenched with water, and the mixture was extracted with ethyl acetate (3 x 15 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and rotary evaporated. The residue was subjected to silica gel column chromatography (petroleum ether (60-90 °C): ethyl acetate, 1:1) to give a crude product. The crude product was slurried with diethyl ether (5 mL) and dried under vacuum (80 °C, 2 h) to give a white solid IV-2 (82 mg, 27%).

[0148] 1 H NMR (600MHz, DMSO-d6) δ9.02(s,1H),8.38(s,1H),7.97(d,J=8.7Hz,1H),7.91(d,J=1.7Hz,1H),7.27(br d,J=8.3Hz,1H),7.18(d,J=1.9Hz,1H),7.12(dd,J=8.3,1.9Hz,1H),7.00(d,J=8.4Hz,1H),6.67– 6.64(m,1H),3.77(s,3H),3.77(s,3H),2.58–2.55(m,1H),0.68–0.64(m,2H),0.45–0.41(m,2H).

[0149] Example 6 Preparation of compound V-1

[0150] Preparation of 7-amino-2-(3,4-dimethoxyphenyl)benzodihydropyran-4-one (N-1)

[0151] At room temperature, polyphosphoric acid (PPA) (9 g) was added to a suspension of F-1 (3 g, 8.8 mmol) in concentrated hydrochloric acid (7 mL) and ethanol (7 mL), and the mixture was stirred at 85 °C for 2 h. The resulting reaction solution was washed successively with ice water (20 mL) and saturated sodium carbonate aqueous solution (20 mL). The aqueous phases were combined and back-extracted with dichloromethane (3 x 15 mL). The organic phases were combined, washed with saturated brine (20 mL), and rotary evaporated. The residue was subjected to silica gel column chromatography (dichloromethane:methanol, 40:1) and vacuum drying (100 °C, 2 h) to give a red solid N-1 (0.67 g, 25%).

[0152] Preparation of N-(2-(3,4-dimethoxyphenyl)-4-oxobenzodihydropyran-7-yl)cyclopropane carboxamide (V-1)

[0153] Under nitrogen protection and reflux, Z-8 (100 mg, 1.01 mmol) was slowly added dropwise to a solution of N-1 (200 mg, 0.67 mmol) and triethylamine (136 mg, 1.34 mmol) in anhydrous dichloromethane (15 mL). After addition, the mixture was refluxed and stirred for 1 h. The residue was extracted by rotary evaporation with ethyl acetate (8 mL). The residue was filtered and then rotary evaporated. The residue was subjected to silica gel column chromatography (dichloromethane) and vacuum drying (100 °C, 3 h) to give a pale yellow solid V-1 (0.11 g, 44%).

[0154] 1 H NMR (400MHz, DMSO-d6) δ10.56(s,1H),7.72(d,J=8.6Hz,1H),7.47(d,J=1.9Hz,1H),7 .19(dd,J=8.6,2.0Hz,1H),7.15(d,J=2.0Hz,1H),7.04(dd,J=8.3,2.0Hz,1H),6.97(d ,J=8.3Hz,1H),5.53(dd,J=12.9,2.9Hz,1H),3.78(s,3H),3.77(s,3H),3.23(dd,J=1 6.9,12.9Hz,1H),2.72(dd,J=16.8,3.0Hz,1H),1.84–1.76(m,1H),0.86–0.81(m,4H).

[0155] ESI-HRMS: calcd for [M+H] + ,m / z:368.14925; found,m / z:368.14903;calcd for[M+Na] + ,m / z:390.13119; found,m / z:390.13084.

[0156] Example of effect test:

[0157] Cells and viruses

[0158] African green monkey kidney cells (Vero) and porcine kidney epithelial cells (LLC-PK1) were deposited at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Human breast cancer cells (MCF-7) were purchased from the American Type Culture Collection (ATCC). Vero cells were cultured in DMEM (catalog number 12677019, Gibco, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS), and LLC-PK1 cells were cultured in MEM (catalog number 11095-080, Gibco, Thermo Fisher Scientific) containing 10% FBS, both containing 100 U / mL penicillin-streptomycin. MCF-7 cells were cultured in EMEM (catalog number 320-006-CL, WISENT) containing 10% FBS, containing 50 U / mL penicillin-streptomycin. Cell culture was performed at 37°C in a cell culture incubator with 5% carbon dioxide.

[0159] Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) strains are preserved at the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0160] Example 1

[0161] The control antiviral drug ribavirin (catalog number BD5387, CAS Registry No. 36791-04-5) was purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd. The CCK-8 (Cell Counting Kit-8) cell proliferation and toxicity assay kit (catalog number NBS1157) was purchased from Shanghai Nuoning Biotechnology Co., Ltd.

[0162] The in vitro anticoronavirus activity of the test compounds was determined by the CCK-8 assay.

[0163] The cytotoxicity (CC) of the test compounds was determined on Vero cells. 50 ), and the antiviral activity of the test compounds against PEDV (EC). 50 ), calculate the selectivity index (SI) (SI = CC) 50 / EC 50 The cytotoxicity (CC) of the test compound was determined in LLC-PK1 cells. 50 ), and the antiviral activity of the tested compounds against PDCoV (EC). 50 SI was calculated. The in vitro anti-PEDV and anti-PDCoV activity data of the test compounds determined by the CCK-8 assay are shown in Tables 1 and 2, respectively. 50 and EC 50 It represents the mean ± standard deviation.

[0164] Half-maximal cytotoxic concentration (CC) 50 Measurement:

[0165] After digesting the cells that had grown into a dense monolayer into single cells using trypsin, 5 × 10 5 Cell suspension at concentrations of cells / mL was added to 96-well cell culture plates, 100 μL per well, and cultured in a cell culture incubator at 37°C and 5% CO2 until cells formed a monolayer. The test compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution; 1000 μM was used as the highest concentration, and the solution was serially diluted to eight concentrations using DMEM containing 2% FBS. The original culture medium in the 96-well plates was discarded, and the plates were washed twice with PBS (phosphate-buffered saline). Different concentrations of the prepared test compound were added, 100 μL per well, with three replicates for each concentration, for three independent experiments. A positive control group (ribavirin), a negative control group (0.1% DMSO), and a blank control group (cell-free, with added DMEM) were also included. Cells were cultured at 37°C and 5% CO2 for 72 h, and cell growth was observed. Discard the culture medium, wash twice with PBS, and incubate at 37°C for 1 hour with CCK-8 reagent (0.1 mL / mL DMEM solution). Measure the absorbance (OD) of each well at 450 nm using a microplate reader. Calculate the cell viability inhibition rate (%) using the formula: (A1–A2) / (A3–A2), where A1 is the average absorbance of the compound group, A2 is the average absorbance of the blank group, and A3 is the average absorbance of the negative control group. Data processing was performed using GraphPad Prism 8 (GraphPad Software, Boston, MA, USA) to calculate the CCK-8 concentration. 50 .

[0166] Half-maximal effective concentration (EC50) 50 Measurement:

[0167] Cells that had grown into a monolayer in a 96-well cell culture plate were inoculated with 100 μL of virus solution (MOI = 1) per well and infected for 1 h. The culture medium was discarded, and different concentrations of the prepared test compound (100 μL) were added. A cell control group (cells + maintenance medium) and a virus control group (virus + cells + maintenance medium) were also set up. Cells were cultured at 37°C and 5% CO2 for 48 h. The supernatant was discarded, and the cells were washed twice with PBS. CCK-8 reagent (0.1 mL / mL DMEM solution) was added to each well at 37°C and incubated for 1 h. The OD of each well was then measured at 450 nm using a microplate reader. The viral replication inhibition rate (%) was calculated using the formula: (A4 – A5) / (A6 – A5) × 100%, where A4 is the average absorbance of the compound plus virus group, A5 is the average absorbance of the virus control group, and A6 is the average absorbance of the cell control group. Data processing was performed using GraphPad Prism 8, and EC5 was calculated. 50The Vero cell maintenance medium was DMEM containing 2% FBS, and the LLC-PK1 cell maintenance medium was MEM containing 2% FBS. In vitro anti-coronavirus activity data are shown in Tables 1 and 2.

[0168] Table 1. In vitro activity against porcine epidemic diarrhea virus (PEDV)

[0169] Table 2. In vitro activity against porcine deltacoronavirus (PDCoV)

[0170] Example 2

[0171] The in vitro anticoronavirus activity of the test compounds was determined by Western blot.

[0172] PEDV N protein antibody (catalog number SD17-103) was purchased from Medgene, PDCoV N protein antibody (catalog number PDCOV11-M) from Alpha Diagnostic International, and β-actin antibody (catalog number 66009-1-Ig) from Proteintech. Horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin G (catalog number 115-035-003) and goat anti-rabbit immunoglobulin G (catalog number 111-035-003) were purchased from Jackson ImmunoResearch Laboratories.

[0173] CC 50 To achieve the highest concentration, the dimethyl sulfoxide stock solution (20 mM) of the test compound was serially diluted to four concentrations using DMEM containing 2% FBS. 5 × 10⁻⁶ 5Cell suspension at 100 μL / mL was added to each well of a 12-well cell culture plate. After the cells grew into a monolayer, 100 μL of virus solution (MOI = 1) was inoculated into each well, and the cells were cultured at 37°C in a 5% CO2 incubator for 1.5 h. The supernatant was discarded, and different concentrations of the test compound (1 mL) were added. A virus control group was also set up, and the cells were cultured at 37°C in a 5% CO2 incubator for 12 h. The supernatant was discarded, and the cells were washed three times with pre-cooled PBS. After discarding the PBS, RIPA buffer (150 μL) was added to each well to lyse the cells, and the cells were transferred to centrifuge tubes and centrifuged at 12,000 rpm for 5 min. The supernatant was mixed with 5×SDS protein loading buffer (37.5 μL), and the mixture was incubated at 100°C in a metal bath for 10 min until the protein denatured. The supernatant was subjected to 10% SDS-PAGE protein electrophoresis, and then transferred to a PVDF membrane. The PVDF membrane was removed and blocked with 5% skim milk at room temperature for 2 h. After blocking, the membrane was washed three times with TBST on a shaker. It was then incubated overnight at 4°C with primary antibodies against PEDV N protein (1:2000 dilution), PDCoV N protein (1:2000 dilution), and β-actin (1:10000 dilution). After incubation, the PVDF membrane was removed and washed three times with TBST on a shaker. PVDF membranes incubated with antibodies against PEDV and PDCoV N protein were incubated with secondary antibodies with 1:8000 dilution of HRP-labeled goat anti-mouse immunoglobulin G, and PVDF membranes incubated with β-actin antibody were incubated with 1:8000 dilution of HRP-labeled goat anti-rabbit immunoglobulin G, and incubated at room temperature for 1 hour, followed by three washes with TBST on a shaker. The PVDF membrane was incubated in ECL luminescent solution for 20 s, and the expression levels of the corresponding proteins were detected using a fully automated chemiluminescence imaging analysis system (Shanghai Tianneng, Tanon 5200Multi). The expression levels of PEDV N protein in Vero cells and PDCoV N protein in LLC-PK1 cells after treatment with different concentrations of compounds I-1 and II-1 are shown in Figure 1, as determined by Western blot.

[0174] Example 3

[0175] Changes in host Vero cell protein synthesis and eIF2α phosphorylation after administration of the test compound

[0176] Puromycin (catalog number X2370-1) was purchased from Inovogen. Puromycin antibody (catalog number MABE343, Sigma-Aldrich) was purchased from Merck. eIF2α antibody (catalog number 9721S) and phospho-eIF2α antibody (catalog number 9722S) were purchased from CST. β-actin antibody (catalog number 66009-1-Ig) was purchased from Proteintech. HRP-labeled goat anti-mouse immunoglobulin G (catalog number 115-035-003) and goat anti-rabbit immunoglobulin G (catalog number 111-035-003) were purchased from Jackson ImmunoResearch Laboratories.

[0177] Vero cells or LLC-PK1 cells were seeded into 6-well plates and cultured at 37°C and 5% CO2 in an incubator until the cell density reached 80-90%. The culture medium was discarded, and 2 mL of dimethyl sulfoxide (DMSO) stock solution (20 mM) of compound I-1 or II-1 was diluted to 30 μM with the corresponding cell maintenance medium and added to each well. At 3, 7, 11, 17, and 23 h post-drug administration, cells were labeled with puromycin solution diluted to 2 mg / mL with the corresponding cell maintenance medium for 1 h. Cells were harvested at 4, 8, 12, 18, and 24 h post-drug administration. After discarding PBS, 150 μL of RIPA buffer was added to each well to lyse the cells, which were then transferred to centrifuge tubes and centrifuged at 12000 rpm for 5 min. The supernatant was mixed with 37.5 μL of 5×SDS protein loading buffer and incubated at 100°C for 10 min until protein denaturation. The supernatant was then subjected to 10% SDS-PAGE protein electrophoresis and transferred to a PVDF membrane. Remove the PVDF membrane and block it with 5% skim milk at room temperature for 2 hours. After blocking, wash three times with TBST on a shaker. Incubate overnight at 4°C with puromycin antibody (1:2000 dilution), P-eIF2α antibody (1:2000 dilution), T-eIF2α antibody (1:2000 dilution), and β-actin antibody (1:10000 dilution) as primary antibodies. After incubation, remove the PVDF membrane and wash three times with TBST on a shaker. PVDF membranes incubated with puromycin antibody are incubated with HRP-labeled goat anti-mouse immunoglobulin G (1:8000 dilution) as secondary antibody, and PVDF membranes incubated with P-eIF2α antibody, T-eIF2α antibody, and β-actin antibody are incubated with HRP-labeled goat anti-rabbit immunoglobulin G (1:8000 dilution) as secondary antibody, and then incubated with TBST on a shaker for 1 hour. The PVDF membrane was incubated in ECL luminescent solution for 20 s, and then detected and developed using a fully automated chemiluminescence imaging analysis system (Shanghai Tianneng, Tanon 5200Multi). Figure 2 shows the results of puromycin labeling experiments and eIF2α phosphorylation at different time points after treatment of Vero cells with compounds I-1 and II-1. Figure 3 shows the results of puromycin labeling experiments and eIF2α phosphorylation at different time points after treatment of LLC-PK1 cells with compounds I-1 and II-1.

[0178] Example 4

[0179] The in vitro antitumor cell proliferation activity of the test compounds was determined by the CCK-8 assay.

[0180] MCF-7 cells were seeded into 384-well cell culture plates at 1500 cells per well and cultured for 24 h. Different concentrations of the test compound (starting at 10 μM, serially diluted 3 times) were added for 72 h, followed by incubation of 2.5 μL of CCK-8 reagent per well for 1.5 h. The absorbance (OD) of each well was measured at 450 and 650 nm using a microplate reader. Cell viability per well was calculated as V% = (A / V) * ... s –A c ) / (A b –A c ) × 100% calculation, where A s It is the calibrated absorbance value (OD) of the compound test well. 450 –OD 650 ), A c This is the corrected absorbance value for the wells without cells and the test compound (blank control), A. b This is the corrected absorbance value for adding only cells and carrier wells. Each experiment was performed in duplicate and independently twice. Data were processed using Graphpad Prism 7 software, and the half-maximal inhibitory concentration (IC50) obtained from the two independent experiments was used. 50 The average value of the in vitro antitumor cell activity data is shown in Table 3.

[0181] Table 3. In vitro activity against human breast cancer cells MCF-7

Claims

1. A compound of Formula I: ###0001### or a pharmaceutically acceptable salt thereof. wherein R 1 independently C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy; or, two R 1 linked, together, are -O(CH2) p O-; p is 1 or 2; m is 1, 2, 3, 4 or 5; Q is or R 3 ; R 2 independently cyano, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl or C 1-6 haloalkoxy; n is 1, 2, 3, 4 or 5; R 3 is C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 haloalkyl, C 3-6 halocycloalkyl, -NH-C 1-6 alkyl, -NH-C 3-6 cycloalkyl, -NH-C 1-6 haloalkyl or -NH-C 3-6 halocycloalkyl; is a single or double bond.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein preferably, it satisfies one or more of the following conditions: (1) R 1 independently C 1-6 alkoxy; (2) m is 2; (3) R 2 independently cyano or C 1-6 haloalkyl; (4) R 3 is C 3-6 cycloalkyl or -NH-C 3-6 cycloalkyl; and (5) n is 2; preferably, it satisfies one or more of the following conditions: (1) R 1 is methoxy; (2) R 2 is cyano or trifluoromethyl; (3) R 3 is cyclopropyl or -NH-cyclopropyl.

3. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein it satisfies one or more of the following conditions: (1) the C 1-6 alkyl is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl; (2) the halogen is independently fluorine, chlorine, bromine or iodine; (3) the C 1-6 alkoxy is independently methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, or t-butoxy; (4) the C 1-6 C in haloalkyl 1-6 alkyl is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, or t-butyl; (5) the C 1-6 halogen in haloalkyl is independently fluorine, chlorine, bromine, or iodine; (6) the C 1-6 C in haloalkoxy 1-6 alkoxy is independently methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, or t-butoxy; (7) said C 3-6 Cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. (8) the C 1-6 halogen in haloalkoxy is independently fluorine, chlorine, bromine or iodine; (9) said C 3-6 C in halocycloalkyl 3-6 Cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; and (10) said C 3-6 The halogen in the halocycloalkyl group is fluorine, chlorine, bromine or iodine. preferably, it satisfies one or more of the following conditions: (1) the C 1-6 alkyl is methyl; (2) the halogen is fluorine; (3) said C 1-6 alkoxy is methoxy; (4) said C 1-6 C in haloalkyl 1-6 alkyl is methyl; (5) the C 1-6 The halogen in the haloalkyl group is fluorine; (6) the C 1-6 C in haloalkoxy 1-6 alkoxy is methoxy; (7) said C 1-6 The halogen in the haloalkoxy group is fluorine; (8) said C 3-6 cycloalkyl is cyclopropyl; (9) said C 3-6 C in haloalkyl 3-6 cycloalkyl is cyclopropyl; and (10) said C 3-6 The halogen in the halocycloalkyl group is fluorine.

4. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein it satisfies one or more of the following conditions: (1) the compound of Formula I is: Preferably: wherein R 1 , m and Q are as defined in any one of claims 1 to 3; (2) For and (3) For preferably, it satisfies one or two of the following conditions: (1) For Preferably and (2) For 5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein The compound of Formula I is any one of the following:

6. A method for preparing a compound of formula I according to any one of claims 1 to 5, which is Method 1 or Method 2: When Q is -NH-C 1-6 alkyl, -NH-C 3-6 cycloalkyl, -NH-C 1-6 haloalkyl or -NH-C 3-6 halocycloalkyl, the preparation method is Method I; when Q is C 1-6 alkyl, C 3-6 cycloalkyl, C 1-6 haloalkyl or C 3-6 halocycloalkyl, the process is Process Two: Scheme one comprises the following steps: nucleophilic addition reaction of compound S1 with compound S2 in the presence of organic solvent, in the absence of base or in the presence of base, to obtain compound of formula I-(1); Method two comprises the following steps: the amidation reaction of compound S3 and compound S2 in an organic solvent in the presence of a base, to obtain a compound of formula I-(2); In the compound S1, the compound S2, and the compound S3, R 1 , R 2 , R 3 , m, n and is independently defined as in any one of claims 1 to 5; X is halogen; preferably, the nucleophilic addition reaction satisfies one or more of the following conditions: (1) the organic solvent is a halogenated hydrocarbon, preferably dichloromethane; (2) the organic solvent is an ester, preferably ethyl acetate; (3) the base is an organic base, preferably triethylamine and / or pyridine; (4) the nucleophilic addition reaction is carried out under inert gas protection, preferably nitrogen; (5) the nucleophilic addition reaction is carried out under refluxing conditions of the solvent; preferably, the amidation reaction satisfies one or more of the following conditions: (1) X is chlorine; (2) the organic solvent is a halogenated hydrocarbon, preferably dichloromethane; (3) the base is an organic base, preferably triethylamine and / or pyridine; (4) the amidation reaction is carried out under inert gas protection, preferably nitrogen; (5) the amidation reaction is carried out under refluxing conditions of the solvent.

7. A compound of formula A, formula B, formula D, formula G, formula K, formula L, formula M, or formula N: R 1 independently as defined in any one of claims 1-5; Preferably, it is the following compound:

8. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. Use of a compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease related to eIF2a kinase, preferably a disease caused by an infection with an alphacoronavirus or a deltacoronavirus, or a malignant tumor; said alphacoronavirus is preferably porcine epidemic diarrhea virus, and said deltacoronavirus is preferably porcine deltacoronavirus; said malignant tumor is preferably a malignant solid tumor or a malignant hematological disease; said malignant solid tumor is preferably breast cancer, and said malignant hematological disease is preferably multiple myeloma.

10. Use of a compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, preferably for the treatment of a disease caused by an infection with an alphacoronavirus or a deltacoronavirus, or a malignant tumor; said alphacoronavirus is preferably porcine epidemic diarrhea virus, and said deltacoronavirus is preferably porcine deltacoronavirus; said malignant tumor is preferably a malignant solid tumor or a malignant hematological disease; said malignant solid tumor is preferably breast cancer, and said malignant hematological disease is preferably multiple myeloma.

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