CN116687932A - The medical use of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate - Google Patents

Abstract

The present invention relates to ((3-carbamoyl-5-fluoropyrazine-2-yl)oxy) methyl isobutyrate, its geometric isomer, pharmaceutically acceptable salt, solvate or hydrate Medicinal use, especially for the treatment of diseases or infections caused by viruses of the order bunyaviruses.

Description

The medical use of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate

technical field

The present invention relates to ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (Isobutyric acid3-carbamoyl-5-fluoro-pyrazin-2-yloxymethyl ester), its geometry Medical use of isomers, pharmaceutically acceptable salts, solvates or hydrates, especially for the treatment of diseases caused by viruses of the order Bunyaviruses, such as Crimean-Congo hemorrhagic fever virus (CCHFV), or Infection use.

Background technique

Crimean-Congo hemorrhagic fever (CCHF) is an acute infectious disease widely prevalent in Africa, Asia, and Europe. It is characterized by fever, gastrointestinal bleeding, epistaxis, hematemesis, and shock. Up to 50%, the causative pathogen of CCHF is Crimean-Congo hemorrhagic fever virus (CCHFV). Crimean-Congo hemorrhagic fever virus belongs to Bunyavirales (Bunyavirales), Nairoviridae (Nairoviridae), or Nairovirus (Orthonairovirus) tick-borne viruses. The clinical symptoms of CCHF patients are extensive, and the typical disease course goes through four stages: incubation period, prebleeding period, bleeding period and recovery period. The length of the incubation period is related to factors such as the route of exposure and viral load; after the incubation period, the pre-bleeding stage manifests as a febrile illness, and the patient is accompanied by severe headache, nausea, diarrhea, muscle aches, and photophobia. When the patient enters the bleeding stage, the body will Petechiae, large area of ecchymosis, and massive hemorrhage appear in turn, and 9% to 50% of cases die at this stage. In addition to these symptoms, the study also found that the onset of CCHF is accompanied by serious damage to the liver, nervous system, respiratory system, and heart. There are currently no anti-Crimea-Congo hemorrhagic fever drugs and vaccines on the market, which seriously threatens human health.

Contents of the invention

The structural formula of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (hereinafter referred to as compound or compound I shown in formula I) is as shown in formula I,

The inventor surprisingly found that the compound ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy) methyl isobutyrate shown in formula I can inhibit Crimean-Congo hemorrhagic fever Virus replication in cells reduces viral nucleic acid load levels in cells infected with Crimean-Congo hemorrhagic fever virus at micromolar concentrations and has a positive effect on Crimean-congo hemorrhagic fever virus-infected mice Significant protective effect, can be used to treat diseases caused by Crimean-Congo hemorrhagic fever virus, such as simple infection, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, epistaxis, hematemesis, shock, etc.

The present invention relates to compounds represented by formula I, their geometric isomers, their pharmaceutically acceptable salts, their solvates or their hydrates in the preparation of medicines for treating diseases or infections caused by viruses of the order Bunyaviruses the use of.

The present invention also relates to the use of the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate in the preparation of a medicament as a bunyavirus inhibitor.

The present invention also relates to the compound shown in formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate in the preparation for inhibiting bunyaviruses in cells (such as mammalian cells) ) for use in medicines reproduced or reproduced.

The present invention also relates to the use of the pharmaceutical composition in the preparation of medicines for the treatment of diseases or infections caused by viruses of the order Bunyavirus, wherein the pharmaceutical composition comprises the compound represented by formula I, its geometric isomer, its pharmaceutical acceptable salts, solvates or hydrates thereof.

The present invention also relates to the use of a pharmaceutical composition in the preparation of a medicine as a bunyavirus inhibitor, wherein the pharmaceutical composition comprises the compound shown in formula I, its geometric isomer, and its pharmaceutically acceptable salt , its solvate or its hydrate.

The present invention also relates to the use of the pharmaceutical composition in the preparation of drugs for inhibiting the replication or reproduction of bunyaviruses in cells (such as mammalian cells), wherein the pharmaceutical composition comprises the compound shown in formula I, its Geometric isomers, pharmaceutically acceptable salts thereof, solvates or hydrates thereof.

The present invention also relates to the pharmaceutical composition comprising the compound represented by the formula I, its geometric isomer or its pharmaceutically acceptable salt and/or its solvate and/or its hydrate or the said formula I The shown compound, its geometric isomer or its pharmaceutically acceptable salt and/or its solvate and/or its hydrate are used in the preparation for the treatment of hemorrhagic fever (comprising simple infection, fever, headache, muscle pain, vomiting , gastrointestinal bleeding, epistaxis, hematemesis, shock, etc.).

The present invention also relates to a method of treating and/or preventing disease or infection in a mammal in need thereof, the method comprising administering a therapeutically and/or preventing effective amount of said formula I comprising said The pharmaceutical composition of the compound shown, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate or the compound shown in the formula I, its geometric isomer, its pharmaceutically acceptable salts, solvates or hydrates thereof, wherein the diseases or infections include diseases or infections caused by viruses of the order Bunyaviridae.

The present invention also relates to a method for inhibiting the replication or reproduction of bunyaviruses in a mammal in need, the method comprising administering to the mammal in need a therapeutically and/or prophylactically effective amount of said The pharmaceutical composition of the compound shown in formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate or the compound shown in formula I, its geometric isomer, its pharmaceutically Acceptable salts, solvates or hydrates thereof.

The present invention also relates to the preparation of the pharmaceutical composition for the treatment of diseases or infections caused by Bunya virus, especially Crimean-Congo hemorrhagic fever virus (such as simple infection, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, epistaxis, hematemesis, shock, etc.), wherein the pharmaceutical composition comprises the compound represented by the formula I, its geometric isomer or its pharmaceutically acceptable salt and/or its solvent compounds and/or their hydrates.

The present invention also relates to the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate, which is used for treating diseases or infections caused by viruses of the order Bunyaviridae.

The present invention also relates to the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate, which is used as a bunyavirus inhibitor.

The present invention also relates to the compound shown in formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate, which is used for inhibiting bunyavirus order virus in cells (such as mammalian cells) ) to copy or reproduce.

The present invention also relates to a pharmaceutical composition for treating diseases or infections caused by viruses of the order Bunyavirus, wherein the pharmaceutical composition comprises the compound represented by the formula I, its geometric isomer, and its pharmaceutically acceptable Accepted salts, solvates or hydrates thereof.

The present invention also relates to a pharmaceutical composition, which is used as a bunyavirus inhibitor, wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt and/or its Solvates and/or hydrates thereof.

The present invention also relates to a pharmaceutical composition, which is used to inhibit the replication or reproduction of bunyaviruses in cells (such as mammalian cells), wherein the pharmaceutical composition comprises the compound represented by formula I, its geometric isomers, Its pharmaceutically acceptable salt and/or its solvate and/or its hydrate.

In certain embodiments, the pharmaceutically acceptable salts of the compounds represented by formula I of the present invention include their inorganic or organic acid salts, and inorganic or organic alkali salts. The present invention relates to all forms of the above salts, including but not limited to: Sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, acetic acid Salt, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, Fumarate, picrate, aspartate, gluconate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate salt etc.

In certain embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier or adjuvant.

In some embodiments, the pharmaceutical composition of the present invention is a solid preparation, injection, external preparation, spray, liquid preparation or compound preparation.

In certain embodiments, the bunyavirales described herein are viruses of the family Neiroviridae.

In certain embodiments, the bunyaviruses of the present invention are viruses of the genus Nerovirus.

In certain embodiments, the bunyaviruses of the present invention are Crimean-Congo hemorrhagic fever virus (CCHFV).

In certain embodiments, the disease caused by the bunyavirales described in the present invention is hemorrhagic fever caused by the bunyavirales.

In certain embodiments, the disease caused by the Bunyaviridae virus described in the present invention is hemorrhagic fever caused by a Neroviridae virus.

In certain embodiments, the disease caused by the bunyaviruses described in the present invention is hemorrhagic fever caused by a virus of the genus Nerovirus.

In some embodiments, the disease caused by the bunyaviruses described in the present invention is hemorrhagic fever caused by Crimean-Congo hemorrhagic fever virus.

In some embodiments, the disease caused by the bunyaviruses described in the present invention is simple infection, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, epistaxis caused by the bunyaviruses , vomiting or shock.

In certain embodiments, the disease caused by the bunyavirales described herein is Crimean-Congo hemorrhagic fever.

In certain embodiments, the disease caused by the bunyaviruses of the present invention is a disease caused by Crimean-Congo hemorrhagic fever virus.

In certain embodiments, the mammals of the present invention include bovines, equines, ovines, porcines, canines, felines, rodents, primates, such as human , cat, dog or pig.

In the present invention, the term "therapeutically effective amount" or "prophylactically effective amount" refers to an amount sufficient to treat or prevent a patient's disease but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment. A therapeutically effective amount of a compound will depend on the particular compound selected (e.g., taking into account the potency, effectiveness, and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, the condition of the patient being treated, Factors such as age, size, weight and physical ailments, medical history of the patient being treated, duration of treatment, nature of concurrent therapy, desired therapeutic effect, etc. vary but can still be routinely determined by one skilled in the art.

In addition, it should be pointed out that the specific dosage and method of use of the compound shown in formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate for different patients depends on many factors, including age, weight, gender, natural health status, nutritional status, drug activity, taking time, metabolic rate, severity of disease and subjective judgment of treating physicians. A dose of 0.001-1000 mg/kg body weight/day is preferably used here.

The pharmaceutical composition of the present invention can be prepared in various forms according to different administration routes.

According to the present invention, the pharmaceutical composition can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal cavity administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular , intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or with the aid of an explanted reservoir. Of these, oral, intraperitoneal or intravenous administration is preferred.

When administered orally, the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate can be made into any orally acceptable preparation form, including but not limited to tablet elixirs, capsules, solutions or suspensions in water. Wherein, the generally used carrier of tablet comprises lactose and cornstarch, also can add lubricating agent such as magnesium stearate in addition. Diluents commonly used in capsule formulations include lactose and dried cornstarch. Aqueous suspensions are usually prepared by mixing the active ingredient with suitable emulsifying and suspending agents. If desired, some sweetening, flavoring or coloring agents may also be added to the above oral preparation forms.

When administered rectally, the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate can generally be made into the form of suppository, which is obtained by combining the drug with a suitable prepared by mixing non-irritating excipients. The excipient is solid at room temperature and melts at rectal temperature to release the drug. Such excipients include cocoa butter, beeswax and polyethylene glycols.

When used locally, especially when treating the affected surface or organs that are easily accessible by local external application, such as eyes, skin or lower intestinal neuropathy, the compound represented by the formula I, its geometric isomers, and its pharmaceutically acceptable The salt, its solvate or its hydrate can be made into different topical preparations according to different affected areas or organs, and the specific instructions are as follows:

For topical administration to the eye, the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate can be formulated into a micronized suspension or solution, The carrier used is isotonic sterile saline at a certain pH with or without the addition of preservatives such as benzyl alkoxide chloride. Additionally for ophthalmic use, the compounds may also be formulated in the form of ointments such as petrolatum ointment.

When the skin is topically applied, the compound represented by the formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate can be made into a suitable ointment, lotion or cream preparation form, In which the active ingredient is suspended or dissolved in one or more carriers. Carriers that can be used for ointments include but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax, and water; carriers that can be used for lotions or creams include but are not limited to: Mineral oil, sorbitan monostearate, Tween 60, cetyl esters wax, cetyl aryl alcohol, 2-octyldodecanol, benzyl alcohol and water.

When locally administered in the lower intestinal tract, the compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate can be made into rectal suppository preparation or suitable enema In formulation form, topically transdermal patches are also available.

The compound represented by formula I, its geometric isomer, its pharmaceutically acceptable salt, its solvate or its hydrate can also be administered in the form of sterile injectable preparations, including sterile injectable water or oil suspension, or sterile injection Bacterial injection solution. Among them, usable vehicles and solvents include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, such as mono- or diglycerides, can be employed as a solvent or suspending medium.

The above-mentioned medicines in various dosage forms can be prepared according to conventional methods in the field of pharmacy.

Description of drawings

Figure 1 shows the effect of compound I anti-CCHFV in vitro, wherein A shows the preliminary screening results of compound I anti-CCHFV; B shows the _EC50 and _CC50 of compound I;

Figure 2 shows that compound I can effectively inhibit the in vivo infection of CCHFV, wherein A shows the body weight change curve and survival rate of mice; B shows the results of virus copy number detection in mouse tissues; C shows EIDD-2801 Inability to suppress in vivo infection of CCHFV;

Figure 3 shows that low-dose compound I can effectively inhibit the in vivo infection of CCHFV, wherein A shows the mouse body weight change curve and survival rate; B shows the virus copy number detection results in mouse tissues;

Figure 4 shows that delayed administration of compound I can effectively inhibit the in vivo infection of CCHFV, wherein A shows the mouse body weight change curve and survival rate; B shows the virus copy number detection results in mouse tissues; C shows Pathological changes of mouse liver and spleen;

Figure 5 shows the ¹ H-NMR spectrum of compound I;

Figure 6 shows the ¹³ C-NMR spectrum of compound I;

FIG. 7 shows the HRMS spectrum of Compound I.

Detailed ways

The following examples are illustrative preferred embodiments of the present invention and do not constitute any limitation to the present invention.

Compound I (compound represented by formula I, ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate) used in the following examples was synthesized by the following method.

Under nitrogen protection, T-705 (1.57g, 10mmol) was dissolved in anhydrous acetonitrile (10mL), after the solution was stirred at room temperature for 15min, triethylamine (3.03g, 30mmol) was added dropwise, and the reaction solution was cooled to -20°C, followed by dropwise addition of bromomethyl isobutyrate (3.41 g, 25 mmol). After the reaction solution continued to react at -20°C for 24h, the reaction solution was poured into cold water (100mL), the mixture was extracted three times with dichloromethane, and the combined organic phases were respectively treated with 1N hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride solution. After the solution was washed twice, it was dried overnight over sodium sulfate. The dried organic phase was concentrated to dryness, mixed with silica gel and then separated and purified by Flash column chromatography (eluted with dichloromethane/methanol) to obtain the target compound I. ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 8.19 (d, J=8.0Hz, 1H, pyrazine H), 7.37 (brs, 1H, CONH ₂ ), 6.21 (s, 2H, CH ₂ ), 6.18-6.05 (brs, 1H, CONH ₂ ), 2.58 (dt, J=14.0, 7.0Hz, 1H, CH), 1.16 (d, J=4.0Hz, 6H, CH3). ¹³ C-NMR (100MHz, CDCl ₃ ) δ176.09(O=CO), 163.08(O=C-NH ₂ ), 154.90(CF, ¹ J _CF ＝247Hz), 154.50(CF, ⁴ J _CF ＝2Hz), 132.19(CCF, J _CCF ＝ 41Hz), 129.20(CF, ³ J _CF =7Hz), 82.83(CH ₂ ), 33.88(CH), 8.72(CH ₃ ).HRMS(ESI ⁺ )m/z[M+H] ⁺ calculated for C ₁₀ H ₁₂ FN ₃ O ₄ : 258.0885; found: 258.0893. HRMS(ESI ⁺ )m/z[M+Na] ⁺ calculated for C ₁₀ H ₁₂ FN ₃ NaO ₄ : 280.0710; found: 280.0713.

The ¹ H-NMR, ¹³ C-NMR and HRMS of compound I are generally shown in Figures 5-7.

Embodiment 1: Compound I reduces the nucleic acid load experiment of Crimea-Congo hemorrhagic fever virus

All CCHFV infection experiments were performed in the BLS-3 laboratory.

1. Materials and Methods

1.1 Test compound: Compound I was made into 100 mM stock solution with DMSO. During the experiment, it was diluted with cell culture solution (MEM medium containing 2% FBS) to the concentration required for the experiment.

1.2 Cells: Vero E6 cells (ATCC number: 1586) are African green monkey kidney cells, which are preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

1.3 Virus: CCHFV YL16070 isolate (GenBank accession number: KY354082) was isolated and subcultured by Wuhan Institute of Virology, Chinese Academy of Sciences.

1.4 Reagents, laboratory supplies and instruments:

1.4.1 Reagents: minimum Eagle's medium (MEM) medium, product of American GIBCO Company; fetal bovine serum, product of American GIBCO Company; sodium bicarbonate, product of Sinopharm Group; penicillin, streptomycin and kanamycin: all in North China Pharmaceutical factory product.

1.4.2 Experimental supplies and instruments: culture flasks, products of Corning Company of the United States; 96-well culture plates, products of Corning Company of the United States; carbon dioxide incubators, products of Thermo Company of the United States;

1.4.3 Preparation of cell culture medium and reagents: 100 ml of MEM culture medium: containing 10% of fetal bovine serum (FBS), 100 U/ml of penicillin, streptomycin and kanamycin, and 5% of NaHCO ₃ . Cell digestion solution: 0.25% trypsin, prepared with Hanks solution, 0.02% EDTA.

1.5. Experimental method:

1.5.1 Vero E6 cell culture: Add 0.1ml of 0.25% trypsin and 5ml of 0.02% EDTA to a culture flask full of cells, digest at 37°C for 5 minutes, discard the digestion solution, add culture solution and pipette gently, pass passage 1:3 , 3 days full, for compound I toxicity test and inhibition test.

1.5.2 Cytotoxicity test

The cytotoxicity of compound I was detected by CCK-8 kit (Beyotime). Specific steps are as follows:

① Inoculate about 2×10 ⁴ Vero-E6 (ATCC) cells in a 96-well plate and culture at 37°C for 8 hours.

②Dilute the mother solution of compound I with cell culture medium (MEM medium containing 2% FBS) to the concentration of the drug, discard the original medium in the 96-well plate, and add 100 μL of the cell culture medium containing compound I to the cells, each Concentrations were repeated in triplicate. Note that a negative control (cell wells with corresponding concentrations of DMSO and no compound I in the culture medium) and a blank control (without cells, only DMSO and cell culture medium) are set. After the dosing was completed, the cells were cultured at 37°C for 24 hours.

③ Add 20 μL of CCK-8 solution (Beyotime) to the well to be tested, mix gently without generating air bubbles, and continue to incubate at 37°C for 2 hours. Read OD450 on a microplate reader and calculate cell viability:

Cell activity (%)=(A (compound I treatment group)-A (blank control))/(A (negative control)-A (blank control))×100%

1.5.3 Anti-virus experiment:

① Inoculate Vero E6 cells into a 48-well plate, about 6×10 ⁴ cells per well, and wait for the experiment the next day. First, 100 μL of cell culture medium containing the corresponding concentration of compound I was added to the cell plate, and the cells were pretreated for 1 hour, then 20 μL of diluted virus (MOI=0.01) was added and incubated in an incubator for 1 hour. Then discard the virus culture solution, wash away the uninfected residual virus with PBS, then add the cell culture solution containing the corresponding concentration of compound I, and then put it in a 37°C, 5% _CO2 incubator to continue culturing for 72 hours, and add the final concentration to the cell control group 0.5% DMSO in cell culture medium.

② RNA extraction, using the kit produced by TaKaRa Company (TaKaRa MiniBEST Viral RNA/DNAExtraction Kit, Cat. No. 9766), the steps are as follows:

1) Take 100 μL of the supernatant from the culture plate to be tested and add it to a nuclease-free EP tube, then add 321 μL of lysate (100 μL PBS, 200 μL buffer VGB, 20 μL proteinase K, 1 μL carrier RNA) to each well, mix well and place in Digest at 56°C for 30 minutes;

2) Add 200 μL of absolute ethanol to the obtained mixture, and mix well;

3) Transfer the above mixed solution into an RNase-free spin column, centrifuge at 12000rpm for 15s, and discard the waste solution;

4) Add 500μL Buffer RW1, centrifuge at 12000rpm for 15s, and discard the waste liquid;

5) Add 650μL Buffer RW2, centrifuge at 12000rpm for 15s, and discard the waste liquid;

6) Add 650μL Buffer RW2, centrifuge at 12000rpm for 2min, and discard the waste liquid;

7) Replace with a new RNase-free 2ml collection tube, and centrifuge at 12000rpm for 1min to dry the spin column;

8) Replace with new 1.5ml collection tubes, add 30 μl RNase-free water to each tube, centrifuge at 12000 rpm for 2 min, and the eluate will contain the corresponding RNA.

③ RNA reverse transcription, the steps are as follows:

In the experiment, the reverse transcription kit (PrimeScript ^TM RT reagent Kit with gDNA Eraser, product number RR047Q) produced by TaKaRa Company was used for RNA reverse transcription. First, remove gDNA: collect RNA samples from each experimental group, and take 3 μL of RNA for reverse transcription. Add 2 μl of 5×gDNA Eraser Buffer to the RNA of each experimental group, make up the reaction system to 10 μl with RNaseFree water, mix well, and remove gDNA that may exist in the sample in a water bath at 42°C for 2 minutes. Then carry out reverse transcription: add an appropriate amount of enzyme, primer Mix and reaction buffer to the obtained sample, make up the volume to 20 μl with RNase Free water, react in a 37°C water bath for 15 minutes, and then put it into 85°C water for 5 seconds to obtain cDNA.

④Real-time PCR. Fluorescent quantitative PCR uses TB Green Premix (Takara, Cat#RR820A) to mix the reaction system, and performs amplification reaction and reading in StepOne Plus Real-time PCR instrument (brand: ABI). Calculate the copy number per milliliter of the original virus solution. Proceed as follows:

1) Establish the standard first: Dilute the plasmid pMT-S to 5×10 ⁸ , 5×10 ⁷ , 5×10 ⁶ , 5×10 ⁵ , 5×10 ⁴ , 5×10 ³ and 5×10 ² copies/ μL. Take 2 μL of standard or cDNA template for qPCR reaction;

2) The primer sequences used in the experiment are as follows (both indicated in the 5'-3' direction):

S-qF:TCAAGTGGAGGAAGGACATAGG

S-qR: TCCACATGTTCACGGCTCACTGGG

3) The reaction procedure is as follows:

Pre-denaturation: 95°C for 5 minutes;

Cycle parameters: 95°C for 15 seconds, 54°C for 15 seconds, 72°C for 30 seconds. A total of 40 cycles.

2. Results

2.1 Toxicity of compound I to Vero E6 cells

Cytotoxicity results showed that the CC ₅₀ of Compound I was 242.50 μM (Table 1).

Table 1. Cytotoxicity test results of compound I

2.2 Antiviral activity of compound I

The results of the antiviral experiment showed that compound I could significantly inhibit the replication of the viral genome in the supernatant of CCHFV YL16070 strain virus infection at an effective concentration (Table 2).

Table 2. Effect evaluation results of compound I against CCHFV YL16070 strain

2.3 Calculation of its EC ₅₀ and CC ₅₀

Formula: cell activity (%)=(A (drug treatment group)-A (blank control))/(A (negative control)-A (blank control))×100%

Under the condition of virus multiplicity of infection MOI=0.01:

The antiviral effect of compound I on CCHFV YL16070 strain is:

EC ₅₀ =8.08 μM, CC ₅₀ =101.4 μM, SI=12.55.

In initial drug screening, infected cells were stained with IFA. The results showed that Compound I significantly inhibited the infection of CCHFV at 20 μM in vitro (A in FIG. 1 ). The calculated EC ₅₀ value of Compound I was 8.08±2.47 μM, CC ₅₀ value was 101.4 μM, and SI was 12.55 (B in FIG. 1 ).

Example 2: Compound I Protects Crimean-Congo Hemorrhagic Fever Virus Infected Mice from Death Experiments

2.1 In vivo experimental design

Type I IFN receptor knockout (IFNAR ^−/− ) mice on a C57BL/6J background were used for all animal experiments. Male or female mice aged 12-18 weeks were infected with CCHFV (3000 or 5000 _TCID50 ) by intraperitoneal injection (IP). Mice were given different doses of Compound I or EIDD-2801 (purchased from Hanxiang Biotechnology Company, Cat. No. BCP32744) by IP administration once a day, and the control group was given similar solvent injection. Body weight and clinical symptoms of mice were monitored daily. Euthanize mice with isoflurane anesthesia when they lose more than 20% of their body weight, are unresponsive to touch stimuli, and have difficulty crawling. After the death of the control group, the mice in the drug treatment group were sacrificed, and the organs and tissues (liver, spleen) of the mice were collected for further viral load determination and pathological analysis.

2.2 Experimental results

IFNAR ^-/- Each mouse was injected intraperitoneally with 3000 TCID ₅₀ CCHFV. The dose of Compound I was injected intraperitoneally into the mice at 300 mg/kg, and the mice in the control group were injected with normal saline intraperitoneally. Administration was continued for 8 days from the day of challenge, once a day. The results showed that severe weight loss occurred in the control group, while only slight weight loss or weight fluctuation occurred in the Compound I treatment group (A in FIG. 2 ). The control group had 80% (6/8) mortality at day 7, while the Compound I treated group showed 100% protection (A in Figure 2). The viral load in liver and spleen was reduced by about 5 log10 copies/g RNA after 300 mg/kg Compound I treatment (P<0.001) ( FIG. 2B ). Compound I at a dose of 150 mg/kg had a similar effect. EIDD-2801 was unable to inhibit CCHFV infection in vivo (C in Figure 2). The results of the virus copy number in CCHFV-infected mouse tissues in this experiment are shown in Table 3.

Table 3. Virus copy number (Copies/g tissue) in CCHFV infected mouse tissue

To characterize the protective effect of Compound I in more detail, the virus infection dose was increased to 5000 TCID ₅₀ while the drug dose was decreased. Similar to the time of 3000 TCID ₅₀ virus infection, the survival rate of the control group was 14% (1/7) (A in FIG. 3 ). Compound I effectively protects mice from weight loss at 150 mg/kg or a lower dose of 75 mg/kg, and the survival rate of mice is 100%. Viral load assays showed that treatment with both doses of compound I significantly reduced the replication of CCHFV in the liver and spleen (B in Figure 3). Thus, a low dose (75 mg/kg) of Compound I effectively protected IFNAR ^-/- mice from lethal CCHFV challenge. The results of the virus copy number in CCHFV-infected mouse tissues in this experiment are shown in Table 4.

Table 4. Virus copy number (Copies/g tissue) in CCHFV infected mouse tissue

To assess whether delayed treatment of Compound I also provided effective protection, mice were administered Compound I (150 mg/kg) once daily 24 or 48 hours after CCHFV challenge until the end of the experiment. Compound I completely protected mice from lethal CCHFV infection for the 24-hour treatment group (A in Figure 4). The average viral load in the liver and spleen of Compound I-treated mice was also significantly reduced (B in FIG. 4 ). However, when the administration time was 48 hours after challenge, the survival rate of the Compound I-treated group was 71% (5/7) (A in FIG. 4 ). The virus copy numbers in the mouse tissues of the Compound I treatment group were significantly decreased at 48 hours (B in FIG. 4 ). These data indicate that at a dose of 150 mg/kg, a 24 hour delayed administration of Compound I fully protects mice infected with CCHFV. The results of the virus copy number in CCHFV-infected mouse tissues in this experiment are shown in Table 5.

Table 5. Virus copy number (Copies/g tissue) in CCHFV infected mouse tissue

Pathological observations were made on the liver and spleen tissues of the mice in the control group and the 24h drug treatment group. Histopathological analysis showed that the liver and spleen were significantly damaged in the control group (C in Figure 4). The livers of CCHFV-infected mice showed extensive coagulation necrosis surrounded by fibroblast proliferation, multifocal punctate necrosis and edema of hepatocytes (C in Figure 4). In addition, hepatic sinusoidal monocyte/lymphocyte infiltration was significantly increased. The boundary between the white pulp and red pulp of the spleen of infected mice was blurred, and the mononuclear cells in the splenic sinus were significantly increased (C in Figure 4). In the compound I treatment group, only a small amount of liver cell necrosis was observed, the inflammatory reaction was mild, and the white pulp and red pulp structure of the spleen were relatively normal and clear. Likewise, in the control group, abundant CCHFV NP protein was detected in the liver and spleen by IFA, whereas in the compound I-treated group, the viral protein was barely detected (C in Fig. 4). Therefore, compound I can effectively protect mice from virus infection and tissue damage.

In summary, compound I can effectively inhibit the Crimean-Congo hemorrhagic fever virus CCHFV, and can efficiently treat the diseases caused by the Crimean-Congo hemorrhagic fever virus CCHFV infection, and significantly improve the survival rate after infection.

SEQUENCE LISTING

<110> Academy of Military Medical Sciences, Chinese People's Liberation Army

<120> Medical use of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate

<130> IDC220073

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 22

<212>DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 1

tcaagtggag gaaggacata gg 22

<210> 2

<211> 24

<212>DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 2

tccacatgtt cacggctcac tggg 24

Claims (10)

1. The use of a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof in the manufacture of a medicament for the treatment of a disease or infection caused by a virus of the order bunyaviridae,

2. use of a pharmaceutical composition comprising a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof in the manufacture of a medicament for the treatment of a disease or infection caused by a virus of the order bunyaviridae,

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material, and specifically, the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation or a compound preparation.

3. Use of a pharmaceutical composition comprising a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof,

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material, and specifically, the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation or a compound preparation.

4. Use of a pharmaceutical composition comprising a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof in the manufacture of a medicament for inhibiting replication or propagation of a virus of the order bunyaviridae in a cell, such as a mammalian cell,

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or auxiliary material, and specifically, the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation or a compound preparation.

5. The use of a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof for the manufacture of a medicament as a virus inhibitor of the order bunyaviridae,

6. the use of a compound of formula I, a geometric isomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof in the manufacture of a medicament for inhibiting replication or propagation of a virus of the order bunyaviridae in a cell, such as a mammalian cell,

7. the use according to any one of claim 1 to 6, wherein the virus of the order bunyaviridae is a virus of the family inner ro viridae,

preferably, the bunyaviridae order virus is a orthoinner rovirus genus virus,

preferably, the bunyaviridae order virus is crimia-congo hemorrhagic fever virus (CCHFV).

8. The use according to claim 1 or 2, wherein the disease caused by the virus of the order bunyaviridae is hemorrhagic fever caused by the virus of the order bunyaviridae,

preferably, the disease caused by the bunyaviridae is simple infection, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, epistaxis, hematemesis or shock caused by the bunyaviridae;

preferably, the disease caused by the bunyaviridae virus is hemorrhagic fever caused by the endoproviridae virus;

preferably, the disease caused by the bunyaviridae virus is hemorrhagic fever caused by the orthoinner rovirus virus;

preferably, the disease caused by the bunyaviridae virus is hemorrhagic fever caused by crimia-congo hemorrhagic fever virus.

9. The use of claim 1 or 2, wherein the disease caused by a virus of the order bunyaviridae is crimia-congo hemorrhagic fever.

10. The use of claim 4 or 6, wherein the mammal comprises a bovine, equine, ovine, porcine, canine, feline, rodent, primate, such as a human, cat, dog or pig.