((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate and preparation method and application thereof

Abstract

The present invention belongs to the field of pharmaceutical chemicals, and relates to ((3-carbamoyl-5-fluoropyrazine-2-yl)oxy)methyl isobutyrate, a preparation method thereof, and its application in antiviral treatment. The compound ((3-carbamoyl-5-fluoropyrazine-2-yl)oxy)methyl isobutyrate involved in the present invention can be hydrolyzed or metabolized by esterase or P450 enzyme in vivo, and converted into the corresponding 6-fluoro-3-oxo-3,4-dihydropyrazine-2-carboxamide (T705) to exert an antiviral effect. Compared with the prototype drug 6-fluoro-3-oxo-3,4-dihydropyrazine-2-carboxamide (T705), the compound shown in formula I involved in the present invention has obvious advantages such as high bioavailability and long in vivo action time.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and relates to a compound ((3-carbamoyl-5-fluoropyrazine-2-yl)oxy)methyl isobutyrate, a pharmaceutical composition thereof, a preparation method thereof and use thereof in antiviral treatment. Background Art

[0002] 6-Fluoro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide (T705, structure as follows) is an inhibitor of RNA polymerase of a class of viruses and has good antiviral effects. It is reported that T705, used alone or in combination with neuraminidase inhibitors, has a good effect against influenza virus (Antimicrobial Agents and Chemotherapy, 2007, Vol. 51, No. 3, 845-851; Antimicrobial Agents and Chemotherapy, 2010, 126-133, PCT patent: WO2000010569). In addition, T705 also has a good therapeutic effect on diseases caused by other RNA viruses. For example, T705 has a therapeutic effect on western equine encephalitis in mouse models (Antiviral Research 82 (2009) 169–171); T705 has a therapeutic effect on yellow fever in hamsters (Antimicrobial Agents and Chemotherapy, 2009, 202–209); T705 has a therapeutic effect on diseases caused by arenavirus and bunyavirus infections in vivo and in vitro (Antimicrobial Agents and Chemotherapy, 2007, 3168–3176); T705 has a therapeutic effect on rodents infected with West Nile virus (Antiviral Research 80 (2008) 377–379); T705 has a therapeutic effect on phlebovirus infections (Antiviral Research 86 (2010) 121–127).

[0003]

[0004] T705 can be converted into the corresponding nucleoside triphosphate form in vivo, and exerts its antiviral effect by competitively inhibiting viral RNA polymerase by simulating guanosine triphosphate (GTP) (Antimicrobial Agents and Chemotherapy, 2005, Vol. 49, No. 3, p. 981-986).

[0005] However, T705 has some adverse pharmacokinetic properties that are not conducive to its efficacy. Specifically, T705 has a serious first-pass effect. After oral administration, it is quickly metabolized by aldehyde oxidase (XO), resulting in rapid metabolic elimination and a short half-life, which leads to a large dosage of 800mg-2400mg per day. Summary of the invention

[0006] After in-depth research, the inventors of the present application obtained a new compound, namely ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (compound shown in Formula I), and surprisingly found that the compound can be converted into T705 in vivo, showing good antiviral effects, and its bioavailability is significantly improved compared to T705, and has the potential to be used as an antiviral drug. The following invention is provided:

[0007] One aspect of the present invention relates to a compound represented by formula I, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof,

[0008]

[0009] The experiments in Examples 4 and 5 demonstrate that the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate shown in Formula I can be converted into T705 in mice and monkeys. Moreover, Example 4 demonstrates that the oral bioavailability of the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (i.e., the compound shown in Formula I) administered orally to mice is significantly higher than that of T705. Example 5 demonstrates that the bioavailability of the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (i.e., the compound shown in Formula I) administered orally to monkeys is significantly higher than that of T705. In addition, since the various antiviral and disease therapeutic effects of T705 are known in the prior art, those skilled in the art can expect that the compound shown in Formula I also has the same antiviral and disease therapeutic (such as prevention and treatment of foot-and-mouth disease) activity.

[0010] In addition, those skilled in the art know that solvates do not change the structure of the compound but only change the physical properties of the compound, and therefore should have the same biological activity.

[0011] Another aspect of the present invention relates to a pharmaceutical composition comprising the compound represented by formula I of the present invention, its pharmaceutically acceptable salt, its hydrate, or its solvate.

[0012] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0013] In some embodiments, the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a compound preparation.

[0014] The pharmaceutical composition usually contains 0.1-90% by weight of the compound of formula I and / or its pharmaceutically acceptable salt and / or its hydrate and / or its solvate. The pharmaceutical composition can be prepared according to methods known in the art. For this purpose, if necessary, the compound of formula I or its pharmaceutically acceptable salt or its hydrate or solvate can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to prepare a suitable administration form or dosage form for mammals.

[0015] The compound of formula I or the pharmaceutical composition containing it can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum. The dosage form is, for example, tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation and various microparticle delivery systems. In order to make the unit dosage form into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the dosing unit into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the administration unit as a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the administration unit as a capsule, the active ingredient Formula I compound or its pharmaceutically acceptable salt, hydrate, or solvate thereof is mixed with the above-mentioned various carriers, and the resulting mixture is placed in a hard capsule or soft capsule. The active ingredient compound of formula I or its pharmaceutically acceptable salt or hydrate or solvate can also be made into microcapsules and suspended in an aqueous medium to form a suspension, or can be loaded into hard capsules or prepared into injections for use.In order to prepare the dosing unit into an injectable preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injectable preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. can also be added.

[0016] In addition, if necessary, colorants, preservatives, perfumes, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations.

[0017] Another aspect of the present invention relates to a method for preparing the compound represented by formula I of the present invention:

[0018]

[0019] X represents Cl, Br or I

[0020] Compound A (6-fluoro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide) is subjected to a nucleophilic substitution reaction with compound B (i.e., chloromethyl isobutyrate, bromomethyl isobutyrate or iodomethyl isobutyrate) to obtain a compound shown in formula I.

[0021] In some embodiments, the nucleophilic substitution reaction is performed in the presence of an organic base or an inorganic base.

[0022] In some embodiments, the nucleophilic substitution reaction is performed in the presence of an organic base.

[0023] In some embodiments, the organic base is selected from triethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diisopropylethylamine, or any combination thereof.

[0024] In some embodiments, the reaction solvent of the nucleophilic substitution reaction is an organic solvent.

[0025] In some embodiments, the organic solvent is selected from dichloromethane, N,N-dimethylformamide (DMF), acetonitrile, dimethyl ether (DME), tetrahydrofuran (THF), or any combination thereof.

[0026] In some embodiments, when the compound B is chloromethyl isobutyrate, the nucleophilic substitution reaction is carried out in the presence of a catalyst.

[0027] In some embodiments, the catalyst is selected from KI, LiBr, or a combination thereof.

[0028] In some embodiments, the method comprises:

[0029] In an aprotic organic solvent and in the presence of an organic base or an inorganic base, 6-fluoro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide reacts with halogenated methyl isobutyrate, and after separation and purification, ((3-carbamoyl-5-fluoropyrazine-2-yl)oxy)methyl isobutyrate is obtained.

[0030]

[0031] wherein X represents Cl, Br or I.

[0032] Another aspect of the present invention relates to the use of the compound of formula I described in the present invention, its pharmaceutically acceptable salt, its hydrate, or its solvate, or the pharmaceutical composition described in the present invention, or the compound of formula I prepared by the method described in the present invention in the preparation of a drug, wherein the drug is used to prevent and / or treat diseases caused by viral infection.

[0033] In some embodiments, the virus is an RNA virus.

[0034] In some embodiments, the virus is Influenza Virus, Hepatitis C Virus, Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus.

[0035] In some embodiments, the influenza virus is an influenza virus of subtype A1 (H1N1).

[0036] In some embodiments, the disease caused by the viral infection is foot-and-mouth disease.

[0037] Another aspect of the present invention relates to the compound of formula I described in the present invention, its pharmaceutically acceptable salt, its hydrate, or its solvate, or the pharmaceutical composition described in the present invention, or the compound of formula I prepared by the method described in the present invention, which is used for preventing and / or treating diseases caused by viral infection.

[0038] In some embodiments, the virus is an RNA virus.

[0039] In some embodiments, the virus is Influenza Virus, Hepatitis C Virus, Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus.

[0040] In some embodiments, the influenza virus is an influenza virus of subtype A1 (H1N1).

[0041] In some embodiments, the disease caused by the viral infection is foot-and-mouth disease.

[0042] Another aspect of the present invention relates to a method for preventing and / or treating diseases caused by viral infection, which comprises administering to a subject in need thereof an effective amount of the compound shown in Formula I of the present invention, its pharmaceutically acceptable salt, its hydrate, or its solvate, or the pharmaceutical composition of the present invention, or the compound shown in Formula I prepared by the method of the present invention.

[0043] In some embodiments, the virus is an RNA virus.

[0044] In some embodiments, the virus is Influenza Virus, Hepatitis C Virus, Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus.

[0045] In some embodiments, the influenza virus is an influenza virus of subtype A1 (H1N1).

[0046] In some embodiments, the disease caused by the viral infection is foot-and-mouth disease.

[0047] Another aspect of the present invention relates to a method for treating and / or preventing foot-and-mouth disease in mammals, comprising the step of administering an effective amount of a compound of formula I of the present invention or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof to a subject in need thereof (such as a mammal), or an effective amount of a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof. The compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof, or an effective amount of a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof can be used alone.

[0048] Another aspect of the present invention relates to the use of the compound of formula I described in the present invention, its pharmaceutically acceptable salt, its hydrate, or its solvate, or the pharmaceutical composition described in the present invention, or the compound of formula I prepared by the method described in the present invention in the preparation of viral inhibitors or in the preparation of drugs for inhibiting viral replication or reproduction in cells.

[0049] In some embodiments, the virus is an RNA virus.

[0050] In some embodiments, the virus is Influenza Virus, Hepatitis C Virus, Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus.

[0051] In some embodiments, the influenza virus is an influenza virus of subtype A1 (H1N1).

[0052] In some embodiments, the cell is a mammalian cell.

[0053] In some embodiments, the mammal includes bovine, equine, ovine, porcine, canine, feline, rodent, primate, such as a human, cat, dog or pig.

[0054] Another aspect of the present invention relates to the compound of formula I described in the present invention, its pharmaceutically acceptable salt, its hydrate, or its solvate, or the pharmaceutical composition described in the present invention, or the compound of formula I prepared by the method described in the present invention, which is used to inhibit viral replication or reproduction in cells.

[0055] In some embodiments, the virus is an RNA virus.

[0056] In some embodiments, the virus is Influenza Virus, Hepatitis C Virus, Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus.

[0057] In some embodiments, the influenza virus is an influenza virus of subtype A1 (H1N1).

[0058] In some embodiments, the cell is a mammalian cell.

[0059] In some embodiments, the mammal includes bovine, equine, ovine, porcine, canine, feline, rodent, primate, such as a human, cat, dog or pig.

[0060] Another aspect of the present invention relates to a method for inhibiting viral replication or reproduction in cells, which comprises administering to cells an effective amount of a compound of formula I of the present invention, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, or a pharmaceutical composition of the present invention, or a compound of formula I prepared by the method of the present invention.

[0061] In some embodiments, the virus is an RNA virus.

[0062] In some embodiments, the virus is Influenza Virus, Hepatitis C Virus, Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus.

[0063] In some embodiments, the influenza virus is an influenza virus of subtype A1 (H1N1).

[0064] In some embodiments, the cell is a mammalian cell.

[0065] In some embodiments, the mammal includes bovine, equine, ovine, porcine, canine, feline, rodent, primate, such as a human, cat, dog or pig.

[0066] Another aspect of the present invention relates to the use of the compound represented by formula I of the present invention, its pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition of the present invention in the preparation of antiviral drugs.

[0067] Specifically, the virus is an RNA virus; specifically, an influenza virus, HCV virus (Hepatitis C Virus), Bunyavirus, Phlebovirus, Foot and Mouth Disease Virus, West Nile virus, Arenavirus, Western Equine Encephalitis Virus, or Yellow Fever Virus; specifically, the influenza virus is subtype A1 (H1N1) influenza virus.

[0068] Another aspect of the present invention relates to an in vivo or in vitro antiviral method, comprising the step of administering to a subject an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof, or an effective amount of a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof or a solvate thereof.

[0069] In the present invention, "subject" refers to a vertebrate. In certain embodiments, a vertebrate refers to a mammal. The mammal includes bovines, equines, ovines, swine, canines, felines, rodents, primates, for example, humans, cats, dogs or pigs. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice and rats. In certain embodiments, a mammal refers to a human.

[0070] In the present invention, the dosage of the compound of formula I or its pharmaceutically acceptable salt or its hydrate or its solvate depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific compound used, the route of administration and the number of administrations, etc. The above dosage can be administered in a single dosage form or divided into several, such as two, three or four dosage forms.

[0071] The actual dosage level of the active ingredient in the pharmaceutical composition can be varied so as to obtain an amount of active compound effective to obtain the desired therapeutic response for a particular patient, composition, and mode of administration. The dosage level must be selected based on the activity of the compound of formula I, the route of administration, the severity of the condition being treated, and the condition and medical history of the patient being treated. However, it is common practice in the art to start the dosage of the compound at a level below that required to obtain the desired therapeutic effect and to gradually increase the dosage until the desired effect is obtained.

[0072] When used for the above-mentioned treatment and / or prevention or adjuvant therapy, a therapeutically and / or preventively effective amount of one of the compounds of the present invention may be applied in pure form, or in the form of a pharmaceutically acceptable ester or prodrug (where these forms exist). Alternatively, the compound may be administered as a pharmaceutical composition containing the compound of interest and one or more pharmaceutically acceptable excipients. The term "preventively and / or therapeutically effective amount" of the compound of the present invention refers to a sufficient amount of the compound to treat the disorder at a reasonable effect / risk ratio applicable to any medical prevention and / or treatment. However, it should be recognized that the total daily dose of the compounds and compositions of the present invention must be determined by the attending physician within the scope of reliable medical judgment. For any specific patient, the specific therapeutically effective dose level must be determined based on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or simultaneously with the specific compound used; and similar factors known in the medical field. For example, the practice in the art is to start the dosage of the compound from a level lower than that required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained. Generally speaking, the dosage of the compound of formula I of the present invention for mammals, especially humans, can be between 0.001-1000 mg / kg body weight / day, for example, between 0.01-100 mg / kg body weight / day, for example, between 0.01-10 mg / kg body weight / day.

[0073] The compounds of the present invention can effectively prevent and / or treat the various diseases or conditions described in the present invention.

[0074] In the present invention, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or disorders described in the present invention in a subject.

[0075] Advantageous Effects of the Invention

[0076] The compound of the present invention can be converted into the form of T705 in vivo to exert an antiviral effect, and at the same time can significantly improve the bioavailability of the compound and prolong the action time of the compound T705 in vivo. In other words, the compound ((3-carbamoyl-5-fluoropyrazine-2-yl)oxy)methyl isobutyrate involved in the present invention can be converted into the corresponding 6-fluoro-3-oxo-3,4-dihydropyrazine-2-carboxamide (T705) in vivo through hydrolysis or metabolism of esterase or P450 enzyme to exert an antiviral effect. Compared with the prototype drug 6-fluoro-3-oxo-3,4-dihydropyrazine-2-carboxamide (T705), the compound shown in formula I involved in the present invention has obvious advantages such as high bioavailability and long action time in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The drug concentration-time curve of T705 in the blood of mice orally administered Compound 1 and T705 according to an embodiment of the present invention is shown.

[0078] Figure 2 The drug concentration-time curve of T705 in the blood of macaques orally administered Compound 1 and T705 according to the example of the present invention is shown. DETAILED DESCRIPTION

[0079] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0080] Example 1: Preparation of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (1)

[0081]

[0082] Under nitrogen protection, 1.57g (10mmol) of T705 was dissolved in 10mL of anhydrous acetonitrile. After stirring at room temperature for 15min, 3.03g (30mmol) of triethylamine was added dropwise, the temperature was lowered to 0℃, and 3.41g (25mmol) of chloromethyl acetate was added dropwise. A catalytic amount of KI or LiBr was added or not added. After reacting at 0℃ for 24 hours, the temperature was raised to room temperature for reaction. The reaction solution was poured into 100mL of cold water, extracted three times with dichloromethane, the organic phases were combined, washed twice with 1N hydrochloric acid, washed with saturated sodium bicarbonate solution, washed with sodium chloride solution, dried with sodium sulfate, concentrated to dryness, mixed with silica gel, and separated by Flash column chromatography (eluted with dichloromethane / methanol) to obtain ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate. 1 HNMR (DMSO) δ1.066 (6H, d, J = 6.96Hz), 2.56 (1H, m), 6.06 (2H, s), 7.83 (1H, bs), 7.96 (1H, brs), 8.45 (1H, d, J = 8.14Hz); ESIMS (m / e) 258 (MH + ).

[0083] Example 2: Preparation of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (2)

[0084]

[0085] Under nitrogen protection, 1.57 g (10 mmol) of T705 was dissolved in 10 mL of anhydrous acetonitrile. After stirring at room temperature for 15 min, 3.03 g (30 mmol) of triethylamine was added dropwise, the temperature was lowered to -20 °C, and 3.41 g (25 mmol) of bromomethyl acetate was added dropwise. After the addition was completed, the reaction mixture was reacted at -20 °C for 24 hours, and then the reaction solution was poured into 100 mL of cold water, extracted three times with dichloromethane, the organic phases were combined, washed twice with 1N hydrochloric acid, washed with saturated sodium bicarbonate solution, washed with sodium chloride solution, dried with sodium sulfate, concentrated to dryness, mixed with silica gel, and separated by Flash column chromatography (eluted with dichloromethane / methanol) to obtain ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate.

[0086] Example 3: Preparation of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate (3)

[0087]

[0088] Under nitrogen protection, 1.57 g (10 mmol) of T705 was dissolved in 10 mL of anhydrous acetonitrile. After stirring at room temperature for 15 min, 3.03 g (30 mmol) of triethylamine was added dropwise, the temperature was lowered to -40 °C, and then 2.12 g (20 mmol) of iodomethyl acetate in acetonitrile was added dropwise. After the addition was completed, the mixture was reacted at -40 °C for 48 hours, and then the temperature was gradually raised to 0 °C. The reaction solution was poured into 100 mL of cold water and extracted three times with dichloromethane. The organic phases were combined, washed twice with 1N hydrochloric acid, washed with saturated sodium bicarbonate solution, washed with sodium chloride solution, dried with sodium sulfate, concentrated to dryness, mixed with silica gel, and separated by Flash column chromatography (eluted with dichloromethane / methanol) to obtain ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate.

[0089] Example 4: Metabolic experiment in mice

[0090] Experimental drugs: Compound 1 [((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate], T705.

[0091] Experimental subjects: KM mice (male, 27±2g) were randomly divided into 15 groups according to body weight, with 3 mice in each group.

[0092] Experimental method: T705 and compound 1 were administered orally at a dose of 0.0637mmol / 10ml / kg (equivalent to 10mg / 10ml / kg for T705). 20μL of blood was collected from the eye socket at different time points of administration, and 20μL of cationic internal standard acetonitrile solution, 20μL of anionic internal standard acetonitrile solution, and 40μL of acetonitrile were added, shaken, centrifuged at 18000g for 10min, and the supernatant was taken for LC / MS / MS injection to determine the concentrations of the corresponding test compounds and T705.

[0093] Experimental results: The drug concentration-time curve of T705 in the blood of mice after oral administration of compound 1 and T705 is shown in Figure 1 The parameter values ​​of bioavailability [AUC (umol / L*h)] and blood drug concentration Cmax of T705 and compound 1 after oral administration in mice are shown in Table 1.

[0094] Experimental conclusion: This experiment proves that the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate can be converted into T705 in mice, and the oral bioavailability of the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate administered orally to mice is significantly higher than that of T705.

[0095] Example 5: Metabolism test in monkeys

[0096] Experimental drugs: Compound [((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate] and T705.

[0097] Experimental subjects: 3 macaques.

[0098] Three macaques were given a test drug orally by gavage at 0.0637mmol / 1ml / kg (the dose was one-tenth of the dose given to mice). 50μL of monkey plasma was taken intravenously at different time points of administration, and 50μL of different concentrations of T705 acetonitrile standard solution, 50μL of anion internal standard acetonitrile solution, and 100μL of acetonitrile were added to make the analytical sample equivalent to plasma concentrations of 100, 500, 1000, 5000, and 10000ng / ml. Oscillate, centrifuge at 18000g for 10min, take the supernatant for LC / MS / MS injection, and determine the T705 concentration. After a wash period of no less than 3 days, another test drug was administered in exactly the same way and the T705 concentration was determined under exactly the same conditions.

[0099] Experimental results: The drug concentration-time curve of T705 in the blood of macaques after oral administration of compound 1 and T705 is shown in Figure 2 The parameter values ​​of bioavailability [AUC (μmol / L*h)] and blood drug concentration Cmax of T705 and compound 1 after oral administration to macaques are shown in Table 1.

[0100] Experimental conclusion: This experiment proves that the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate can be converted into T705 in monkeys, and the bioavailability of the compound (3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate after oral administration to monkeys is significantly higher than that of T705.

[0101] Table 1: Parameter values ​​of bioavailability [AUC (μmol / L*h)] and plasma concentration Cmax of T705 and compound 1 [((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate] in mice and monkeys

[0102]

[0103]

[0104] Summary: The above research results show that compound 1 [((3-carbamyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate] can effectively improve the pharmacokinetic properties of T705 under the condition of oral administration to mice, and can increase the blood concentration (Cmax) calculated by T705 in mice, and the ratio of Cmax (compound 1) / Cmax (T705) is 1.05. It can also significantly increase the oral bioavailability [AUC (μmol / L*h)] calculated by T705 in mice, and the value of AUC (compound 1) / AUC (T705) is 1.57. The research results also showed that compound 1 [((3-carbamyl-5-fluoropyrazin-2-yl)oxy)methyl isobutyrate] can effectively improve the pharmacokinetic properties of T705 under the condition of oral administration in monkeys, and can significantly increase the blood drug concentration (Cmax) calculated by T705 in monkeys, with the ratio of Cmax(compound 1) / Cmax(T705) being 2.74, and can significantly increase the oral bioavailability [AUC(μmol / L*h)] calculated by T705 in monkeys, with the value of AUC(compound 1) / AUC(T705) being 3.49.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, Formula I.

2. A pharmaceutical composition comprising the compound represented by formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 2, further comprising a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition according to claim 3, wherein The pharmaceutical composition is a solid preparation or a liquid preparation.

5. The pharmaceutical composition according to claim 3, wherein The pharmaceutical composition is an injection, an external preparation or a spray.

6. The pharmaceutical composition according to claim 3, wherein The pharmaceutical composition is a compound preparation.

7. A method for preparing the compound of formula I according to claim 1, comprising: X represents Cl, Br or I, Compound A and compound B undergo a nucleophilic substitution reaction to obtain a compound shown in formula I, wherein the compound A is 6-fluoro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide, and the compound B is chloromethyl isobutyrate, bromomethyl isobutyrate or iodomethyl isobutyrate.

8. The method according to claim 7, wherein: The nucleophilic substitution reaction is carried out in the presence of an organic base or an inorganic base.

9. The method according to claim 7, wherein: The nucleophilic substitution reaction is carried out in the presence of an organic base.

10. The method according to claim 8, wherein: The organic base is selected from triethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, diisopropylethylamine or any combination thereof.

11. The method according to claim 7, wherein: The reaction solvent of the nucleophilic substitution reaction is an organic solvent.

12. The method according to claim 11, wherein: The organic solvent is selected from dichloromethane, N,N-dimethylformamide, acetonitrile, dimethyl ether, tetrahydrofuran or any combination thereof.

13. The method according to any one of claims 7 to 12, wherein: When the compound B is chloromethyl isobutyrate, the nucleophilic substitution reaction is carried out in the presence of a catalyst.

14. The method according to claim 13, wherein: The catalyst is selected from KI, LiBr or a combination thereof.

15. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 2 to 6, or the compound prepared by the method according to any one of claims 7 to 14 in the preparation of a medicament for preventing and / or treating diseases caused by viral infection.

16. The use according to claim 15, wherein The virus is an RNA virus.

17. The use according to claim 15, wherein The virus is influenza virus, HCV virus, Bunyavirus, Bering virus, foot-and-mouth disease virus, West Nile virus, arenavirus, Western equine encephalitis virus, or yellow fever virus.

18. The use according to claim 17, wherein The influenza virus is subtype A1 influenza virus.

19. The use according to claim 15, wherein The disease caused by the viral infection is foot-and-mouth disease.

20. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of any one of claims 2 to 6, or the compound prepared by the method of any one of claims 7 to 14 in the preparation of a viral inhibitor or in the preparation of a medicament for inhibiting viral replication or reproduction in a cell.

21. The use according to claim 20, wherein The virus is an RNA virus.

22. The use according to claim 20, wherein The virus is influenza virus, HCV virus, Bunyavirus, Bering virus, foot-and-mouth disease virus, West Nile virus, arenavirus, Western equine encephalitis virus, or yellow fever virus.

23. The use according to claim 22, wherein The influenza virus is subtype A1 influenza virus.

24. The use according to claim 20, wherein The cells are mammalian cells.

25. The use according to claim 24, wherein The mammals include bovines, equines, ovines, porcines, canines, felines, rodents, and primates.

26. The use according to claim 24, wherein The mammal is a human, a cat, a dog or a pig.

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