KR20100066142A - Composition for preventing or treating picornavirus and coronavirus induced diseases containing 3,5-diaryl-4,5-dihydro pyrazol derivatives or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

PURPOSE: A composition is provided to prevent or treat diseases caused by picornavirus and coronavirus by suppressing activations of picornavirus and coronavirus. CONSTITUTION: A composition for preventing or treating diseases caused by picornavirus and coronavirus contains 3,5-diaryl-4,5-dihydro pyrazole derivative of chemical formula 1 and pharmaceutically acceptable salt as an active ingredient. The diseases caused by picornavirus and coronavirus is respiratory disease included coiling or severe acute respiratory syndrome(SARS). The daily dose of the 3,5-diaryl-4,5-dihydro pyrazole derivative is 0.1-1,500 mg.

Description

Composition for the prevention or treatment of diseases caused by picornaviruses and coronaviruses containing 3,5-diaryl-4,5-dihydropyrazole derivatives or pharmaceutically acceptable salts thereof as active ingredients for preventing or treating picornavirus and coronavirus induced diseases containing 3,5-diaryl-4,5-dihydro pyrazol derivatives or 黄 acceptable salts conjugate as an active ingredient}

The present invention relates to a composition for the prevention or treatment of diseases caused by picornavirus and / or coronavirus.

The definitions for some abbreviations used herein are as follows.

PV, Picornaviruses; RV, rhinovirus; CV, coxsackievirus; EV, enteroviruses; CoV. Coronaviruses; 3CL, 3C-like; Dabcyl, 4- (4-dimethylaminophenylazo) benzoic acid (4- (4-dimethylaminophenylazo) benzoic acid); Edans, 6-[(2-aminoethyl) amino] naphthalene-1 sulfonic acid (5-[(2-aminoethyl) amino] naphthalene-1 sulfonic acid); FRET, fluorescence resonance energy transfer.

Picornavirus, an RNA virus, is a virus that causes various diseases such as colds, foot and mouth disease, Apollo eye disease, aseptic meningitis, myocarditis, hepatitis A, respiratory diseases, digestive diseases, circulatory diseases, and skin diseases. However, there is no therapeutic agent developed to treat it to date. Most of the drugs under development are uncoating inhibitors. Picornavirus is a positive single stranded RNA virus of 7.2-8.5 Kb, a very small spherical virus of about 22-30 nm, which is unenveloped and the oldest known virus. Viruses belonging to the Picornavirus family cause a variety of diseases, such as the above-mentioned respiratory diseases, which cause health, social and economic problems. Picornaviruses form the backbone of tap water viruses and are very stable and not easy to disinfect, causing persistent viral diseases.

The Picornavirus family is the smallest known animal RNA virus, and there are seven genera, namely the genus Reinovirus, Enterovirus, Aptovirus, Carthiovirus, Hepatovirus and Dual Ecovirus. There are some other unclassified picor and viruses. Picorna virus causes various diseases such as respiratory disease, hand and foot disease, meningitis / encephalitis, acute myelitis, cardiovascular disease, hemorrhagic conjunctivitis and hepatitis.

Currently, a variety of picornaviruses are used to prevent vaccines against polyviruses (polioviruses), and only therapeutic hepatitis A vaccines have been developed. It's urgent. Since most picornaviruses cause acute diseases, it is desirable to develop them as short-term or topical treatments with relatively few side effects.

On the other hand, the corona virus is a virus that causes respiratory diseases such as nasal cold. It was first discovered in chickens in 1937, and then in humans in 1965 through animals such as dogs, pigs, and birds. Corona virus has been recognized as a pathogen that is hardly infected by humans but mainly by livestock such as dogs, pigs and cattle. Even when infected with humans, one of several viruses that cause respiratory symptoms may cause a simple cold or a bowel disease such as diarrhea in children. In the middle of March 2003, the first strain of severe acute respiratory syndrome, which first occurred in the world, causing more than 100 deaths and 3,000 patients, became known as the new (variant) coronavirus.

Severe Acute Respiratory Syndrome (SARS) is a new infectious disease caused by a new coronavirus that infects humans (TG Ksiazek, et al., N. Eng. J. Med. 348 (2003). ), 1953-1966; C. Drosten, et al., N. Eng. J. Med. 348 (2003), 1967-1976; RA Fouchier, et al., Nature 423 (2003), 240). Severe acute respiratory syndrome was classified as fatal in about 4-10% of cases reported so far. In mid-February 2003, atypical pneumonia was first discovered in Guangdong, China. With the death of 263 patients diagnosed with severe acute respiratory syndrome symptoms by mid-April 2003, more than 4400 cases have been reported from Canada, China, Hong Kong, Indonesia, Philippines, Singapore, Thailand, Vietnam, and the United States. Due to the rapid spread of the disease in many countries in a short time, the WHO issued alerts around the world and issued emergency bulletins to travelers and aircraft. Severe acute respiratory syndrome, first perceived months after infection, threatened global health and the global economy.

There is no known cure for this and no vaccine is available. Therefore, there is an urgent need to develop antiviral agents that can control or prevent severe acute respiratory syndromes in infected individuals and prevent their transmission. Nevertheless, the development of drugs capable of inhibiting both or at the same time picornavirus and coronavirus has not made much progress.

Accordingly, the present inventors are developing drugs capable of simultaneously inhibiting picornavirus and coronavirus, and 3,5-diaryl-4,5-dihydropyrazole derivatives can strongly inhibit these viruses. Find out that it can be useful for preventing or treating diseases such as colds, foot and mouth disease, apollo eye disease, aseptic meningitis, myocarditis, hepatitis A, severe acute respiratory syndrome (SARS), etc. The present invention has been completed.

An object of the present invention to provide a composition for the prevention or treatment of diseases caused by picornavirus and coronavirus.

In order to achieve the above object, the present invention is picornavirus containing a 3,5-diaryl-4,5-dihydropyrazole derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient And compositions for the prevention or treatment of diseases caused by coronaviruses:

[Formula 1]

Wherein X, Y and Z are as defined in the specification.

The 3,5-diaryl-4,5-dihydropyrazole derivatives of the general formula (1) according to the present invention are compounds which can strongly inhibit the activity of picornavirus and the activity of coronavirus at the same time. It can be usefully used to prevent or treat diseases such as colds, foot and mouth disease, apollo eye disease, aseptic meningitis, myocarditis, hepatitis A and severe acute respiratory syndrome (SARS) caused by viruses.

Hereinafter, the present invention will be described in detail.

The present invention is induced by picornaviruses and coronaviruses containing 3,5-diaryl-4,5-dihydropyrazole derivatives represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient: Provided are compositions for the prophylaxis or treatment of the disease being:

.

.

Where

X is independently or optionally selected from the group consisting of hydrogen, halogen, hydroxy, C ₁ -C ₄ straight or branched chain alkyl, C ₁ -C ₄ straight or branched chain alkoxy, cyano, amino and nitro One,

Y is hydrogen; C ₁ -C ₄ straight or branched alkyl; C ₅ -C ₇ aryl unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ straight or branched alkyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy and C ₁ -C ₄ linear or branched alkyl, one or more independently or optionally selected from the group consisting of nitrogen, oxygen and sulfur Any one selected from the group consisting of 5- or 6-membered heterocycloaryl including a hetero atom,

Z is hydrogen or halogen,

A is hydrogen; halogen; C ₅ -C ₇ aryl unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ straight or branched alkyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ straight or branched alkyl, one or more independently or selectively selected from the group consisting of nitrogen, oxygen and sulfur Is selected from the group consisting of 9 or 10-membered heterocycloarylamino C ₁ -C ₄ alkyl including heteroatoms

Preferably,

X is independently or optionally selected from the group consisting of hydrogen, fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, cyano, amino and nitro Which one is

Y is hydrogen; Methyl, ethyl, propyl, isopropyl, butyl; Phenyl unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, independent of the group consisting of nitrogen, oxygen and sulfur Any one selected from the group consisting of 5 or 6-membered heterocycloaryl including one or more selected heteroatoms, or

Z is hydrogen, fluoro, chloro or bromo,

A is hydrogen; Fluoro, chloro, bromo; Phenyl unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, independently from the group consisting of nitrogen, oxygen and sulfur or It is any one selected from the group consisting of 9 or 10 membered heterocycloarylamino C ₁ -C ₄ alkyl including one or more heteroatoms optionally selected.

More preferably,

The aryl of C ₅ -C ₇ is phenyl;

The 5- or 6-membered heterocycloalkyl is any one selected from the group consisting of pyrazolyl, imidazolyl, pyrrolyl, furyl, thienyl, pyrimidinyl and pyridyl;

In the 9- or 10-membered heterocycloarylamino C ₁ -C ₄ alkyl, the 9- or 10-membered heterocycloaryl is benzimidazolyl or indolyl.

Specific examples of 3,5-diaryl-4,5-dihydropyrazole derivatives represented by Formula 1 according to the present invention are as follows:

1) 3- (4-Bromo-phenyl) -5- (4-chloro-phenyl) -1- (3,4-dichloro-phenyl) -4-imidazol-1-yl-4,5-dihydro -1H-pyrazole;

2) [4- (3,5-diphenyl-4,5-dihydro-pyrazol-1-yl) -benzyl]-(1-propyl-1H-benzoimidazol-2-yl) -amine;

3) [4- (3,5-Diphenyl-4,5-dihydro-pyrazol-1-yl) -benzyl]-(1-ethyl-1H-benzoimidazol-2-yl) -amine; And

4) (1-Butyl-1H-benzoimidazol-2-yl)-[4- (3,5-diphenyl-4,5-dihydro-pyrazol-1-yl) -benzyl] -amine.

In addition, the composition according to the present invention as an active ingredient 3,5-diaryl-4,5-dihydropyrazole derivative represented by the formula (1) or a pharmaceutically acceptable salt thereof, as well as isomers or preparations thereof It includes all possible solvates or hydrates that may be employed.

The 3,5-diaryl-4,5-dihydropyrazole derivatives of the general formula (1) of the present invention may be used in the form of a pharmaceutically acceptable salt, and the salt may be added to the pharmaceutically acceptable free acid. Acid addition salts formed by these are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.

The acid addition salts according to the invention can be dissolved in conventional methods, for example, by dissolving a derivative of formula 1 in an excess of an aqueous solution of an acid, which salts are immiscible with a water miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. Can be prepared by precipitation. It may also be prepared by evaporating the solvent or excess acid from the mixture and then drying or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

The composition according to the present invention can be usefully used to prevent or treat diseases caused by picornavirus, and preferred examples of these diseases include respiratory diseases such as cold, foot and mouth disease, Apollo eye disease, aseptic meningitis, and the like. Diseases or digestive diseases, circulatory diseases such as myocarditis, liver diseases such as hepatitis A, and the like.

In addition, the composition according to the present invention can be usefully used for preventing or treating diseases caused by coronaviruses, and preferred examples of these diseases include cold, severe acute respiratory syndrome (SARS), and the like.

Accordingly, the present invention is directed to picornaviruses and coronaviruses containing 3,5-diaryl-4,5-dihydropyrazole derivatives represented by Formula 1 or pharmaceutically acceptable salts thereof as an active ingredient. Provided are compositions for preventing or treating a disease caused at the same time.

The 3,5-diaryl-4,5-dihydropyrazole derivatives of Formula 1 according to the present invention may simultaneously inhibit picornavirus and coronavirus.

Referring to Example 1 in which the picornavirus or coronavirus inhibitory activity of the compounds 1) to 4) corresponding to the derivative of Chemical Formula 1 according to the present invention is measured, the compounds 1) to 4) are all of the coronaviruses. It can be seen that SARS 3CL and 229E 3CL proteases and picornaviruses effectively inhibit CVB3 3C, EV71 3C and RV14 3C protease simultaneously at low concentrations of 10 μM or less. In particular, the inhibitory activity of compound 1) was excellent among these compounds. In addition, referring to Example 2 in which the inhibition constant for Compound 1) was measured, the inhibition constant K _i for Compound 1) was 0.85 ± 0.15 mM (SARS-CoV 3CL ^pro , FIG. 1), 0.56 ± 0.08 mM (HCoV -229E 3CL ^pro , FIG. 2), 0.82 ± 0.13 mM (CVB3 3C ^pro , FIG. 3), 0.3 ± 0.05 mM (EV71 3C ^pro , FIG. 4) and 0.67 ± 0.14 mM (RV14 3C ^pro , FIG. 5) All Lineweaver-Burk graphs intersect on the longitudinal axis, and the slope tends to increase with increasing compound concentration (inhibitor concentration). From this, it can be seen that the compound of the present invention and each protease perform a typical competitive inhibition reaction on the substrate.

Thus, the compounds of the present invention simultaneously inhibit the activity of picornaviruses and coronaviruses, such that diseases caused by these viruses, such as colds, foot and mouth disease, Apollo's eye disease, aseptic meningitis, myocarditis, hepatitis A, severe acute It can be usefully used for preventing or treating respiratory syndrome. Preferably they can be usefully used to treat cold or severe acute respiratory syndrome.

The composition for the prevention or treatment of diseases caused by picornavirus and coronavirus containing the derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient is various oral or parenteral dosage forms described below. It may be formulated as, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like. One or more diluents or excipients, such as extenders, wetting agents, disintegrating agents, lubricants, binders, and surfactants may be used. As a disintegrant, agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydride, etc. may be used, and as lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. As the binder, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low-substituted hydroxypropyl cellulose, and the like may be used. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, and the like may be used as diluents, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavoring agents, sweeteners, and the like may be used together.

In addition, the composition for the prevention or treatment of diseases caused by picornavirus and coronavirus containing the derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient can be parenterally administered, parenteral administration By subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. At this time, the derivative of Formula 1 or a pharmaceutically acceptable salt thereof is mixed in water with a stabilizer or buffer to prepare a parenteral formulation, and then prepared as a solution or suspension, which is a unit dosage form of ampoules or vials. It can manufacture.

The composition may be sterile or contain preservatives, stabilizers, hydrating or emulsifiers, salts for controlling osmotic pressure, adjuvants such as buffers, and other therapeutically useful substances, and conventional methods of mixing, granulating or coating. It can be formulated according to the method.

If necessary, the composition for the prevention or treatment of diseases caused by picornavirus and coronavirus containing a derivative of Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient may be prepared by other pharmaceutical agents, such as For example, it may be administered in combination with other diabetes treatments.

When formulating a composition for the prevention or treatment of diseases caused by picornavirus and coronavirus containing the derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, in the form of a unit dose, It is preferable that the active ingredient contains a derivative of Formula 1 or a pharmaceutically acceptable salt thereof in a unit dose of about 0.1 to 1,500 mg. Dosage depends on the doctor's prescription depending on factors such as the patient's weight, age and the specific nature and severity of the disease. However, the dosage required for adult treatment typically ranges from about 1 to 500 mg per day, depending on the frequency and intensity of administration. A total dosage of about 5 to 300 mg per day, usually separated by a single dose for intramuscular or intravenous administration to adults, will be sufficient, but for some patients a higher daily dosage may be desirable.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

<Reference Example>

3,5-Diaryl-4,5-dihydropyrazole derivatives of the general formula (1) according to the present invention were screened by the following procedure.

1. Reagents and Apparatus :

Dabcyl-KTSAVLQSGFRK-Edans obtained from BioGenesis (Taiwan) was used as a fluorogenic peptide substrate. Plasmid mini-prep kit, DNA gel extraction kit and NINTA resin were used from Qiagen. FXa and protein expression kits (including pET32Xa / LIC vectors and components JM109 and BL21 cells) were purchased from Novagen. All buffers and reagents used the highest grades.

2. Compound Library :

Approximately 6,800 compounds were provided by the Korea Compound Bank, all of which are low molecular organic materials.

3. Expression and Purification of Proteases :

Human coronavirus derived 3CL ^pro (including SARS-CoV and HCoV-22) and picornavirus derived 3C ^pro (including CVB3, EV71, and RV) were used for inhibitor analysis. SARS-CoV 3CL ^pro and EV71 3C ^pro were prepared as reported in the literature. For expression of CV, RV and 229E proteases, genes were cloned by PCR from viral cDNAs obtained from Chang-Gung Memorial Hospital (Tao-Yuan, Taiwan). Forward primer 5'-CATG CCATGG GCCCTGCCTTTGAGTT-3 'and backward primer 5'-GCG CTCGAG TCAATGATGATGATGATGATGTTGCTCATCATTGAAGTAGTG-3'), and forward primer 5'-CATG CCATGGG ACCAAACACAGAATTT-3 'and reverse primer 5' -GCG CTCGAG TCAATGATGATGATGATGATGTTGTTTCTCTACAAAATATTG-3 'was used in addition to C-terminal hexa-His for CV and RV proteases, respectively. These primers were designed to include Nco I and Xho I restriction portions (underlined). PCR products were digested with Nco I and Xho I, and DNA fragments cloned into pET16b (Novazen).

For the expression of 229E protease, forward primer 5'-GGTATTGAGGGTCGCGCTGGTTTGCGCAAAATGGCA-3 'and reverse primer 5'-AGAGGAGAGTTAGAGCCTTATTGCAGGTTAACACCAAACAT-3' were used. PCR products were ligation with the pET32Xa / Lic vector. Plasmids containing the respective protease genes were converted to E. coli JM109 cells, and the converted cells were Luria-Bertani (LB) agar plates containing 100 μg / mL ampicillin. Streaking.

Ampicillin-resistant colonies were selected from Luria-Bertani (LB) agar plates and incubated at 37 ° C. for 12 hours in 5 mL LB medium containing 100 μg / mL epicillin.

Gene sequence determination confirmed correct structure, and then transformed into E. coli Origami B (DE3) (Novazen) for protein expression. 5 ml of monocolon overnight culture was used in 500 ml fresh LB medium containing 25 μg / ml kanamycin and 50 ug / ml ampicillin. Cells were grown to A600 = 0.6 and induced with 1 mM isopropyl-β-thiogalactopyranoside. After 4-5 hours the cells were collected by centrifugation at 7000 g for 15 minutes.

Protein purification procedures are reported in our report: Kuo CJ, Shie JJ, Fang JM, Yen GR, Hsu JT, Liu HG, Tseng SN, Chang SC, Lee CY, Shih SR, Liang PH, Bioorg. Med. Chem. 16, 7388-7398, 2008 ; Kuo, CJ, Chi, YH, Hsu, TA, Liang, PH, Biophys. Biochem. Res. Commun. 318, 862-867, 2004 ]. Purified protein was confirmed by N-terminal sequencing and mass spectrometry. The protein concentration used in all experiments was determined by absorbance at 280 nm.

4. Primary screening

Fluorescent protein substrates (Dabcyl-KTSAVLQSGFRKME-Edans) and SARS-CoV 3CL ^pro used for ultrafast screening were prepared as described in the literature [21]. Protease was stored and stored in a buffer containing 12 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA, 7.5 mM b-ME, and 1 mM DTT at −70 ° C. prior to use. During the analysis the protease stock was diluted with the buffer to a final concentration of 50 nM and placed in a 96-well plate. For primary screening, compounds in DMSO (5 mM stock solution) were added to the wells (1 compound per well) and diluted to a final concentration of 50 μΜ. After incubation with enzyme for 10 minutes, the fluorescent substrate (final concentration 6 μM in wells) was added to initiate the reaction. The fluorescence of the fluorescent peptide substrate digested by the protease was read under a 355 nm (excitation) / 538 nm (radiation) condition with a fluorescence plate reader (ThermoLabsytems, Finland). Compounds 1) to 4) where 50% or more of enzymatic activity was inhibited at 50 μM were selected and IC ₅₀ values at 10 μM for these compounds were measured as in Example 1 below.

Example 1 Measurement of Picornavirus and Coronavirus Inhibitory Activity (IC ₅₀ )

1-1. IC ₅₀ Determination for Inhibition of SARS Protease :

For compounds 1) to 4) selected by primary screening, to determine coronavirus inhibitory activity, the effective IC ₅₀ value at 10 μM was 0.05 μM SARS protease and various inhibitor compounds ranging from 0-50 μM. The concentration was measured in a reaction mixture containing 6 μΜ fluorescent substrate in 20 mM Bis-Tris at pH 7.06. Fluorescence changes due to reaction results were tracked over time using a 96-well fluorescent plate reader. Initial rates of inhibition reactions were constructed for different inhibitory concentrations to obtain IC ₅₀ according to the following equation.

A (I) = A (0) × {1- [I / (I + IC ₅₀ )]} (1)

Wherein A (I) is the enzymatic activity of inhibitor concentration I; A (0) is enzymatic activity without inhibitor; I is the inhibitor concentration.

1-2. IC ₅₀ Determination for Other Proteases :

Using compounds 1) to 4) that inhibit SARS-CoV 3CL ^pro , to find picornavirus inhibitory activity against HCoV-229E 3CL ^pro for further coronavirus inhibitory activity, EV71 3C ^pro IC ₅₀ was measured for CVB3 3C ^pro and RV14 3C ^pro . In the assay solution, 10 mM MES at pH 6.5 and 6.0 (optimum pH for EV71 and RV14 protease, respectively) and 10 in 10 mM Hepes buffer at pH 7.5 (optimum pH for HCoV-229E and CVB3 protease). The activity of proteolytic enzymes (0.5 μM) with μM fluorescent substrates was measured at various inhibitor compound concentrations in the range of 0-50 μM. The results of Examples 1-1 and 1-2 are shown in Table 1 below.

Referring to Table 1, it can be seen that Compounds 1) to 4) simultaneously inhibited SARS 3CL and 229E 3CL protease of coronavirus and CVB3 3C, EV71 3C and RV14 3C protease of picornavirus at the same time. have. In particular, it can be seen that Compound 1) effectively inhibits the protease activity even at very low concentrations of 2.5 μM or less.

Example 2 Fikornavirus and Coronavirus Inhibition Constant (K _i ) Measurement

Inhibition constant K _i was determined at substrate and inhibitor concentrations ranging from 5-40 μM in reaction mixtures containing 0.05 mM SARS 3CL protease or 0.5 mM 229E 3CL, CVB3 3C, EV71 3C and RV14 3C protease. Fluorescence was used for this purpose. To obtain the K _i value for competitive inhibition using Equation 2 below, a Lineweaver-Burk graph of the kinetic data was obtained with a KinetAsyst II (IntelliKinetics, PA State University) computer program by nonlinear recession.

1 / V = K _m / V _m (1 + [I] / K _i ) 1 / [S] + 1 / V _m (2)

Where K _m is the Michaelis constant of the substrate, K _i is the inhibition constant, V _m is the maximum velocity, and [I] and [S] represent the concentrations of inhibitor and substrate in the reaction mixture.

For the compound 1), which was found to have the highest inhibitory activity in Example 1, a Lineweaver-Burk graph according to Equation (2) is shown in FIGS. 1 to 5.

1 to 5, the inhibition constant K _i for Compound 1) was 0.85 ± 0.15 mM (SARS-CoV 3CL ^pro , FIG. 1), 0.56 ± 0.08 mM (HCoV-229E 3CL ^pro , FIG. 2), 0.82 ± Measured at 0.13 mM (CVB3 3C ^pro , FIG. 3), 0.3 ± 0.05 mM (EV71 3C ^pro , FIG. 4) and 0.67 ± 0.14 mM (RV14 3C ^pro , FIG. 5), with all Lineweaver-Burk graphs intersecting at the longitudinal axis. As the concentration of the compound (inhibitor concentration) increased, the slope tended to increase. From this, it can be seen that the compound of the present invention and each protease perform a typical competitive inhibition reaction on the substrate. Thus, it can be seen that the compounds of the present invention significantly inhibit the activity of picornavirus and coronavirus.

Examples of preparations for compositions for the prevention or treatment of diseases caused by picornaviruses and coronaviruses containing the derivative of formula 1 according to the present invention or a pharmaceutically acceptable salt thereof as an active ingredient are exemplified below.

Preparation Example 1 Preparation of Tablet (Direct Pressing)

Active ingredient 5.0 mg

Lactose 14.1 mg

Crospovidone USNF 0.8 mg

Magnesium Stearate 0.1mg

According to a conventional method for preparing tablets, the tablets were prepared by adding the above ingredients in the contents indicated, mixing them uniformly and pressing.

Preparation Example 2 Preparation of Tablet (Wet Granulation)

Active ingredient 5.0 mg

Lactose 16.0 mg

Starch 4.0 mg

Presorbate 80 0.3 mg

Silicon dioxide 2.7 mg

Magnesium Stearate 2.0mg

After sifting the active ingredient, lactose and starch were mixed. Thereafter, the polysorbate was dissolved in pure water, and then an appropriate amount was added to the active ingredient, lactose and starch mixture, followed by atomization. After drying, the granules were granulated and then mixed with colloidal silicon dioxide and magnesium stearate. Tablets were prepared by pressing the granules.

Preparation Example 3 Preparation of Capsule

Active ingredient 5.0 mg

Lactose 14.8 mg

Polyvinylpyrrolidone 10.0 mg

Magnesium Stearate 0.2mg

According to a conventional method for preparing a capsule, the desired components were prepared by adding the above components in the amounts shown in the present invention, mixing them uniformly, and filling the gelatine capsules with an appropriate size.

Preparation Example 4 Preparation of Injection

Active ingredient 100 mg

Mannitol 180 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

Distilled water 2974 mg

According to a conventional method of preparing an injection, an injection was prepared by containing the above components in the contents shown.

1 is a Lineweaver-Burk graph of SARS 3CL protease of Compound 1), which is a kind of derivative of Formula 1 according to Example 2 of the present invention;

2 is a Lineweaver-Burk graph of 229E 3CL protease of Compound 1), which is a kind of derivative of Formula 1 according to Example 2 of the present invention;

3 is a Lineweaver-Burk graph of CVB3 3C protease of Compound 1), which is a kind of derivative of Formula 1 according to Example 2 of the present invention;

4 is a Lineweaver-Burk graph of EV71 3C protease of Compound 1), which is a kind of derivative of Formula 1 according to Example 2 of the present invention;

5 is a Lineweaver-Burk graph of RV14 3C protease of Compound 1), which is a kind of derivative of Formula 1 according to Example 2 of the present invention.

Brief description of the symbols in the drawings

◆: absence of compound 1) (inhibitor)

▲: concentration of compound 1) 5 μM

■: Concentration of Compound 1) 2.5 μM

●: Concentration of compound 1) 1 μM

□: Concentration of compound 1) 0.5 μM

△: concentration of compound 1) 0.25 μM

Claims (6)

Prevention of diseases caused by picornaviruses and coronaviruses containing 3,5-diaryl-4,5-dihydropyrazole derivatives represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient Or therapeutic compositions: [Formula 1]

(Wherein X is independently or optionally selected from the group consisting of hydrogen, halogen, hydroxy, C ₁ -C ₄ straight or branched chain alkyl, C ₁ -C ₄ straight or branched chain alkoxy, cyano, amino and nitro One, Y is hydrogen; C ₁ -C ₄ straight or branched alkyl; C ₅ -C ₇ aryl unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ straight or branched alkyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy and C ₁ -C ₄ linear or branched alkyl, one or more independently or optionally selected from the group consisting of nitrogen, oxygen and sulfur Any one selected from the group consisting of 5- or 6-membered heterocycloaryl including a hetero atom, Z is hydrogen or halogen, A is hydrogen; halogen; C ₅ -C ₇ aryl unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ straight or branched alkyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, C ₁ -C ₄ straight or branched alkyl, one or more independently or selectively selected from the group consisting of nitrogen, oxygen and sulfur 9 membered or 10 membered heterocycloarylamino C ₁ -C ₄ alkyl including a heteroatom. The compound of claim 1, wherein X is independently or optionally hydrogen, fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, cyano, amino and Any one selected from the group consisting of nitro, Y is hydrogen; Methyl, ethyl, propyl, isopropyl, butyl; Phenyl unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, independently from the group consisting of nitrogen, oxygen and sulfur or Any one selected from the group consisting of 5- or 6-membered heterocycloaryl including one or more heteroatoms optionally selected, Z is hydrogen, fluoro, chloro or bromo, A is hydrogen; Fluoro, chloro, bromo; Phenyl unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl; And unsubstituted or substituted with one or more substituents selected from the group consisting of fluoro, chloro, bromo, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, independently from the group consisting of nitrogen, oxygen and sulfur or By picornavirus and coronavirus, characterized in that it is any one selected from the group consisting of 9 or 10 atom heterocycloarylamino C ₁ -C ₄ alkyl comprising one or more heteroatoms optionally selected. A composition for preventing or treating a disease caused. The compound of claim 1 or 2, wherein the aryl of C ₅ -C ₇ is phenyl; 5- or 6-membered heterocycloalkyl is any one selected from the group consisting of pyrazolyl, imidazolyl, pyrrolyl, furyl, thienyl, pyrimidinyl and pyridyl; In alkyl of 9 or 10 membered heterocycloarylamino C ₁ -C ₄ , 9 or 10 membered heterocycloaryl is selected by picornavirus and coronavirus characterized in that it is benzimidazolyl or indolyl. A composition for preventing or treating a disease caused. According to claim 1, wherein the 3,5-diaryl-4,5-dihydropyrazole derivative represented by Formula 1 is: 1) 3- (4-Bromo-phenyl) -5- (4-chloro-phenyl) -1- (3,4-dichloro-phenyl) -4-imidazol-1-yl-4,5-dihydro -1H-pyrazole; 2) [4- (3,5-diphenyl-4,5-dihydro-pyrazol-1-yl) -benzyl]-(1-propyl-1H-benzoimidazol-2-yl) -amine; 3) [4- (3,5-Diphenyl-4,5-dihydro-pyrazol-1-yl) -benzyl]-(1-ethyl-1H-benzoimidazol-2-yl) -amine; And 4) (1-butyl-1H-benzoimidazol-2-yl)-[4- (3,5-diphenyl-4,5-dihydro-pyrazol-1-yl) -benzyl] -amine; Composition for the prevention or treatment of diseases caused by picornavirus and coronavirus, characterized in that any one selected from the group consisting of. The method of claim 1, wherein the 3,5-diaryl-4,5-dihydropyrazole derivatives of Formula 1 inhibit picornavirus and coronavirus at the same time. A composition for preventing or treating a disease caused by. The disease caused by picornavirus and coronavirus according to claim 1, wherein the disease caused by picornavirus and coronavirus is a respiratory disease including a cold or severe acute respiratory syndrome (SARS). Composition for the prevention or treatment of

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