Dimethylphosphoryl substituted compound, and preparation method therefor, pharmaceutical composition thereof and use thereof

Abstract

Disclosed in the present invention are a dimethylphosphoryl substituted compound, and a preparation method therefor, a pharmaceutical composition thereof and the use thereof. Provided in the present invention is a compound of formula (I) or a pharmaceutically acceptable salt thereof. The compound of the present invention can target a host eIF2α kinase, eIF2α phosphorylation and protein synthesis. The compound of the present invention has great biological activity for treating breast cancer. Particularly, compound SIPI-27 and compound SIPI-28 of the present invention has inhibitory activity against breast cancer significantly superior to that of the control compounds BTdCPU and neratinib; and / or the compound of the present invention also has biological activity against coronaviruses and / or orthopoxviruses, in particular, compound SIPI-25 of the present invention has broad-spectrum anti-coronavirus activity against porcine epidemic diarrhea virus (PEDV), mouse hepatitis virus (MHV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV), and also has activity against the Taterapox virus (TTV) orthopoxvirus.

Description

Dimethylphosphoryl-substituted compounds, methods of making, pharmaceutical compositions, and uses thereof

[0001] This application claims priority to Chinese Patent Application No. 2024112654700, filed on September 10, 2024, and Chinese Patent Application No. 2024119407920, filed on December 26, 2024. This application incorporates the entirety of the above-mentioned Chinese Patent Applications. TECHNICAL FIELD

[0002] The present application relates to dimethylphosphoryl-substituted compounds, methods of making, pharmaceutical compositions, and uses thereof. BACKGROUND

[0003] Tumor cells increase the synthesis of oncogenic proteins at the level of translation by interfering with translation initiation factors to meet their need for vigorous proliferation. Therefore, the translation initiation step plays an extremely important role in many oncogenic pathways and can be used as a target for anti-tumor drugs. By pharmacologically activating eIF2a (the a subunit of eukaryotic translation initiation factor 2) kinase, the integrated stress response (ISR) of cells can be triggered, resulting in upregulation of eIF2a phosphorylation. Upregulation of eIF2a phosphorylation downregulates the synthesis of somatic proteins while selectively upregulating genes such as activating transcription factor 4 (ATF4) to resist, for example, malignant tumor invasion. eIF2a kinases include four serine / threonine kinases, namely HRI (heme-regulated inhibitor kinase, EIF2AK1), PKR (double-stranded RNA-activated protein kinase / protein kinase R, EIF2AK2), PERK (pancreatic eIF2a kinase / PKR-like endoplasmic reticulum kinase, EIF2AK3), and GCN2 (general control nonderepressible-2, EIF2AK4).

[0004] According to the data released by the World Health Organization in March 2024, in 2022, 2.3 million women worldwide were diagnosed with breast cancer, and 670,000 people died of breast cancer; in 185 countries, breast cancer is the most common female cancer in 157 countries. Although the drug treatment of breast cancer has made great progress, there are still significant shortcomings in effectiveness and safety. Therefore, it is urgent to develop new drugs for breast cancer treatment with new mechanisms of action.

[0005] Ting Chen et al. reported that the prototype eIF2a kinase HRI activator BTdCPU showed significant anti-tumor activity against MCF-7 human breast cancer xenografts on female nude mice with mild toxicity. However, BTdCPU has the defect of poor in vivo stability. Revital Yefidoff-Freedman et al. reported that the new HRI activator 3r is stable in vivo and can significantly and dose-dependently inhibit the growth of CRL-2813 human melanoma xenografts on male nude mice. However, the physicochemical properties of 3r are not ideal, with too high lipophilicity and insufficient solubility. Its CLogP is as high as 6.20, and according to Lipinski's '5 rules', it is predicted to be poorly druggable. The lipophilic electron-withdrawing trifluoromethyl group is considered to be the best substituent at the 4-position of the new generation of HRI activators represented by 3r. Jinsook Kwak et al. reported that the bioisosteric squaramide was used to replace the urea functional group in the aforementioned new generation of HRI activators, and tried to use nitro, cyano and methylsulfonyl to replace trifluoromethyl as the 4-position substituent, obtaining target compounds 19 and 40 that can up-regulate eIF2a phosphorylation and its downstream effectors ATF4 and CHOP.

[0006] Coronaviruses are RNA viruses belonging to the family Coronaviridae in the order Nidovirales. According to the serotype and genomic characteristics, they are divided into four genera, i.e., alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. Porcine epidemic diarrhea virus (PEDV) belongs to the genus alphacoronavirus and can cause mass mortality of piglets, resulting in huge losses to the global pig industry. Mouse hepatitis virus (MHV) belongs to the genus betacoronavirus and is highly infectious to mice. Infectious bronchitis virus (IBV) belongs to the genus gammacoronavirus and is the pathogen of chicken infectious bronchitis, which is one of the important diseases in the chicken industry. Porcine deltacoronavirus (PDCoV) belongs to the genus deltacoronavirus and has caused great losses to the global pig industry. In addition, PDCoV has the risk of infecting humans. Vaccine immunization is the main means for preventing and controlling the above-mentioned coronaviruses, and there is no specific drug. However, the existing vaccines cannot meet the prevention and control needs in terms of protection efficacy, safety, etc., especially lacking broad-spectrum activity. Therefore, it is urgent to develop broad-spectrum and efficient therapeutic drugs.

[0007] Orthopoxviruses are DNA viruses belonging to the genus Orthopoxvirus in the family Poxviridae. They include variola virus, monkeypox virus, ectromelia virus, vaccinia virus, cowpox virus and TTV (taterapox virus). Viruses in the genus Orthopoxvirus can cause smallpox, monkeypox and other viral infections that seriously endanger human health. Since September 20, 2023, the National Health Commission of China has included monkeypox in the management of Category B infectious diseases and taken prevention and control measures for Category B infectious diseases. On August 14, 2024, the World Health Organization (WHO) announced that the monkeypox outbreak constitutes an “international public health emergency”. SUMMARY

[0008] The present application aims to solve the technical problems of the prior art, i.e., the single structure of eIF2a kinase modulators and / or the lack of breast cancer and viral infection treatment drugs with new mechanisms of action. The present application provides a dimethyl phosphoryl substituted compound, a preparation method, a pharmaceutical composition and a use thereof. The compound of the present application can target host eIF2a kinase, eIF2a phosphorylation and protein synthesis. The compound of the present application has excellent biological activity in treating breast cancer. In particular, the inhibitory activity of the compound SIPI-27 and the compound SIPI-28 of the present application on breast cancer is significantly better than that of the control compound BTdCPU and neratinib; and / or, the compound of the present application also has biological activity against coronavirus and / or orthopoxvirus, in particular, the compound SIPI-25 of the present application has broad-spectrum anti-coronavirus activity against porcine epidemic diarrhea virus (PEDV), mouse hepatitis virus (MHV), infectious bronchitis virus (IBV) and porcine delta coronavirus (PDCoV), and also has activity against orthopoxvirus taterapox virus (TTV).

[0009] The present application provides a compound of formula I or a pharmaceutically acceptable salt thereof,

[0010] wherein R 1 is cyano, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0011] R 2 is hydrogen, cyano, halogen or C 1-6 haloalkyl;

[0012] Q is

[0013] The present application also provides a compound of formula I or a pharmaceutically acceptable salt thereof,

[0014] wherein R 1 is cyano, halogen or C 1-6 haloalkyl;

[0015] R 2 is cyano, halogen or C 1-6 haloalkyl;

[0016] Q is

[0017] In a certain aspect, in the compound of formula I, the definition of certain groups can be as follows, and the definition of other groups can be as described in any aspect of the present application (hereinafter referred to as "in a certain aspect"): R 1 is halogen, for example, chloro.

[0018] In one particular scheme, R 1 C 1-6 Alkyl, for example, tert-butyl.

[0019] In one particular scheme, R 2 It is a halogen, such as chlorine.

[0020] In one particular scheme, R 2 It is hydrogen.

[0021] In one particular scheme, R 1 and R 2 same.

[0022] In one of the solutions, for For example,

[0023] In one of the solutions, for For example,

[0024] 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.

[0025] In one scheme, the C 1-6 The halogen in the haloalkyl group is independently fluorine, chlorine, bromine or iodine, for example fluorine.

[0026] In one scheme, the C 1-6 The alkyl halogroup is trifluoromethyl.

[0027] 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, tert-butyl.

[0028] In one embodiment, in compound I, the relative configurations of the two substituents at the 1 and 4 positions of the cyclohexyl group can be trans (compound IA) or cis (compound IB); preferably, compound IA has the trans configuration.

[0029] Among them, R 1 R 2 And Q is as described in any of the previous schemes.

[0030] In one embodiment, the compound of formula I is either a compound of formula IU or a compound of formula IS:

[0031] Among them, R 1and R 2 as in any preceding scheme.

[0032] In a certain embodiment, the compound of Formula I is a compound of Formula I-AU, a compound of Formula I-AS, a compound of Formula I-BU, or a compound of Formula I-BS; preferably a compound of Formula I-AU or a compound of Formula I-AS.

[0033] wherein R 1 , R 2 and Q are as in any preceding scheme.

[0034] In a certain embodiment, the compound of Formula I is selected from any one of the following compounds:

[0035] The present application provides a method for preparing the compound of Formula I, which is Method I or Method II:

[0036] when Q is , the method is Method I; the Method I comprises the following steps: reacting a compound of Formula IV or a compound of Formula V with a compound P in the presence of a base in an organic solvent to obtain a compound of Formula I-U;

[0037] when Q is , the method is Method II; the Method II comprises the following steps: reacting a compound of Formula III with a compound P in the presence of a base in an organic solvent to obtain a compound of Formula I-S;

[0038] wherein R 1 and R 2 are as in any preceding scheme; R 3 is hydrogen or an electron-withdrawing substituent, such as a nitro group.

[0039] In a certain embodiment of the Method I, the base is an organic base, such as triethylamine.

[0040] In a certain embodiment of the Method I, the organic solvent is an ether solvent, such as tetrahydrofuran.

[0041] In a certain embodiment of the Method II, the base is an inorganic base, such as potassium carbonate.

[0042] In a certain embodiment of the Method II, the organic solvent is an alcohol solvent, such as methanol.

[0043] In a certain embodiment, the compound P is a compound P-1

[0044] The present application provides a compound P-1, a compound of formula GP-4, or a compound P-4:

[0045] wherein G is an amino protecting group, for example tert-butyloxycarbonyl (compound P-4). The relative configuration of the cyclohexyl 1,4 substituents in compound P-1, compound of formula GP-4, and compound P-4 are all trans.

[0046] The present application provides a pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0047] The present application provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating breast cancer.

[0048] In one aspect, the compound is

[0049] The present application provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating an eIF2a kinase related disease, for example breast cancer.

[0050] In one aspect, the compound is

[0051] The present application provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of an eIF2a kinase modulator; the eIF2a kinase modulator is an eIF2a kinase activator.

[0052] The present application provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of an anti-coronavirus medicament and / or an anti-orthopoxvirus medicament; the coronavirus is an alphacoronavirus, a betacoronavirus, a gammacoronavirus, or a deltacoronavirus.

[0053] In one aspect, the alphacoronavirus is porcine epidemic diarrhea virus (PEDV).

[0054] In one aspect, the betacoronavirus is mouse hepatitis virus (MHV).

[0055] In one aspect, the gammacoronavirus is infectious bronchitis virus (IBV).

[0056] In one aspect, the deltacoronavirus is porcine deltacoronavirus (PDCoV).

[0057] In certain embodiments, the orthopoxvirus is a taterapox virus (TTV).

[0058] In certain embodiments, the compound of Formula I is

[0059] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings. A particular term or phrase should not be construed to be indefinite or unclear in the absence of a specific definition, but should be interpreted in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0060] The term "alkyl" refers to saturated aliphatic hydrocarbon groups; for example, alkyl groups of 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and the like, as well as various branched isomers thereof, and the like.

[0061] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halogens. Non-limiting examples include trifluoromethyl.

[0062] The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0063] The term "pharmaceutically acceptable salt" means a salt of a compound of this application prepared from a pharmaceutically acceptable acid or base. If the compound of the application contains a relatively acidic moiety, e.g., a carboxylic acid group, base addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of the desired pharmaceutically acceptable base, either neat or in a suitable inert solvent. If the compound of the application contains a relatively basic moiety, e.g., a pyridine group, acid addition salts can be prepared by contacting the neutral form of such compounds with a sufficient amount of the desired pharmaceutically acceptable acid, either neat or in a suitable inert solvent. Salts of both basic and acidic moieties can be formed by using a suitable inert solvent that will dissolve either the base or the acid, but not both. See, e.g., 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).

[0064] The term "treatment" refers to therapeutic treatment. With respect to a particular condition, treatment refers to: (1) relieving the disease or condition, or one or more of the symptoms associated with the disease or condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more of the symptoms associated with the condition, (3) ameliorating 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 of the symptoms associated with the condition.

[0065] The term "prevention" refers to a reduction in the risk of acquiring or developing a disease or disorder.

[0066] The term "therapeutically effective amount" refers to the amount of a compound that, when administered to a patient, is sufficient to effect treatment for a disease or condition as described herein. The "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted by those skilled in the art as needed.

[0067] The term "pharmaceutically acceptable excipient" refers to an excipient and an additional agent used in the manufacture of pharmaceutical products and dispensing of prescriptions, and is all substance other than the active ingredient that is contained in a pharmaceutical preparation. See the People's Republic of China Pharmacopoeia (2020 Edition) Volume IV or Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009 Sixth Edition).

[0068] The term "patient" refers to any animal, preferably a mammal, and most preferably a human, to whom the compounds are administered according to the embodiments of the present application. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like, with humans being most preferred.

[0069] The above-mentioned preferred conditions can be combined in any manner without departing from the common general knowledge of the skilled person, i.e. to obtain preferred embodiments of the present application.

[0070] The above-mentioned preferred conditions can be combined in any manner without departing from the common general knowledge of the skilled person, i.e. to obtain preferred embodiments of the present application.

[0071] The reagents and materials used in the present application are commercially available.

[0072] The positive progress effect of the present application is that the compounds of the present application have a new mechanism of action, which can target host eIF2a kinase, eIF2a phosphorylation and protein synthesis. The compounds of the present application have excellent biological activity in treating breast cancer. In particular, the compounds SIPI-27 and SIPI-28 of the present application have significantly better inhibitory activity on breast cancer than the control compounds BTdCPU and neratinib; and / or, the compounds of the present application also have biological activity against coronaviruses and / or orthopoxviruses, in particular, the compound SIPI-25 of the present application has broad-spectrum anti-coronavirus activity against porcine epidemic diarrhea virus (PEDV), mouse hepatitis virus (MHV), infectious bronchitis virus (IBV) and porcine delta coronavirus (PDCoV), and also has activity against orthopoxvirus taterapox virus (TTV). Therefore, SIPI-25 has a broad-spectrum antiviral activity across two major categories of viruses, DNA and RNA. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 is the Western blotting data of compound SIPI-25 against PEDV.

[0074] Figure 2 is the Western blotting data of compound SIPI-25 against MHV.

[0075] Figure 3 is the Western blotting data of compound SIPI-25 against IBV.

[0076] Figure 4 is the Western blotting data of compound SIPI-25 against PDCoV.

[0077] Figure 5 is the RT-qPCR data of compound SIPI-25 against PEDV.

[0078] Figure 6 is the RT-qPCR data of compound SIPI-25 against MHV.

[0079] Figure 7 is the RT-qPCR data of compound SIPI-25 against IBV.

[0080] Figure 8 is the RT-qPCR data of compound SIPI-25 against PDCoV.

[0081] Figure 9 is the changes of protein synthesis and eIF2a phosphorylation in Vero cells after treatment with 35 mM concentration of SIPI-25 at different time points, Time refers to the time after treatment of the test compound. β-actin is used as the loading control. The intensity of Western blotting is quantified by ImageJ.

[0082] Figure 10 is the changes of protein synthesis and eIF2a phosphorylation in LLC-PK1 cells after treatment with 35 mM concentration of SIPI-25 at different time points, Time refers to the time after treatment of the test compound. β-actin is used as the loading control. The intensity of Western blotting is quantified by ImageJ.

[0083] Figure 11 is the changes of protein synthesis and eIF2a phosphorylation in Vero cells after treatment with 50 mM concentration of SIPI-27 at different time points, Time refers to the time after treatment of the test compound. β-actin is used as the loading control. The intensity of Western blotting is quantified by ImageJ.

[0084] Figure 12 is the changes of protein synthesis and eIF2a phosphorylation in Vero cells after treatment with 15 mM concentration of SIPI-28 at different time points, Time refers to the time after treatment of the test compound. β-actin is used as the loading control. The intensity of Western blotting is quantified by ImageJ. DETAILED DESCRIPTION

[0085] The present application is further illustrated by the following examples without limiting the present application to the examples described. The experimental methods in the following examples, if not specified, are selected according to the conventional methods and conditions, or according to the product instructions.

[0086] NMR was determined on a Bruker Avance III 400 MHz or a Bruker Avance Neo 600 MHz instrument. High resolution mass spectrometry was determined on a Thermo Scientific Q Exactive Plus instrument, electrospray ionization (ESI), positive ionization mode, using an external calibrant.

[0087] General preparation methods are described:

[0088] The relative configuration of the two substituents in the cyclohexyl 1,4-positions of the compounds of the present application does not affect the synthesis of the compounds of formula I. Thus, the preparation of the compounds of formula I is illustrated with the example of the compound of formula I-A.

[0089] Preparation of the key intermediate P-1. P-2 and P-3 with the relative configuration cis are subjected to a Mitsunobu reaction to give P-4 with the relative configuration trans inverted. P-4 is subjected to removal of the tert-butyloxycarbonyl (BOC) protecting group to give P-1.

[0090] The compound of formula II and P-1 are subjected to a urea-forming reaction to give the compound of formula I-AU. The compound of formula III and P-1 are subjected to a nucleophilic substitution reaction to give the compound of formula I-AS.

[0091] For the urea-forming reaction of the compound of formula II and P-1 to give the compound of formula I-AU, the compound of formula II is first activated. For example, the compound of formula II is first converted into the activated compound of formula IV (isocyanates); or the compound of formula II is first converted into the activated compound of formula V (phenyl carbamates), R 3 is hydrogen or an electron withdrawing substituent such as a nitro group.

[0092] For the preparation of the compound of formula I-AS from the compound of formula III and P-1, the compound of formula III can be prepared from the compound of formula II and P-5 by a nucleophilic substitution reaction.

[0093] In the above preparation methods, R 1 and R 2 as described in any of the previous options.

[0094] Example 1

[0095] Preparation of 1-(3-cyano-5-trifluoromethylphenyl)-3-((1r,4r)-4-(4- (dimethylphosphoryl)phenoxy)cyclohexyl)urea (SIPI-23)

[0096] P-2 (1.37 g, 6.36 mmol), P-3 (1.3 g, 7.64 mmol) and triphenylphosphine (2.51 g, 9.57 mmol) were added to dry tetrahydrofuran (THF) (15 mL) under room temperature and nitrogen protection, and a solution of diisopropyl azodicarboxylate (DIAD) (2 g, 9.89 mmol) in dry THF (10 mL) was added dropwise slowly, then stirred at room temperature for 24 h. The resulting reaction solution was rotary evaporated, and the resulting yellow oily residue was subjected to silica gel H column chromatography (dichloromethane:methanol, 20:0-20:1), ethyl acetate slurry and vacuum drying (80 °C, 2 h) in sequence to obtain white solid P-4 (0.82 g, 35%).

[0097] 1 H NMR (400 MHz, DMSO-d6) δ 7.64 (dd, J = 11.0, 8.6 Hz, 2H), 7.04 (dd, J = 8.8, 2.1 Hz, 2H), 6.83 (d, J = 7.7 Hz, 1H), 4.40 - 4.29 (m, 1H), 3.32 - 3.22 (m, 1H), 2.10 - 1.98 (m, 2H), 1.88 - 1.75 (m, 2H), 1.59 (d, J = 13.3 Hz, 6H), 1.46 - 1.39 (m, 2H), 1.38 (s, 9H), 1.36 - 1.31 (m, 2H).

[0098] 31 P NMR (162 MHz, DMSO-d6) δ 31.80.

[0099] A solution of trifluoroacetic acid (6.4 g, 56.1 mmol) in dry dichloromethane (10 mL) was added dropwise slowly to a solution of P-4 (0.8 g, 2.18 mmol) in dry dichloromethane (10 mL) under room temperature and nitrogen protection, then stirred at room temperature for 2.5 h. Saturated aqueous sodium carbonate solution (20 mL) was added to the resulting reaction solution, and dichloromethane (3 x 10 mL) was extracted. The combined organic phase was washed with saturated brine (15 mL) and dried over anhydrous sodium sulfate. Rotary evaporation and vacuum drying (65 °C, 2 h) gave yellow semi-solid P-1 (0.42 g, 72%).

[0100] 1H NMR (400 MHz, DMSO-d6) δ 7.65 (dd, J = 11.0, 8.6 Hz, 2H), 7.06 (dd, J = 8.8, 2.1 Hz, 2H), 4.42 - 4.33 (m, 1H), 3.05 - 2.83 (m, 1H), 2.13 - 2.05 (m, 2H), 1.95 - 1.87 (m, 2H), 1.59 (d, J = 13.2 Hz, 6H), 1.43 - 1.40 (m, 2H), 1.25 - 1.23 (m, 2H).

[0101] P-1 (0.26 g, 0.97 mmol) and triethylamine (2 g, 19.8 mmol) were dissolved in anhydrous THF (15 mL) under nitrogen protection, and a solution of IV-1 (0.25 g, 1.18 mmol) in anhydrous THF (10 mL) was added dropwise at room temperature, followed by stirring at reflux for 2 h. The resulting reaction solution was rotary evaporated, and the residue was dissolved in dichloromethane (10 mL), washed with water (20 mL) and saturated brine (20 mL) in turn, and dried over anhydrous sodium sulfate. Rotary evaporation was performed, and the resulting yellow oily residue was subjected to silica gel H column chromatography (dichloromethane:methanol, 15:1-10:1) and vacuum drying (80 °C, 2 h) to obtain white solid SIPI-23 (182 mg, 39%).

[0102] 1 H NMR (600 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.18 - 8.14 (m, 1H), 8.01 - 7.97 (m, 1H), 7.79 - 7.75, 7.65 (dd, J = 11.1, 8.6 Hz, 2H), 7.07 (dd, J = 8.7, 2.1 Hz, 2H), 6.57 (d, J = 7.5 Hz, 1H), 4.47 - 4.40 (m, 1H), 3.59 - 3.51 (m, 1H), 2.10 - 2.02 (m, 2H), 1.96 - 1.90 (m, 2H), 1.60 (d, J = 13.2 Hz, 6H), 1.52 - 1.45 (m, 2H), 1.44 - 1.37 (m, 2H).

[0103] 31 P NMR (243 MHz, DMSO-d6) δ 32.06.

[0104] 19 F NMR (565 MHz, DMSO-d6) δ -61.86.

[0105] ESI-HRMS: calcd for [M+H] +m / z: 480.16584; found, m / z: 480.16495; calcd for [M+Na] + m / z: 502.14778; found, m / z: 502.14673.

[0106] Example 2

[0107] Preparation of 3-((2-(((1r,4r)-4-(4-(dimethylphosphoryl)phenoxy)cyclohexyl)amino)-3,4- dioxocyclobut-1-en-1-yl)amino)-5-trifluoromethylbenzonitrile (SIPI-24)

[0108] A solution of II-1 (0.5 g, 2.69 mmol) in methanol (10 mL) was added slowly dropwise to a solution of P-5 (0.42 g, 2.96 mmol) in methanol (10 mL) at room temperature, and then stirred at room temperature for 10 h. The resulting reaction solution was rotary evaporated, and the resulting yellow solid residue was recrystallized with methanol and dried in vacuum (110 °C, 8 h) to give III-1 (0.32 g, 40%) as a yellow solid.

[0109] 1 H NMR (400 MHz, DMSO-d6) d 11.19 (s, 1H), 8.05 - 8.02 (m, 1H), 8.02 - 7.97 (m, 2H), 4.41 (s, 3H).

[0110] A solution of III-1 (0.32 g, 1.08 mmol) in methanol (10 mL) was added slowly dropwise to a suspension of P-1 (0.26 g, 0.97 mmol) and potassium carbonate (1.37 g, 9.9 mmol) in methanol (10 mL) at room temperature, and then stirred at room temperature for 12 h. The resulting reaction suspension was filtered, and the filter cake was washed with methanol, and the filtrate was rotary evaporated. The resulting residue was successively column chromatographed on silica gel H (dichloromethane:methanol, 10:0 - 10:1), recrystallized from ethyl acetate, and dried in vacuum (110 °C, 3 h) to give SIPI-24 (0.2 g, 39%) as a white solid.

[0111] 1H NMR (400 MHz, DMSO-d6) δ 10.11 (br s, 1H), 8.12 - 8.05 (m, 1H), 8.03 - 8.00 (m, 1H), 7.95 - 7.86 (m, 2H), 7.67 (dd, J = 11.0, 8.6 Hz, 2H), 7.10 (dd, J = 8.8, 2.1 Hz, 2H), 4.54 - 4.43 (m, 1H), 4.03 - 3.92 (m, 1H), 2.14 - 2.02 (m, 4H), 1.61 (d, J = 13.3 Hz, 6H), 1.58 - 1.45 (m, 4H).

[0112] 31 P NMR (162 MHz, DMSO-d6) δ 32.93.

[0113] 19 F NMR (376 MHz, DMSO-d6) δ -61.86.

[0114] ESI-HRMS: calcd for [M+H] + , m / z: 532.16075; found, m / z: 532.16025; calcd for [M+Na] + , m / z: 554.14270; found, m / z: 554.14212.

[0115] Example 3

[0116] Preparation of 1-(3-tert-butylphenyl)-3-((1r,4r)-4-(4-(dimethylphosphoryl)phenoxy)cyclohexyl)urea (SIPI-25)

[0117] P-1 (0.318 g, 1.19 mmol) and triethylamine (2.4 g, 23.7 mmol) were dissolved in anhydrous THF (10 mL) under nitrogen protection, and a solution of IV-2 (0.25 g, 1.4 mmol) in anhydrous THF (15 mL) was slowly added dropwise at room temperature, and then stirred at reflux for 2.5 h. The resulting reaction solution was rotary evaporated, water (15 mL) was added to the residue, and ethyl acetate (3 x 10 mL) was extracted. The organic phase was combined, washed with saturated brine (10 mL), and dried over anhydrous sodium sulfate. The resulting yellow oily residue was column chromatographed on silica gel H (dichloromethane:methanol, 10:0-10:1), recrystallized from ethyl acetate, and vacuum dried (80 °C, 2 h) to obtain white solid SIPI-25 (0.22 g, 42%).

[0118] 1H NMR (600 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.66 (t, J = 9.6 Hz, 2H), 7.40 (s, 1H), 7.21 - 7.16 (m, 1H), 7.13 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 7.7 Hz, 1H), 6.09 (d, J = 7.6 Hz, 1H), 4.49 - 4.41 (m, 1H), 3.56 - 3.49 (m, 1H), 2.08 - 2.01 (m, 2H), 1.96 - 1.89 (m, 2H), 1.60 (d, J = 13.2 Hz, 6H), 1.52 - 1.45 (m, 2H), 1.39 - 1.32 (m, 2H), 1.24 (s, 9H).

[0119] 31 P NMR (243 MHz, DMSO-d6) δ 31.99.

[0120] ESI-HRMS: calcd for [M+H] + , m / z: 443.24581; found, m / z: 443.24562; calcd for [M+Na] + , m / z: 465.22775; found, m / z: 465.22704.

[0121] Example 4

[0122] 3-((3-tert-Butylphenyl)amino)-4-(((1r,4r)-4-(4-(dimethylphosphoryl)phenoxy)cyclohexyl)amino)cyclobut-3-ene-1,2-dione (SIPI-26)

[0123] A solution of II-2 (0.5 g, 3.35 mmol) in methanol (10 mL) was added slowly dropwise to a solution of P-5 (0.523 g, 3.68 mmol) in methanol (10 mL) at room temperature, then stirred at room temperature for 12 h. The resulting reaction suspension was filtered, the filter cake was washed with methanol, and dried under vacuum (110 °C, 2 h) to give white solid III-2 (0.6 g, 69%).

[0124] 1 H NMR (400 MHz, DMSO-d6) δ 10.71 (br s, 1H), 7.44 (br s, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.16 - 7.10 (m, 2H), 4.37 (s, 3H), 1.27 (s, 9H).

[0125] P-1 (0.515 g, 1.93 mmol) and potassium carbonate (2.66 g, 19.0 mmol) were added to methanol (10 mL), a solution of III-2 (0.6 g, 2.31 mmol) in methanol (10 mL) was added dropwise slowly at room temperature, and then stirred at room temperature for 12 h. The resulting reaction suspension was filtered, the filter cake was washed with methanol, and the filtrate was rotary evaporated. The resulting residue was successively subjected to silica gel H column chromatography (dichloromethane:methanol, 10:0-10:1) and vacuum drying (110 °C, 3 h) to give off-white solid SIPI-26 (0.31 g, 32%).

[0126] 1 H NMR (400 MHz, DMSO-d6) δ 9.64 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.68 (dd, J = 11.1, 8.6 Hz, 2H), 7.55 - 7.50 (m, 1H), 7.27 - 7.20 (m, 2H), 7.10 (dd, J = 8.8, 2.1 Hz, 2H), 7.05 (dt, J = 7.1, 1.8 Hz, 1H), 4.53 - 4.44 (m, 1H), 4.01 - 3.88 (m, 1H), 2.08 (dd, J = 15.6, 9.9 Hz, 4H), 1.62 (d, J = 13.2 Hz, 6H), 1.56 - 1.46 (m, 4H), 1.28 (s, 9H).

[0127] ESI-HRMS: calcd for [M+H] + , m / z: 495.24072; found, m / z: 495.23962; calcd for [M+Na] + , m / z: 517.22267; found, m / z: 517.22166.

[0128] Example 5

[0129] 1-(3,4-Dichlorophenyl)-3-((1r,4r)-4-(4-(dimethylphosphoryl)phenoxy)cyclohexyl)urea (SIPI-27) Preparation

[0130] P-1 (0.237 g, 0.886 mmol) and triethylamine (0.9 g, 8.86 mmol) were dissolved in anhydrous THF (15 mL) under nitrogen protection, and a solution of IV-3 (0.2 g, 1.06 mmol) in anhydrous THF (10 mL) was slowly added dropwise at room temperature, and then stirred at reflux for 2 h. To the resulting reaction solution was added 1 mol / L aqueous hydrochloric acid (30 mL), and extracted with dichloromethane (3 x 15 mL). The combined organic phase was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. The resulting yellow oily residue was column chromatographed on silica gel H (dichloromethane:methanol, 10:0-10:1), recrystallized from ethyl acetate, and vacuum dried (100 °C, 8 h) to obtain white solid SIPI-27 (0.16 g, 39%).

[0131] 1 H NMR (600 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.83 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 11.0, 8.6 Hz, 2H), 7.44 (d, J = 8.8 Hz, 1H), 7.22 (dd, J = 8.8, 2.6 Hz, 1H), 7.09 - 7.02 (m, 2H), 6.30 (d, J = 7.6 Hz, 1H), 4.47 - 4.40 (m, 1H), 3.56 - 3.47 (m, 1H), 2.07 - 2.02 (m, 2H), 1.95 - 1.89 (m, 2H), 1.59 (d, J = 13.2 Hz, 6H), 1.51 - 1.44 (m, 2H), 1.41 - 1.34 (m, 2H).

[0132] ESI-HRMS: calcd for [M+H] + , m / z: 455.10526; found, m / z: 455.10450; calcd for [M+Na] + , m / z: 477.08721; found, m / z: 477.08633.

[0133] Example 6

[0134] Preparation of 3-((3,4-dichlorophenyl)amino)-4-(((1r,4r)-4-(4- (dimethylphosphoryl)phenoxy)cyclohexyl)amino)cyclobut-3-ene-1,2-dione (SIPI-28)

[0135] A solution of II-3 (1.0 g, 6.17 mmol) in methanol (10 mL) was added slowly dropwise to a solution of P-5 (0.964 g, 6.78 mmol) in methanol (10 mL) at room temperature, and then stirred at room temperature for 12 h. The resulting reaction suspension was filtered, the filter cake was washed with methanol, and dried under vacuum (110 °C, 3 h) to give yellow solid III-3 (1.2 g, 72%).

[0136] 1 H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 7.65 (d, J = 2.6 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.33 (dd, J = 8.8, 2.7 Hz, 1H), 4.39 (s, 3H).

[0137] P-1 (0.49 g, 1.83 mmol) and potassium carbonate (2.54 g, 18.4 mmol) were added to methanol (10 mL), and a solution of III-3 (0.6 g, 2.2 mmol) in methanol (10 mL) was added slowly dropwise at room temperature, and then stirred at room temperature for 12 h. The resulting reaction suspension was filtered, the filter cake was washed with methanol, and the filtrate was rotary evaporated. The resulting residue was successively column chromatographed on silica gel H (dichloromethane:methanol, 10:0 - 10:1), recrystallized from ethyl acetate, and dried under vacuum (110 °C, 3 h) to give off-white solid SIPI-28 (0.3 g, 32%).

[0138] 1 H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 7.89 (br d, J = 7.5 Hz, 1H), 7.86 (br s, 1H), 7.66 (dd, J = 11.2, 8.5 Hz, 2H), 7.57 (d, J = 8.7 Hz, 1H), 7.31 (dd, J = 8.8, 2.7 Hz, 1H), 7.09 (dd, J = 8.8, 2.1 Hz, 2H), 4.57 - 4.41 (m, 1H), 3.98 - 3.93 (m, 1H), 2.15 - 2.02 (m, 4H), 1.60 (d, J = 13.3 Hz, 6H), 1.58 - 1.44 (m, 4H).

[0139] ESI-HRMS: calcd for [M+H] + , m / z: 507.10018; found, m / z: 507.09971; calcd for [M+Na] + , m / z: 529.08212; found, m / z: 529.08162.

[0140] Example 1

[0141] In vitro anti-tumor cell proliferation activity of the test compounds was determined by CCK-8 method

[0142] Human breast cancer cell MCF-7 was purchased from American Type Culture Collection (ATCC). MCF-7 cell culture medium was EMEM (Cat. No. 320-006-CL, purchased from WISENT) containing 10% fetal bovine serum (FBS) and 50 U / mL penicillin-streptomycin. Cell culture was performed in a cell culture incubator at 37°C and 5% carbon dioxide. CCK-8 cell viability assay reagent (Cat. No. B34302) was purchased from Selleck.

[0143] MCF-7 cells were seeded in 384-well cell culture plates at 1500 cells per well and incubated for 24 h. Different concentrations of test compounds (starting concentration of 10 mM, 3-fold serial dilution) were added for 72 h, then 2.5 pL of CCK-8 reagent was added to each well and incubated for 1.5 h. The absorbance value (OD) of each well was measured at 450 and 650 nm using a microplate reader. The cell survival rate of each well was calculated as V% = (A s –A c ) / (A b –A c ) x 100%, where A s is the corrected absorbance value (OD 450 – OD 650 ) of the compound test well, A c is the corrected absorbance value of the well without cells and test compound (blank control), and A b is the corrected absorbance value of the well with cells and vehicle only. Each experiment was set up in duplicate and repeated independently twice. The data were processed using Graphpad Prism 7 software, and the average of the half maximal inhibitory concentration (IC 50 ) values from two independent experiments was taken. The in vitro anti-tumor cell activity data are shown in Table 1.

[0144] Table 1. In vitro activity against human breast cancer cell MCF-7

[0145] The positive control eIF2a kinase HRI activator BTdCPU was synthesized according to the literature (Ting Chen et al. Nature Chemical Biology. 2011, 7(9): 610-616). The positive control eIF2a kinase GCN2 activator neratinib (Colin P. Tang et al. Nature Chemical Biology. 2022, 18(2): 207-215) (Cat. No. N126132) was purchased from Selleck.

[0146] Example 2

[0147] In vitro anti-coronavirus activity of the test compounds was determined by CCK-8 method

[0148] African green monkey kidney cells Vero and pig kidney epithelial cells LLC-PK1 were preserved in the Shanghai Institute of Animal Husbandry, Chinese Academy of Agricultural Sciences, and mouse hepatocytes NCTC 1469 were purchased from Wuhan Punsun Life Science and Technology Co., Ltd. The Vero cell culture solution was DMEM containing 10% FBS (item number 12677019, Gibco, purchased from Thermo Fisher Scientific), the NCTC 1469 cell culture solution was DMEM containing 10% horse serum (HS), and the LLC-PK1 cell culture solution was MEM containing 10% FBS (item number 11095-080, Gibco, purchased from Thermo Fisher Scientific), all containing 100 U / mL penicillin-streptomycin. Cell culture was carried out in a cell culture incubator at 37°C and 5% carbon dioxide. The cell maintenance solution was DMEM containing 2% FBS for Vero cells, MEM containing 2% FBS for LLC-PK1 cells, and DMEM containing 2% HS for NCTC 1469 cells.

[0149] Porcine epidemic diarrhea virus (PEDV), mouse hepatitis virus (MHV), infectious bronchitis virus (IBV), and porcine delta coronavirus (PDCoV) strains were preserved in the Shanghai Institute of Animal Husbandry, Chinese Academy of Agricultural Sciences.

[0150] The CCK-8 (Cell Counting Kit-8) cell proliferation and toxicity detection kit (item number NBS1157) was purchased from Shanghai Nonin Biological Technology Co., Ltd.

[0151] In Vero cells, the cytotoxicity (half cytotoxic concentration, CC 50 ) of the test compounds was determined, as well as the antiviral activity (half effective concentration, EC 50 ) of the test compounds against PEDV and IBV, and the selectivity index (SI) (SI = CC 50 / EC 50 ) was calculated. In NCTC 1469 cells, the CC 50 of the test compounds was determined, as well as the EC 50 of the test compounds against MHV, and the SI was calculated. In LLC-PK1 cells, the CC 50 of the test compounds was determined, as well as the EC 50 of the test compounds against PDCoV, and the SI was calculated. The in vitro anti-PEDV, MHV, IBV, and PDCoV activity data of the test compounds determined by the CCK-8 method are shown in Tables 2 to 5, CC 50 and EC50 All are mean ± standard deviation.

[0152] Half maximal inhibitory concentration (IC 50 ) assay: Cells grown into a dense monolayer were trypsinized into single cells, and 5 x 104 5 cells / well of cell suspension were added into 96-well cell culture plates in 100 μL per well, and incubated at 37 °C in a cell culture incubator with 5% carbon dioxide. After the cells grew into a monolayer, the test was performed. The test compound was dissolved with dimethyl sulfoxide (DMSO) to prepare a 20 mM stock solution. The highest concentration was 1000 μM, which was diluted to 8 concentrations by the corresponding cell maintenance solution. The original culture solution in the aforementioned 96-well plate was discarded, and washed twice with phosphate buffered saline (PBS). Different concentrations of the prepared test compound were added to each well in 100 μL, 3 replicates were set for each concentration, and 3 independent experiments were set as the compound group. A negative control group (0.1% DMSO was added) and a blank control group (without cells) were also set. After incubation at 37 °C in a cell culture incubator with 5% carbon dioxide for 72 h, the growth of the cells was observed. The culture solution was discarded, and washed twice with PBS. CCK-8 reagent (diluted to 0.1 mL / mL) was added to each well, and incubated at 37 °C for 1 h. The absorbance value (OD) of each well was measured at 450 nm using a microplate reader. The half maximal cytotoxic concentration (IC 50 ) of the test compound was calculated according to IC 50 = (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 IC 50 .

[0153] Half maximal effective concentration (EC 50 ) assay: The corresponding IC 50For the highest concentration, the test compound DMSO stock solution (20 mM) was serially diluted to 6 concentrations with the corresponding cell maintenance medium. The cells grown in monolayer in 96-well cell culture plates were inoculated with 100 μL of virus solution (multiplicity of infection (MOI) = 1) per well, and incubated for 1 h. The supernatant was discarded, and the prepared test compound at different concentrations (100 μL) was added, with 3 replicates for each concentration, and 3 independent experiments, set as the compound plus virus group. Another cell control group (with cells and 0.1% DMSO) and virus control group (with virus, cells and 0.1% DMSO) were set. After incubation at 37°C in a cell culture incubator with 5% carbon dioxide for 48 h, the supernatant was discarded, and the cells were washed twice with PBS, and CCK-8 reagent (diluted to 0.1 mL / mL) was added to each well (Vero cells, NCTC 1469 cells and LLC-PK1 cells were diluted with the corresponding cell maintenance medium), and incubated at 37°C for 1 h, and then the OD of each well was measured at 450 nm using a microplate reader. The half maximal effective concentration (EC 50 ) of the test compound against the virus was calculated according to EC 50 = (A4–A5) / (A6-A5) x 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 the EC 50 was calculated.

[0154] Table 2. Anti-PEDV activity of the compounds in Vero cells

[0155] Table 3. Anti-MHV activity of the compounds in NCTC-1469 cells

[0156] Table 4. Anti-IBV activity of the compounds in Vero cells

[0157] Table 5. Anti-PDCoV activity of the compounds in LLC-PK1 cells

[0158] Effect Example 3

[0159] In vitro anti-orthopoxvirus TTV activity of the test compounds was determined by the plaque method

[0160] African green monkey kidney cells Vero were preserved by the China CDC. Orthopoxvirus TTV-EGFP was preserved by the China CDC. The in vitro activity of the test compounds against TTV-EGFP was determined in Vero cells.

[0161] Cell cytotoxicity (CC 50 ) of the test compounds was determined by CCK-8 method. 50 Anti-TTV-EGFP activity (EC 50 ) of the test compounds was determined by plaque assay. 50 The selectivity index (SI) was calculated (SI = CC 50 / EC 4 ). The in vitro anti-TTV-EGFP activity data of the test compounds are shown in Table 6.

[0162] Half cytotoxic concentration (CC 50 ) determination: Vero cells were seeded at 2 x 105cells / well in 96-well plates and cultured overnight in DMEM medium containing 10% FBS, then replaced with DMEM medium containing 2% FBS. Dimethyl sulfoxide solution (20 mM) of the test compounds was serially diluted with DMEM containing 2% FBS to a total of 6 concentrations, added to the cells, and 3 replicate wells were set for each concentration, with blank control and cell control groups. Continue to culture in a 37°C and 5% carbon dioxide cell incubator for 72 h, then discard the supernatant, add CCK-8 working solution (Dojindo, item number CK04-1000T) (100 μL) to each well, incubate at 37°C for 1 to 4 h, and use a multifunctional enzyme label instrument to determine the absorbance value (OD) at 450 nm. Calculate according to the formula: cell activity inhibition rate (%) = (drug group - blank control group) / (cell control group - blank control group) x 100%. Use GraphPad Prism 8 (GraphPad Software, Boston, MA, USA) for data processing to calculate CC 50 .

[0163] Half effective concentration (EC 50 ) determination of anti-TTV-EGFP: EC 50 of anti-TTV-EGFP was determined by plaque assay. Vero cells were treated with serially diluted compounds for 2 h, then TTV-EGFP (100 PFU / well) was added and cultured at 37°C for 72 h. Fix the cells with 4% paraformaldehyde solution for 30 min, then stain with 0.1% crystal violet for 15 min to show plaques.

[0164] Table 6. Anti-TTV-EGFP activity of compounds in Vero cells

[0165] Effect Example 4

[0166] Determination of in vitro anti-coronavirus activity of test compounds by Western blotting

[0167] Cells and viruses refer to the effect of Example 2.

[0168] The PEDV N protein antibody (product number SD17-103) is a product of Medgene, the MHV nsp 9 protein antibody (product number 200-301-A56) is a product of Rockland Immunochemicals, the IBV N protein antibody is a gift from Professor Liu Dingxiang of South China Agricultural University, the PDCoV N protein antibody (product number PDCOV11-M) is a product of Alpha Diagnostic International, and the β-actin antibody (product number 66009-1-Ig) is a product of Proteintech. The horseradish peroxidase (HRP) labeled goat anti-mouse immunoglobulin G (product number 115-035-003) and goat anti-rabbit immunoglobulin G (product number 111-035-003) are products of Jackson ImmunoResearch Laboratories. The lumiQ universal ECL luminescent solution (product number SB-WB012) is a product of Sainter.

[0169] The CC 50 The dimethyl sulfoxide stock solution (20 mM) of the test compound was diluted into four concentrations with the corresponding cell maintenance fluid. 5×10 5Cell suspension was added to 12-well cell culture plates at 100 μL per well. After the cells grew into a monolayer, 100 μL of virus liquid (PEDV, IBV and PDCoV, MOI = 1; MHV, MOI = 0.1) was inoculated in each well, and incubated in a 37°C and 5% carbon dioxide cell incubator for 1.5 h. The supernatant was discarded, and different concentrations of the prepared test compounds (1 mL) were added, while setting up cell control groups (no virus, loading body) and virus control groups (with virus, loading body), and incubated in a 37°C and 5% carbon dioxide cell incubator for 12 h. The supernatant was discarded, and the cells were washed with pre-cooled PBS 3 times. After the PBS was discarded, RIPA buffer (150 μL) was added to each well to lyse the cells, and transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was mixed with 5x SDS protein loading buffer (37.5 μL), and denatured at 100°C metal bath for 10 min. The supernatant was subjected to protein electrophoresis, except that 15% SDS-PAGE protein electrophoresis was used for MHV nsp 9 and its precursor protein, and 10% SDS-PAGE protein electrophoresis was used for other proteins. Then, it was 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 PVDF membrane was washed with TBST on a shaker for 3 times. Anti-PEDV N protein antibody (1:2000 dilution), anti-MHV nsp 9 protein antibody (1:1000 dilution), anti-IBV N protein antibody (1:5000 dilution), anti-PDCoV N protein antibody (1:2000 dilution), and β-actin antibody (1:10000 dilution) were used as primary antibodies, and incubated at 4°C overnight. After incubation, the PVDF membrane was removed and washed with TBST on a shaker for 3 times. The PVDF membrane incubated with MHV nsp 9 protein antibody and PDCoV N protein antibody was incubated with HRP-labeled goat anti-mouse immunoglobulin G (1:8000 dilution) as a secondary antibody, and the PVDF membrane incubated with PEDV N protein antibody, IBV N protein antibody, and β-actin antibody was incubated with HRP-labeled goat anti-rabbit immunoglobulin G (1:8000 dilution) as a secondary antibody, and incubated at room temperature for 1 h, and then washed with TBST on a shaker for 3 times. The PVDF membrane was incubated in lumiQ universal ECL luminescent liquid for 20 s, and then the expression level of the corresponding protein was detected on a full-automatic chemiluminescence image analysis system (Shanghai Tianni, Tanon5200 Multi). The Western blotting intensity was quantified by ImageJ. After treatment with different concentrations of various test compounds, the expression of PEDV N protein and IBV N protein in Vero cells, MHV nsp 9 and its precursor protein in NCTC 1469 cells, and PDCoV N protein in LLC-PK1 cells were determined by Western blot, as shown in FIGS. 1 to 4.

[0170] Example 5

[0171] The in vitro anticoronavirus activity of the test compounds was determined by RT-qPCR (real-time quantitative PCR).

[0172] For cells and viruses, see Effect Example 2.

[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 the appropriate cell maintenance medium. 5 × 10⁻⁶ 5 Cell suspension was added to 12-well cell culture plates at 100 μL per well. After the cells grew into a monolayer, 100 μL of virus solution (MOI = 1) was inoculated into each well, and the cells were incubated at 37°C with 5% CO2 for 1 h. The supernatant was discarded, and 1 mL of the prepared test compound at different concentrations was added. The cells were then incubated at 37°C with 5% CO2 for 12 h. Viral RNA was extracted according to the TransZol Up kit (TransZol, catalog number ET111-01-V2) manufacturer's instructions. The One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, catalog number AE311-02) was used to reverse transcribe the extracted RNA according to the instructions. Quantification was performed using the Green miRNA Two-Step qRT-PCR SuperMix Kit (TransGold, catalog number AQ202-01) according to the manufacturer's instructions. Primers used for RT-qPCR experiments are shown in Table 7. RT-qPCR data for SIPI-25 against PEDV, MHV, IBV, and PDCoV are shown in Figures 5 to 8.

[0174] Table 7 Primers used for RT-qPCR experiments Note: The primer sequences for glyceraldehyde-3-phosphate dehydrogenase for PEDV and IBV are GAPDH-F and GAPDH-R, for MHV they are M-GAPDH-F and M-GAPDH-R, and for PDCoV they are β-actin primer sequences β-actin-F and β-actin-R. Statistical analysis was performed using GraphPad Prism 8. Data are presented as mean ± standard deviation of three independent experiments. **** indicates p < 0.0001.

[0175] Example 6

[0176] Changes in host cell protein synthesis and eIF2α phosphorylation after administration of the test compound

[0177] Cells and viruses refer to the effect of Example 2.

[0178] Puromycin (Cat. No. X2370-1) was purchased from Inovogen. Puromycin antibody (Cat. No. MABE343, Sigma-Aldrich) was purchased from Merck. eIF2a antibody (Cat. No. 9722S) and phospho-eIF2a antibody (Cat. No. 9721S) were purchased from CST. b-actin antibody (Cat. No. 66009-1-Ig) was purchased from Proteintech. HRP-labeled goat anti-mouse immunoglobulin G (Cat. No. 115-035-003) and goat anti-rabbit immunoglobulin G (Cat. No. 111-035-003) were purchased from Jackson ImmunoResearch Laboratories.

[0179] Vero cells or LLC-PK1 cells were seeded into 6-well plates and cultured at 37°C and 5% carbon dioxide in a cell incubator to a cell density of 80-90%. The culture solution was discarded, and dimethyl sulfoxide stock solutions (20 mM) of compounds SIPI-25, SIPI-27, and SIPI-28 were diluted to 35 μM, 50 μM, and 15 μM, respectively, with the corresponding cell maintenance solution to a volume of 2 mL per well; 2 μg / mL of a puromycin solution diluted with the corresponding cell maintenance solution was added to label the cells for 1 h at 3, 7, 11, 17, and 23 h after administration, and the cells were collected at 4, 8, 12, 18, and 24 h after administration. After the PBS was discarded, the cells were lysed by adding RIPA buffer (150 μL) per well, and the lysate was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 5 min. The supernatant was mixed with 5x SDS protein loading buffer (37.5 μL), and the mixture was denatured at 100°C for 10 min. 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% non-fat milk at room temperature for 2 h. After blocking, the PVDF membrane was washed with TBST on a shaker for 3 times. Puromycin antibody (1:2000 dilution), P-eIF2α antibody (1:2000 dilution), T-eIF2α antibody (1:2000 dilution), and β-actin antibody (1:10000 dilution) were used as primary antibodies, and the PVDF membrane was incubated at 4°C overnight. After incubation, the PVDF membrane was removed and washed with TBST on a shaker for 3 times. The PVDF membrane incubated with puromycin antibody was incubated with HRP-labeled goat anti-mouse immunoglobulin G diluted 1:8000 as a secondary antibody, and the PVDF membrane incubated with P-eIF2α antibody, T-eIF2α antibody, and β-actin antibody was incubated with HRP-labeled goat anti-rabbit immunoglobulin G diluted 1:8000 as a secondary antibody at room temperature for 1 h, and then washed with TBST on a shaker for 3 times. The PVDF membrane was incubated in ECL luminescent developing solution for 20 s, and then detected for development on an automatic chemiluminescence image analysis system (Shanghai Tianyi, Tanon 5200 Multi). The results of the puromycin labeling experiment and eIF2α phosphorylation determined by Western blotting after treatment of Vero cells or LLC-PK1 cells with compounds SIPI-25, SIPI-27, or SIPI-28 are shown in FIG. 9 (Vero cells), FIG. 10 (LLC-PK1 cells), FIG. 11 (Vero cells), and FIG. 12 (Vero cells).

Claims

1. A compound of Formula I or a pharmaceutically acceptable salt thereof, wherein R 1 is cyano, halogen, C 1-6 alkyl or C 1-6 haloalkyl; R 2 haloalkyl; and n is 1 or 2. 1-6 haloalkyl; and n is 1 or 2. Q is 2. A compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, wherein, R 1 is cyano, halogen or C 1-6 haloalkyl; R 2 is cyano, halogen or C 1-6 haloalkyl; Q is 3. The compound of formula I according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein which satisfies at least one of the following conditions (1)-(4): (1) R 1 is halogen, preferably chlorine; (2) R 2 halogen, preferably chlorine; (3) R 1 and R 2 are the same; (4) For preferably 4. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein which satisfies at least one of the following conditions (1)-(3): (1) R 1 is C 1-6 alkyl, preferably tert-butyl; (2)R 2 It is hydrogen; (3) For preferably 5. The compound of formula I according to claim 1 or 2, wherein the compound is ###0002### or a pharmaceutically acceptable salt thereof. which satisfies at least one of the following conditions (1)-(3): (1) 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, preferably methyl; (2) said C 1-6 The halogen in haloalkyl is independently fluorine, chlorine, bromine or iodine, preferably fluorine, again preferably said C 1-6 The haloalkyl is trifluoromethyl. (3) the C 1-6 alkyl is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl, preferably t-butyl.

6. The compound of formula I according to claim 1 or 2, wherein the compound is ###0002### or a pharmaceutically acceptable salt thereof. which satisfies at least one of the following conditions (1)-(3): (1) the relative configuration of the two substituents in the 1,4-positions of the cyclohexyl group is trans or cis; preferably the relative configuration is trans; wherein R 1 , R 2 and Q are as described in any one of claims 1 to 5; (2) the compound of Formula I is a compound of Formula I-U or a compound of Formula I-S: wherein R 1 and R 2 as in any of claims 1-5; (3) the compound of Formula I is a compound of Formula I-AU, a compound of Formula I-AS, a compound of Formula I-BU, or a compound of Formula I-BS; preferably a compound of Formula I-AU or a compound of Formula I-AS; wherein R 1 , R 2 and Q are as described in any one of claims 1 to 5.

7. The compound of claim 1 or pharmaceutically acceptable salt thereof, wherein The compound of Formula I is selected from any one of the following:

8. A process for the preparation of a compound of formula I according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, characterized in that, which is the method one or the method two: When Q is In some embodiments, the preparation method is Method I; the Method I comprises the following steps: the compound of formula IV or the compound of formula V reacts with the compound P in the presence of a base in an organic solvent to obtain the compound of formula I-U; When Q is In some embodiments, the preparation method is Method Two; the Method Two comprises the following steps: the compound of formula III reacts with compound P in an organic solvent in the presence of a base to obtain the compound of formula I-S; wherein R 1 and R 2 as in any of claims 1-7; R 3 is hydrogen or an electron withdrawing substituent; Preferably, the preparation method satisfies at least one of the following conditions (1)-(6): (1) in the method one, the base is an organic base, preferably triethylamine; (2) in the method one, the organic solvent is an ether solvent, preferably tetrahydrofuran; (3) in the method two, the base is an inorganic base, preferably potassium carbonate; (4) in the method two, the organic solvent is an alcohol solvent, preferably methanol; (5) R 3 is hydrogen or nitro; (6) the compound P is compound P-1 9. The compound P-1 or a compound of formula GP-4: wherein, G is an amino protecting group, preferably the compound of formula GP-4 is compound P-4 10. A pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, and a pharmaceutically acceptable excipient.

11. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-7 in the preparation of a medicament for treating breast cancer.

12. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-7 in the preparation of a medicament for treating an eIF2a kinase-related disease, preferably breast cancer.

13. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1-7 in the preparation of an anti-coronavirus medicament or an anti-orthopoxvirus medicament, the coronavirus is an alphacoronavirus, a betacoronavirus, a gammacoronavirus, or a deltacoronavirus; Preferably, the use satisfies at least one of the following conditions (1)-(5): (1) the alphacoronavirus is porcine epidemic diarrhea virus; (2) the betacoronavirus is mouse hepatitis virus; (3) the gammacoronavirus is infectious bronchitis virus; (4) the deltacoronavirus is porcine deltacoronavirus; (5) the orthopoxvirus is taterapox virus.

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