Bunyavirus inhibitor and its preparation method and use

Abstract

This application relates to a compound represented by Formula I, and its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, hydrates, polymorphs, prodrugs, or isotope-labeled compounds. These compounds can be used as bunyavirus inhibitors to prepare medicaments for preventing and / or treating bunyavirus infection or a disease caused by a bunyavirus infection in a subject, or for inhibiting the replication or reproduction of a bunyavirus in a subject. #imgabs0#.

Description

Technical Field

[0001] The present application relates to a Bunyavirus inhibitor and a preparation method and use thereof. Background Art

[0002] Bunyaviruses are a group of negative-sense, segmented, single-stranded RNA viruses encompassing nine families, 13 genera, and over 500 species. In my country, the main prevalent Bunyaviruses are Crimean-Congo hemorrhagic fever virus (CCHFV) and severe fever with thrombocytopenia syndrome virus (SFTSV). Infection with CCHFV and SFTSV causes severe hemorrhagic fever with a mortality rate of 10%-40%. Both viruses pose a significant threat to human health and socioeconomic security. The World Health Organization has designated CCHF and SFTSV as high-priority diseases requiring high-risk research, and the development of vaccines and treatments is urgently needed. Summary of the Invention

[0003] The first aspect of the present application provides a compound represented by formula I, a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, hydrate, polymorph, prodrug or isotope-labeled compound thereof, wherein R1, R2, R3, R4, and R5 are each independently selected from the group consisting of: H, F, Cl, Br, I, cyano, hydroxyl, nitro, C 1-4 Alkyl, halogenated C 1-6 Alkyl, -O-(C 1-6 alkyl), -NH2, -NH-(C 1-6 alkyl), -N-(C 1-6 Alkyl)2, -C(O)(C 1-6 alkyl), -OC(O)( C 1-4 alkyl), -C(O)O(C 1-4 alkyl) and -CO2H, or

[0004] R1, R4, and R5 are each independently H, and R2 and R3 together form -O-(CH2)nO-, wherein n is 1, 2 or 3.

[0005] In certain embodiments, R1, R2, R3, R4, and R5 in Formula I are each independently selected from the group consisting of: H, F, Cl, Br, I, cyano, hydroxy, nitro, C 1-4 Alkyl, halogenated C 1-4 Alkyl, -O-(C 1-4 alkyl), -NH2, -NH-(C 1-4alkyl), -N-(C 1-4 Alkyl)2, -C(O)(C 1-4 alkyl), -OC(O)( C 1-4 alkyl), -C(O)O(C 1-4 alkyl) and -CO2H.

[0006] In certain embodiments, R1, R2, R3, R4, and R5 in Formula I are not H at the same time.

[0007] In certain embodiments, R1, R2, R3, R4, and R5 in Formula I are each independently selected from the group consisting of: H, F, Cl, Br, I, cyano, hydroxy, nitro, C 1-4 Alkyl, halogenated C 1-4 Alkyl and -O-(C 1-4 alkyl).

[0008] In certain embodiments, in Formula I, R1 is H, R4 is H, and R2, R3, and R5 are each independently selected from the group consisting of: H, F, Cl, Br, I, cyano, hydroxy, nitro, C 1-4 Alkyl, halogenated C 1-4 Alkyl and -O-(C 1-4 alkyl).

[0009] In certain embodiments, in Formula I, R1 is H, R2 is H, R4 is H, and R3 and R5 are each independently selected from the group consisting of: H, F, Cl, Br, I, cyano, hydroxy, nitro, C 1-4 Alkyl, halogenated C 1-4 Alkyl and -O-(C 1-4 alkyl).

[0010] In certain embodiments, R1, R2, R3, R4, and R5 in Formula I are each independently selected from the group consisting of H, F, Cl, Br, I, cyano, hydroxy, nitro, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, propoxy, and butoxy.

[0011] In certain embodiments, in Formula I, R1 is H, R4 is H, and R2, R3, and R5 are each independently selected from the group consisting of H, F, Cl, Br, I, cyano, hydroxy, nitro, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, propoxy, and butoxy.

[0012] In certain embodiments, in Formula I, R1 is H, R2 is H, R4 is H, and R3 and R5 are each independently selected from the group consisting of H, F, Cl, Br, I, cyano, hydroxy, nitro, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, tert-butyl, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, propoxy, and butoxy.

[0013] In certain embodiments, in Formula I, R1 is H, R2 is H, R4 is H, and R3 and R5 are each independently selected from the group consisting of F, Cl, Br, methyl, ethyl, n-propyl, and n-butyl.

[0014] In certain embodiments, in Formula I, R1 is H, R2 is H, R4 is H, and R3 and R5 are each independently selected from the group consisting of F, Cl, Br, and methyl.

[0015] In certain embodiments, the compound of Formula I is selected from:

[0016] .

[0017] The second aspect of the present application provides a pharmaceutical composition comprising a compound described in any embodiment of the first aspect of the present application, a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, hydrate, polymorph, prodrug or isotope-labeled compound thereof, and optionally one or more pharmaceutically acceptable carriers and / or excipients.

[0018] In certain embodiments, the compound, its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, hydrate, polymorph, prodrug or isotopically labeled compound in the pharmaceutical composition is present in a preventive and / or therapeutically effective amount (e.g., an amount effective for preventing and / or treating bunyavirus infection or a disease caused by bunyavirus infection).

[0019] In certain embodiments, the bunyavirus infection is a Crimean-Congo hemorrhagic fever virus infection. In certain embodiments, the disease caused by the bunyavirus infection is a disease caused by the Crimean-Congo hemorrhagic fever virus infection. In certain embodiments, the disease caused by the Crimean-Congo hemorrhagic fever virus infection is hemorrhagic fever. In certain embodiments, the disease caused by the Crimean-Congo hemorrhagic fever virus infection is fever, headache, muscle pain, vomiting, gastrointestinal or other bleeding, hematemesis, or shock caused by the Crimean-Congo hemorrhagic fever virus infection.

[0020] The third aspect of the present application provides the use of the compound described in any embodiment of the first aspect of the present application, its pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, hydrate, polymorph, prodrug or isotope-labeled compound in the preparation of a medicament, wherein the medicament is used to prevent and / or treat bunyavirus infection or a disease caused by bunyavirus infection in a subject, or to inhibit the replication or reproduction of bunyavirus in a subject.

[0021] In certain embodiments, the bunyavirus infection is a Crimean-Congo hemorrhagic fever virus infection. In certain embodiments, the disease caused by the bunyavirus infection is a disease caused by the Crimean-Congo hemorrhagic fever virus infection. In certain embodiments, the disease caused by the Crimean-Congo hemorrhagic fever virus infection is hemorrhagic fever. In certain embodiments, the disease caused by the Crimean-Congo hemorrhagic fever virus infection is fever, headache, muscle pain, vomiting, gastrointestinal or other bleeding, hematemesis, or shock caused by the Crimean-Congo hemorrhagic fever virus infection.

[0022] In certain embodiments, the subject comprises a mammal, such as a human. In certain embodiments, the subject (eg, a human) suffers from a bunyavirus infection or a disease caused by a bunyavirus infection, or is at risk of suffering from the above disease.

[0023] When this application uses the terms "for example," "such as," "including," "including" or variations thereof, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0024] The terms "a" and "an" and "the" and similar referents in the context of describing this application (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0025] As used herein, the term "alkyl" refers to a saturated linear or branched monovalent hydrocarbon group, preferably having 1-6, 1-4 or 1-3 carbon atoms. For example, "C 1-6 "Alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group having 1 to 6 carbon atoms. Typical examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, and the like.

[0026] As used herein, the term "pharmaceutically acceptable" means that the substance it describes is not only physiologically acceptable to a subject but also pharmaceutically useful. For example, when describing a "pharmaceutically acceptable salt," it means that the salt is not only physiologically acceptable to a subject but also pharmaceutically useful. For example, a salt formed as an intermediate in a chiral resolution may be useful in obtaining the final product of the present application, even though the salt of such an intermediate cannot be directly administered to a subject.

[0027] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include acid addition salts formed with inorganic or organic acids, such as salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, stearic acid, and tannic acid. Other acids, such as oxalic acid, although not pharmaceutically acceptable in themselves, may be used to prepare salts used as intermediates to obtain compounds of the present invention and pharmaceutically acceptable salts thereof. Alternatively, the salt formed when the acidic protons present on the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions, is, for example, a sodium salt, potassium salt, magnesium salt or calcium salt. Alternatively, the coordination compound formed by the parent compound and an organic base, such as ethanolamine, diethanolamine, triethanolamine or N-methylglucamine, is, for example, an ammonium salt.

[0028] Methods for preparing pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art. References herein to the compounds of the present application include compounds of Formula I and pharmaceutically acceptable salts thereof.

[0029] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including but not limited to binders, diluents, fillers, disintegrants, wetting agents, lubricants, colorants, flavorings, solubilizers, osmotic pressure regulators or other conventional additives. Typical pharmaceutically acceptable carriers and / or excipients include, for example, microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfate or magnesium stearate.

[0030] As used herein, the term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including meso-, racemic-, enantiomers, diastereomers, cis- and trans-isomers, conformers, or mixtures thereof.

[0031] The compound of the present application can exist with two or more mixtures (commonly referred to as tautomers) of different forms of structures in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imines-enamine tautomers etc. It is to be understood that the scope of the present application encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0032] The present application encompasses all possible crystalline forms or polymorphs of the compounds, which may be single polymorphs or mixtures of more than one polymorph in any ratio.

[0033] The compounds of the present application may exist in the form of solvates (e.g., hydrates), wherein the compounds of the present application contain a solvent as a structural element of the crystal lattice of the compound, such as water, methanol, ethanol, propanol, or acetonitrile. The amount of the solvent may be present in a stoichiometric ratio or a non-stoichiometric ratio.

[0034] As used in the present application, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or undergo other reactions under biological conditions (in vitro or in vivo) to provide compounds described herein. Prodrugs only become active compounds through this reaction under biological conditions, or they do not have or only have relatively low activity in their unreactive forms. Conventionally, known methods can be used to prepare prodrugs, such as those methods described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff compiles, the 5th edition).

[0035] As used herein, the term "isotopically labeled compound" refers to a compound in which one or more atoms are replaced with atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature. Examples of suitable isotopes for inclusion in the compounds of the present invention include, but are not limited to, hydrogen isotopes such as 2 H, 3 H; carbon isotopes such as 11 C, 13 C and 14 C; chlorine isotopes such as 36 Cl; fluorine isotopes such as 18 F; iodine isotopes such as 123 I and 125 I; Nitrogen isotopes such as 13 N and 15 N; oxygen isotopes such as 15 O, 17 O and 18 O; and sulfur isotopes such as 35 S.

[0036] The pharmaceutical compositions described herein can be prepared into various dosage forms according to conventional methods in the art, including but not limited to tablets, capsules, solutions, suspensions, granules or injections, and administered via oral or parenteral routes.

[0037] As used herein, the term "prevention" refers to methods performed to prevent or delay the onset of a disease, condition, or symptom (e.g., a viral infection or a disease caused by a viral infection) in a subject. "Prevention" does not necessarily mean completely preventing the onset of a disease or disease-related symptoms. For example, if the administration of a drug reduces the risk of a subject developing a particular disease or disease-related symptom, or reduces the severity of symptoms that subsequently develop, the onset or development of the disease can be considered "prevented."

[0038] As used herein, the term "treat" refers to a method performed to achieve a beneficial or desired clinical outcome. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and relief of symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treat" may also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0039] As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. A "prophylactically effective amount" refers to an amount sufficient to prevent, inhibit, or delay the onset of a disease (e.g., a viral infection or a disease caused by a viral infection). A "therapeutically effective amount" refers to an amount sufficient to alleviate or eliminate the disease and its complications in a patient already suffering from the disease. The pharmaceutical compositions of the present application may include a "therapeutically effective amount" or a "prophylactically effective amount" of a compound described herein, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, hydrate, polymorph, prodrug, or isotopically labeled compound thereof. Determining such an effective amount is within the capabilities of those skilled in the art.

[0040] It should also be noted that the dosage and method of use of the compound of Formula I, its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, hydrates, polymorphs, prodrugs, or isotope-labeled compounds described herein depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, potency of the compound, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.001 and 1000 mg / kg body weight / day.

[0041] In the present application, the dosage regimen may be adjusted to obtain the optimal treatment or prevention of the intended use. For example, a single dose may be administered, multiple doses may be administered over a period of time, or the dosage may be proportionally reduced or increased as the exigencies of the therapeutic situation warrant.

[0042] As used herein, the term "optionally" means that the event or circumstance it describes occurs or does not occur.

[0043] Beneficial technical effects of this application

[0044] 1. The compound of formula I described in the present application, its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, hydrates, polymorphs, prodrugs or isotope-labeled compounds have strong inhibitory activity against Bunyaviruses such as Crimean-Congo hemorrhagic fever virus.

[0045] 2. The compound of formula I described in the present application, its pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, hydrates, polymorphs, prodrugs or isotope-labeled compounds have a significant protective effect on mice infected with Crimean-Congo hemorrhagic fever virus (CCHFV). BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The results show that the compound LK-E-01 of the present application has a protective effect on CCHFV-infected mice. DETAILED DESCRIPTION

[0047] The substantive content of the present application is further described below in conjunction with the specific examples of the present application. It should be understood that the following examples are only used to illustrate the present application and are not intended to limit the scope of protection of the present application. In the following examples, if no specific conditions are specified, the conventional conditions or manufacturer's recommendations are used. The raw materials used, if the manufacturer is not specified, are all conventional products that can be obtained through commercial purchase.

[0048] Although many materials and operating methods used in the following examples are well known in the art, this application is still described in as much detail as possible. It will be clear to those skilled in the art that, unless otherwise specified, the materials and operating methods used in the following examples are well known in the art.

[0049] Example 1: (3 R ,11a S )-N-(2,4-difluorophenethyl)-6-hydroxy-3-methyl-5,7-dioxo-2,3,5,7,11,11a-hexahydrooxazolo[3,2- a ]pyrido[1,2- d Preparation of pyrazine-8-carboxamide (LK-E-01)

[0050]

[0051] first step:

[0052] 1-(2,2-Dimethoxyethyl)-5-methoxy-6-(methoxycarbonyl)-4-oxo-1,4-dihydropyridine-3-carboxylic acid (LK-E-01-1) (20.0 g, 0.063 mol) was dissolved in acetonitrile (150 mL), and acetic acid (15 mL) and methanesulfonic acid (5 mL) were added. The mixture was heated to 70 °C and reacted for 4 h. The reaction was complete after monitoring by TLC. The mixture was cooled to room temperature and dried under reduced pressure to obtain a yellow solid LK-E-01-2, which was used directly in the next reaction without purification.

[0053] Step 2:

[0054] Dissolve LK-E-01-2 in acetonitrile (150 mL) and add ( R )-2-aminopropanol (7.13 g, 0.095 mol) and K2CO3 (25.9 g, 0.188 mol) were heated to 75°C for 4 h. The reaction was terminated and concentrated by distillation under reduced pressure. 200 mL of water was added and the mixture was extracted with dichloromethane (150 mL × 3). The organic layer was collected, dried over anhydrous sodium sulfate, and evaporated to dryness to afford a yellow solid, LK-E-01-3 (8.9 g). The combined yield of the first and second steps was 48.1%. MS: m / z = 295.1 [M+H] + .

[0055] Step 3:

[0056] LK-E-01-3 (0.50 g, 0.0017 mol) was dissolved in 5 mL of DMF, and HATU (0.66 g, 0.0017 mol), DIPEA (592 μL, 0.0034 mol), and 2-(2,4-difluorophenyl)ethylamine (0.27 g, 0.0017 mol) were added. The reaction was allowed to react at room temperature for 12 h. The reaction was stopped, and 100 mL of water was added. The mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain LK-E-01-4 (0.49 g, 66.9%) as a white solid. MS: m / z = 434.2 [M+H] + .

[0057] Step 4:

[0058] LK-E-01-4 (0.43 g, 0.001 mol) was dissolved in 10 ml of tetrahydrofuran, and anhydrous LiBr (0.26 g, 0.003 mol) was added. The mixture was heated to 60°C and allowed to react for 4 h. The reaction was terminated and concentrated by distillation under reduced pressure. 50 ml of 1N hydrochloric acid was added, and the mixture was extracted with dichloromethane (50 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to remove the solvent. The crude product was purified by silica gel column chromatography (DCM:MeOH = 15:1 v / v) to obtain LK-E-01 (0.22 g, 52.1%) as a white solid. 1 H NMR (600 MHz, DMSO- d 6) δ 11.45 (s, 1H), 9.95 (t, J = 5.8 Hz, 1H), 8.42 (s, 1H), 7.36(m, 1H), 7.17 (m, 1H), 7.01 (m, 1H), 5.38 (dd, J = 10.1, 4.1 Hz, 1H), 4.87(dd, J = 12.1, 4.2 Hz, 1H), 4.39 (dd, J = 8.6, 6.8 Hz, 1H), 4.29 (m, 1H), 3.99 (dd, J = 12.1, 10.1 Hz, 1H), 3.66 (dd, J = 8.6, 6.7 Hz, 1H), 3.55 (q, J= 6.6 Hz, 2H), 2.84 (t, J = 6.9 Hz, 2H), 1.34 (d, J = 6.3 Hz, 3H); MS: m / z =420.1 [M+H] + .

[0059] With reference to Example 1, the compounds in the following table were prepared:

[0060] Example 2:

[0061] This example tests the in vitro anti-CCHFV activity and cytotoxicity of the compounds prepared in the examples of this application. The CCHFV-IbAr10200-eGFP and HUVEC cells used were both from the National Virus Resource Bank of the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0062] 2.1 In vitro anti-Bunyavirus activity assay

[0063] HUVEC cells in good condition were cultured at a rate of 1×10 4 100 μL of drug-containing medium was added to each well 1 hour before infection. Three replicates were made for each drug concentration. The remaining drug-containing medium was placed in a 4°C refrigerator for use. The virus stock solution with the measured titer was diluted to 5×10 4 At a TCID of 50 / mL, 20 μL of virus dilution was added to each well and cells were infected at an MOI of 0.1. Incubate at 37°C for 1 hour. Discard the supernatant after infection, rinse with PBS, and add 200 μL of fresh drug-containing medium to each well. Incubate at 37°C for 72 hours. Harvest the supernatant and cells 72 hours after infection. The supernatant can be used for qPCR analysis. Cells were fixed in 4% paraformaldehyde. After fixation, the cells were stained with Hoechst 33258 nuclear stain. Fluorescence counts per well were calculated using a high-content analyzer and converted to inhibition rate.

[0064] 2.2 Determination of cytotoxicity of compounds

[0065] HUVEC cells were cultured at a rate of 1×10 4 Cells were seeded into a 96-well plate with a white wall and a transparent bottom at a concentration of 100 μL per well. The test compound was diluted in cell maintenance medium and added to the 96-well plate at a volume of 100 μL per well. The cells were incubated at 37°C for 72 hours. Following the instructions for the CCK-8 kit (MCE), 10 μL of CCK8 solution was added to each well. After 1 hour, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0066] Table 1 Anti-CCHFV activity and cytotoxicity of the compound represented by formula 1

[0067]

[0068] The results showed that the compounds LK-E-01 to LK-E-34 and compound LK-B-01 prepared in the examples of the present application had inhibitory activity against CCHFV.

[0069] Example 3: Experiment on the protection of CCHFV-infected mice from death by compound LK-E-01

[0070] All animal experiments used type I IFN receptor knockout (IFNAR) mice on a C57BL / 6J background. - / -)Mice. 7-12 week old mice were infected with CCHFV-IbAr10200-eGFP (provided by the National Virus Resource Library, Wuhan Institute of Virology, Chinese Academy of Sciences) via intraperitoneal injection (IP). Compounds LK-E-01 and T-705 were administered IP to the mice, while a blank control group received a similar solvent injection. The body weight of the mice was monitored daily, and their condition was observed and recorded. When symptoms such as a 20% weight loss, a significant decrease in activity, a hunched back, and ruffled fur appeared, the mice were euthanized using isoflurane anesthesia. Statistical analysis was performed using a two-way analysis of variance (ANOVA). The livers of the mice were collected for viral load and pathological analysis. The P value for survival percentage was determined by the log-rank test. Statistical analysis was performed using an unpaired t-test.

[0071] This example evaluates the in vivo efficacy of compound LK-E-01 against CCHFV. Compound LK-E-01 was administered at doses of 25 mg / kg and 50 mg / kg once daily via intraperitoneal injection for 7 days and observed for 13 consecutive days. T-705 was administered at a dose of 300 mg / kg once daily via intraperitoneal injection for 7 days and observed for 13 consecutive days. The results showed that both compounds LK-E-01 and T-705 showed protective effects against CCHFV-infected mice. Figure 1 The study showed that the overall survival rate of LK-E-01 at a once-daily dose of 25 mg / kg was 50% (n=6 / group), the once-daily dose of 50 mg / kg was 100% (n=6 / group), and the once-daily dose of T-705 at a dose of 300 mg / kg was 100% (n=6 / group). The survival rate of the blank control group was 16.6% (n=6 / group). Compared with the blank control group, once-daily treatment with LK-E-01 at both 25 mg / kg and 50 mg / kg significantly improved the overall survival rate of CCHFV-infected mice, and once-daily treatment with 50 mg / kg achieved 100% protection against CCHFV infection. Compared with the T-705-treated group, LK-E-01 at a dose of 50 mg / kg was equally effective as T-705 at a dose of 300 mg / kg in CCHFV-infected mice, both achieving a 100% survival rate.

[0072] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that, based on all the teachings disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A compound of formula I, a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1, R2, R3, R4, and R5 are each independently selected from: H, F, Cl, Br, or C 1-4 alkyl.

2. The compound according to claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1, R2, R3, R4, and R5 are not H at the same time.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is H, R4 is H, and R2, R3 and R5 are each independently selected from: H, F, Cl, Br or C 1-4 alkyl.

4. The compound of claim 3, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is H, R2 is H, R4 is H, and R3 and R5 are each independently selected from: H, F, Cl, Br or C 1-4 alkyl.

5. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1, R2, R3, R4, and R5 are each independently selected from the group consisting of H, F, Cl, Br, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

6. The compound of claim 5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is H, R4 is H, and R2, R3, and R5 are each independently selected from the group consisting of H, F, Cl, Br, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

7. The compound of claim 6, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is H, R2 is H, R4 is H, and R3 and R5 are each independently selected from the group consisting of H, F, Cl, Br, methyl, ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

8. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from:

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8, a pharmaceutically acceptable salt or stereoisomer thereof, and optionally one or more pharmaceutically acceptable carriers or excipients.

10. Use of a compound according to any one of claims 1 to 8, a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a medicament for preventing and / or treating a bunyavirus infection or a disease caused by a bunyavirus infection in a subject, or for inhibiting the replication or reproduction of a bunyavirus in a subject.

11. The use according to claim 10, wherein the bunyavirus infection is Crimean-Congo hemorrhagic fever virus infection, and the disease caused by the bunyavirus infection is a disease caused by Crimean-Congo hemorrhagic fever virus infection.

12. The use according to claim 11, wherein the disease caused by Crimean-Congo hemorrhagic fever virus infection is hemorrhagic fever.

13. The use according to claim 11, wherein the disease caused by Crimean-Congo hemorrhagic fever virus infection is fever, headache, muscle pain, vomiting, bleeding in the gastrointestinal tract or other parts, hematemesis or shock caused by Crimean-Congo hemorrhagic fever virus infection.

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