WO2020040343A1

WO2020040343A1 - Isoxazole derivatives and preparation process thereof

Info

Publication number: WO2020040343A1
Application number: PCT/KR2018/010557
Authority: WO
Inventors: Dong Yeon Kim; Jae Soo Shin; Dae Jin Cho; Gong Yeal Lee; Hong Youb Kim; Hea Un Lee; Choong Am Ahn; Song Ei SONG; Jong Seong Park; Soung Young KANG; Man Seong Park
Original assignee: Il-Yang Pharm. Co., Ltd.
Priority date: 2018-08-24
Filing date: 2018-09-10
Publication date: 2020-02-27
Also published as: KR20200023034A

Abstract

The present invention relates to an isoxazole derivative compound of Formula (1) useful as a substance for treating respiratory viral infectious disease caused by coronavirus, in particular, Middle East respiratory syndrome-coronavirus (MERS-CoV); a pharmaceutically acceptable derivative thereof; a preparation process of the same; and a pharmaceutical composition for the treatment of coronavirus infection comprising the above comopund as an active ingredient.

Description

ISOXAZOLE DERIVATIVES AND PREPARATION PROCESS THEREOF

The present invention relates to novel isoxazole derivatives having antiviral activity against coronaviruses, especially being useful for the prevention and treatment of respiratory diseases caused by Middle East respiratory syndrome-coronavirus (MERS-CoV). The present invention also relates to a method of using the above compounds, the use of the above compounds and a pharmaceutical composition comprising the above compounds, for the treatment or prevention of Middle East respiratory syndrome-coronavirus (MERS-CoV) infection; and a process for preparing the above compounds; and synthetic intermediates used in the above preparation process.

The present invention has been completed by Project No. 2016M3A9B6916828 under the support of the Korean Ministry of Science and ICT. The Research Management Agency is National Research Foundation of Korea, and the Research Name is Biomedical Technology Development Business. The Project Name is Development of Original Technology for Treatment of Middle East Respiratory Syndrome-Coronavirus, and the Study Managing Department is Industry Academic Cooperation Foundation, Hallym University. The Research Period is from July 1, 2016 to March 31, 2021.

Coronaviruses are enveloped, positive-sense single-strand RNA viruses and have 25 to 32 kb of genome size, and so they belong to a relatively large virus among RNA viruses known up to now. Because spike proteins－which are club-shaped bumps－are embedded in a membrane, the name "coronavirus" is derived from the Latin corona, meaning halo or crown and refers to the characteristic appearance. After the first discovery from chickens in 1937, coronaviruses have been found in various birds and mammals such as bat, cat, dog, cow, pig and mouse. Coronaviruses are divided into four (4) groups (Alpha-, Beta-, Gamma- and Deltacoronavirus). Alphacoronavirus and Betacoronavirus groups primarily infect mammals, and Gammacoronavirus and Deltacoronavirus groups are discovered from birds. It has been known that coronaviruses cause various diseases such as gastrointestinal diseases and respiratory diseases.

Human coronaviruses－which infect human beings－are HCoV-229E and HCoV-OC43, discovered in the 1960s, and HCoV-NL63 (2004) and HCoV-HKU1 (2005), discovered after the severe acute respiratory syndrome (SARS) pandemic. It is known that they generally relate to upper respiratory tract infections, but may cause serious lung diseases in patients with immune deficiencies. It has been reported that coronavirus infection is increased primarily in the winter and early spring seasons, and it has been known that coronaviruses cause a significant percentage of common colds in human adults. SARS coronavirus (SARS-CoV), causing severe acute respiratory syndrome, was first discovered in 2003. According to a report of the World Health Organization (WHO), there were 8,273 patients and 775 deaths (fatality rate: about 10%) all over the world in 2002 and 2003. There were additional cases and deaths until 2004.

In September 2012, severe respiratory disease patients showing respiratory symptoms such as hyperthermia, cough, dyspnea and the like which are similar to SARS occurred, and a pathogen causing this disease was identified as a new type of coronavirus (HCoV-EMC) which is different from known viruses.

In May 2013, this novel coronavirus was classified by the name of "Middle East respiratory syndrome-coronavirus (MERS-CoV)" by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses. Because the genetic sequencing of this virus is similar to Pipistrellus Bat CoV-HKU5 and HKU4 found in bats, it was assumed that bats are the most probable infection source. However, recently an article published in The Lancet Infectious Diseases reported that all of 50 sera from Omani dromedary camels had protein-specific antibodies against MERS-CoV spike. However, in the Canary Islands 15 of 105 camels showed an antibody-positive reaction. Although the virus itself was not found, such study results mean that those camels were infected by the MERS virus or a similar virus at some time, and it is highly likely that camels are a host of the virus.

The first identified case of infection by MERS-CoV occurred in Saudi Arabia in September 2012. After that, there were 808 cases and 313 deaths (fatality rate: 34.5%) officially reported to the WHO by June 2014.

At the time of initial occurrence in patients in which infection by MERS-CoV was diagnosed definitely, the clinical symptoms of patients were very similar to those of SARS. Therefore, there was a concern that MERS-CoV had considerable association with SARS-CoV. However, after analysis of the whole genome, it turned out that MERS-CoV is a novel virus which is considerably different from SARS-CoV in terms of molecular genetics.

First of all, MERS-CoV shows a low nucleotide identity of about 55% with SARS-CoV, and these two viruses show evolutionary differences from each other in a phylogenetic analysis. SARS-CoV belongs to the B group of Betacoronavirus, and MERS-CoV belongs to the C group of Betacoronavirus. As those belonging to the Betacoronavirus group, SARS-CoV, and HCoV-HKU1 and HCoV-OC43, which are human coronaviruses, had been known.

In addition, SARS-CoV and MERS-CoV are different as for the route used to enter into cells. SARS-CoV uses ACE2 receptors distributed on ciliated cells of the human respiratory tract, and MERS-CoV uses dipeptidyl peptidase 4 (DDP4, also known as CD26) expressed in non-ciliated cells. Both receptors are mainly distributed on the human lower respiratory tract rather than the upper respiratory tract. In addition, although MERS-CoV and SARS-CoV show high fatality rates, they must be transmitted via large droplets after progression to pneumonia. As a result, it is believed that they are less contagious as compared with disease spread via aerosol such as influenza or measles. However, in the case of outbreak of SARS patients, when closely exposed to SARS patients or under the circumstance of evoking aerosolization of respirable droplets, their contagiousness can be quite high. As such, it can be said that the risk of infection is still high.

What is a first infection source of MERS-CoV and how it was transmitted to human beings and caused a disease are still unknown. However, in two (2) cases of confirmed patients with novel coronavirus infection, the patients visited a farm and contacted animals. This shows a possibility of zooanthroponosis.

The major clinical symptoms of MERS are those of pneumonia such as fever (87%), cough (89%), shortness of breath and the like, and vomiting and diarrhea (35%) also occur in some patients. Renal failure has also been reported in patients having lowered immune function, and fatality rate (34.5%) is very high. Because many cases occurred in the Middle East region (Saudi Arabia, Qatar, etc.), it is assumed that this is an infection region, but the precise infection route is still unclear. There was no evidence of the disease widely spreading between humans, but it has been confirmed that transmission can occur when family members or medical personnel are in close contact with patients. It is assumed that the incubation period is 9 to 12 days, but this varies considerably depending on patients. The Middle East Journal of Management (MEJM, February 2008) reported that the incubation period is 1.9 to 14.7 days (average 5.2 days).

Up to now, there has been no medicament with a treatment effect on MERS-CoV and vaccine. Accordingly, there is no specific method for prevention and treatment other than avoiding contact with patients suspected to have viral infection and paying close attention to personal hygiene. At present, antibiotics, ribavirin, interferon and serum of patients have been used for the treatment of MERS-CoV infection, but those therapeutic agents did not show specific effects.

Hence, there is an urgent need for developing an alternative medicament for the treatment and prevention of MERS-CoV diseases.

The present invention is intended to provide novel isoxazole derivatives having potent antiviral activity against coronaviruses, especially Middle East respiratory syndrome-coronaviruses (MERS-CoV).

The present invention is also intended to provide a pharmaceutical composition for the prevention or treatment of Middle East respiratory syndrome-coronavirus (MERS-CoV) infection, comprising the above compounds as active ingredients; and a process for preparing the above compounds.

The inventors of the present invention have studied how to solve the above-mentioned problems, and as a result they have found a compound of Formula (1) having a completely different structure from those of compounds that have been developed so far, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide an isoxazole derivative represented by the following Formula (1), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof:

[Formula 1]

wherein,

R₁ and R₂ each independently represent hydrogen; lower alkyl optionally substituted with halogen; lower alkoxy optionally substituted with halogen; or halogen; and

R₃, R₄ and R₅ each independently represent hydrogen, lower alkoxy or halogen.

The present invention further relates to a pharmaceutical composition for the prevention or treatment of coronavirus infection, comprising the above compounds of Formula (1), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof, and a pharmaceutically acceptable carrier or excipient.

The present invention further relates to the use of the above compounds of Formula (1), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof, for the prevention or treatment of coronavirus infection.

The present invention further relates to a method of preventing or treating coronavirus infection in mammals including human beings, comprising administering a therapeutically effective amount of the above compounds of Formula (1), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof, to said mammals in need thereof.

The present invention provides a compound of Formula (1) having antiviral activity against coronaviruses, especially being useful for the prevention and treatment of respiratory diseases caused by Middle East respiratory syndrome-coronaviruses (MERS-CoV); and a process for preparing the same.

The present invention also provides a composition for the prevention and treatment of respiratory viral diseases, comprising a compound of Formula (1) or a pharmaceutically acceptable salt as an active ingredient.

The compounds and compositions of the present invention can be effectively used in the prevention and treatment of respiratory diseases caused by Middle East respiratory syndrome-coronavirus (MERS-CoV).

Figure 1 is a photograph showing the results of the antiviral efficacy evaluation of the compounds of the present invention using a plaque formation inhibition test (plaque reduction assay), obtained by first infecting cells with MERS-CoV (strain isolated in Korea, 002) at the same titer and then treating the cells with the compounds at respective concentrations. EC₅₀ values can be obtained from this experiment.

Figure 2 is a graph showing the results of evaluating whether the compounds which have been selected by the antiviral efficacy evaluation screening against MERS-CoV (strain isolated in Korea, 002) affect the cell viability depending on the concentration of the compounds as effective substances. CC₅₀ values can be obtained from this experiment.

The present inventors have discovered isoxazole derivatives, compounds of the following Formula (1), exhibiting high antiviral activity against Middle East respiratory syndrome-coronavirus, for which no therapeutic agent currently exists.

Thus, in a preferred embodiment, the present invention provides the compounds of the following Formula (1) and pharmaceutically acceptable derivatives thereof:

[Formula 1]

wherein,

In one embodiment of the present invention, R₁ and R₂ each independently represent hydrogen; C₁-C₆alkyl optionally substituted with halogen; C₁-C₆alkoxy optionally substituted with halogen; or halogen; and

R₃, R₄ and R₅ each independently represent hydrogen, C₁-C₆alkoxy or halogen.

As used herein, the term "a compound of Formula (I)" (or "a compound of the present invention," etc.), unless otherwise indicated, is understood to encompass pharmaceutically acceptable derivatives of the compounds of Formula (1), including pharmaceutically acceptable salts, hydrates, solvates, prodrugs, complexes, stereoisomers or enantiomers of the compounds of Formula (1).

As used herein, the term "lower alkyl" preferably refers to straight or branched, saturated aliphatic hydrocarbon radicals containing from 1 to 12, alternatively from 1 to 8, or alternatively from 1 to 6 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl and n-henyl. The alkyl group of the present invention may be optionally substituted.

In addition, the term "alkoxy" refers to an oxygen moiety that additionally has an alkyl substituent. The alkoxy group of the present invention may be optionally substituted.

In addition, the term "lower halo alkyl" refers to straight or branched, saturated aliphatic radicals, preferably containing from 1 to 12, alternatively from 1 to 8, alternatively from 1 to 6 carbon atoms, wherein the hydrogen is substituted with halogen.

In addition, the term "halogen" means fluoro, chloro, bromo or iodo, preferably fluoro or chloro. The halogen group of the present invention may be optionally substituted.

The compounds of the present invention also include salts that are within the scope of the present invention. The compounds of the present invention, for example, the compounds of Formula (I), are understood to include salts thereof unless indicated otherwise.

As used herein, the term "salt" refers to an acidic and/or basic salt formed with inorganic and/or organic acids and bases. Salts of the compounds of the present invention may be formed, for example, by reacting a compound of the present invention with an equivalent amount of an acid or base in an aqueous medium or in a medium such as one in which a salt precipitates.

Non-limiting examples of such salts include the following: For example, acetic acid, adipic acid, benzenesulfonic acid, benzoic acid, camphoric acid, camphorsulfonic acid, citric acid, cyclamic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, bromic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, malic acid, maleic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthalene acid, nicotinic acid, trifluoroacetic acid, oxalic acid, p-toluenesulfonic acid, propionic acid, glycolic acid, succinic acid, tartaric acid, amino acid (e.g., lysine), salicylic acid, 2,2-chloroacetic acid, L-asparti acid, (+)-(1S)-camphor-10-sulfonic acid, 4-acetaamidobenzoic acid, caproic acid, cinnamic acid, gentisic acid, glutaric acid, malonic acid, mandelic acid, orthic acid, pamoic acid, aminosalicylic acid and the like may be used to form an acid addition salt. Specifically, the pharmaceutically acceptable salt of the compound of Formula (I) of the present invention is a hydrochloride salt. When a plurality of basic groups are present, they may form mono- or poly-acid addition salts.

As used herein, the term "pharmaceutically acceptable derivative" is intended to refer to pharmaceutically acceptable salts, hydrates, solvates, prodrugs, complexes, stereoisomers or enantiomers or the compounds of the present invention, that maintain the desired biological activity of the compounds and minimally exhibit or do not exhibit undesirable toxicological effects.

In addition, the present invention includes prodrugs of the compounds of the present invention. The term "prodrug" is intended to indicate a compound that is covalently bonded to a carrier (vehicle), and when the prodrug is administered to a mammalian subject, an active ingredient may be released. Release of the active ingredient may occur in vivo. Prodrugs may be prepared by techniques that would be known to those skilled in the art. An appropriate functional group in a certain compound is modified by these techniques. Such a modified functional group regenerates the original functional group in routine manipulation or in vivo. Examples of prodrugs include, but are not limited to, esters (e.g., acetate, formate and benzoate derivatives) and the like.

The compounds of the present invention show potent antiviral activity against coronavirus and thus exhibit an excellent effect for the treatment and prevention of infection of Middle East respiratory syndrome-coronavirus (MERS-CoV) and similar viruses.

In one embodiment of the present invention, one of R₁ and R₂ represents hydrogen, and the other represents lower alkyl optionally substituted with halogen, lower alkoxy optionally substituted with halogen, or halogen, preferably fluoro or chloro; and one or two of R₃, R₄ and R₅ represents each independently hydrogen, lower alkoxy or halogen.

In one embodiment of the present invention, R₁ represents methyl substituted with halogen, or methoxy substituted with halogen, R₂ represents hydrogen, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, chloro or methoxy; or R₁ represents hydrogen, R₂ represents halogen, preferably fluoro, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, chloro or methoxy.

In another embodiment of the present invention, more preferably, one of R₁ and R₂ represents hydrogen, and the other represents trifluoromethyl, fluoro, or trifluoromethoxy, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, methoxy or chloro.

In one embodiment of the present invention, R₁ represents trifluoromethyl or trifluoromethoxy, R₂ represents hydrogen, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, methoxy or chloro; or R₁ represents hydrogen, R₂ represents fluoro, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, methoxy or chloro. Compounds with the above structures are preferable.

The compounds of Formula (I) according to the present invention can be prepared by the following synthesis process, comprising the steps of:

a) reacting a compound of the following Formula (2) preferably with a hydroxylammonium chloride in the presence of a base to produce a compound of the following Formula (3);

b) chlorinating the compound of Formula (3) to produce a compound of the following Formula (4);

c) cyclizing the compound of Formula (4) to produce a compound of the following Formula (5), which is an isoxazole compound;

d) reducing the compound of Formula (5), which is an ester compound, with an alcohol to form a compound of the following Formula (6);

e) oxidizing the compound of Formula (6) with an aldehyde to form a compound of the following Formula (7); and

f) reacting the compound of Formula (7) with a compound of the following Formula (8) to form a compound of Formula (1):

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 1]

wherein R₁ to R₅ are as defined in the above.

In one embodiment of the present invention, the compounds of Formula (1) may be prepared by the following Reaction Scheme 1:

[Reaction Scheme 1]

Specifically, the starting material is a phenylaldehyde compound of Formula (2) wherein R₁ and R₂ are substituted on the benzene ring, and a commercially available one was purchased and used. The phenylaldehyde compound of Formula (2) is reacted with preferably hydroxylammonium chloride or its equivalent in the presence of a base to synthesize the benzaldehyde oxime of Formula (3), which is followed by a chlorination reaction to produce benzimidonyl chloride compound of Formula (4). Subsequently, the compound of Formula (5) can be obtained through a cyclization reaction, a commonly used synthetic method, using alkyl acetoacetate or alkyl cyanoacetate. The ester compound of Formula (5) is reduced to obtain an alcohol compound of Formula (6), which is then oxidized to obtain an aldehyde compound of Formula (7). The synthesized aldehyde compound of Formula (7) is reacted with a compound of Formula (8) to produce the final compound isoxazole derivative of Formula (1).

Isomers and solvates (e.g., hydrates) of the compounds of Formula (1) are also within the scope of the present invention. Methods of solvation are generally known in the art. Thus, the compounds of the present invention may be in the form of free hydrates or salts.

The above preparation process according to the present invention can be preferably carried out in a solvent in the presence of a base or an acid. Such solvents, acids and bases may be any conventional solvent, base or acid that does not adversely affect the reaction. For example, as a solvent, one or more selected from the group consisting of tetrahydrofuran, 1,2-dichloroethane, methylene chloride, chloroform, methanol, ethanol, isopropyl alcohol, n-butanol, tert-butanol, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, diethyl ether, toluene and dioxane can be used. In addition, for example, as a base, one or more selected from the group consisting of pyridine, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, lithium aluminum hydride, lithium borohydride, sodium nitrate and cesium carbonate can be used. In addition, for example, as an acid, one or more selected from the group consisting of trifluoroacetic acid, hydrochloric acid, nitric acid, sulfuric acid, bromic acid, zinc bromide and acetic acid can be preferably mentioned.

The starting materials used in the preparation of the compounds of the present invention according to the above method are either commercially available or readily purchasable for easy use. The reaction can be generally carried out under cooling or heating, and after completion of the reaction, the final compound may be purified through a conventional post-treatment process such as column chromatography, recrystallization and the like.

The present invention also relates to a pharmaceutical composition, treatment method and use for the treatment or prevention of coronavirus infection, comprising an effective amount of the compounds of Formula (I) or pharmaceutically acceptable derivatives thereof; and said pharmaceutical composition, treatment method and use are particularly effective against Middle East respiratory syndrome-coronavirus (MERS-CoV) infection and thus can be effectively used for the treatment of such diseases.

For the compounds of the present invention whose effects are confirmed as described above, a typical suitable dosage required for treatment as a single dose or separation dosage is in the range of about 0.01 to 750 mg per kg of body weight per day, preferably 0.1 to 100 mg, most preferably in the range of 0.5 to 25 mg. However, a specific dose level for an individual patient may vary depending on the particular compound to be used, the body weight, sex and diet of the patient, time of administration of a drug, method of administration, rate of excretion, drug mix, condition and age of the patient, and the like.

The compounds of the present invention may be directly administered for the treatment without further processing, but it is preferable to provide the active ingredient as a pharmaceutical preparation.

Accordingly, the present invention provides pharmaceutical preparations (such as pharmaceutical compositions) comprising a compound of Formula (I) or a pharmaceutically acceptable derivative thereof in a mixture with a pharmaceutically acceptable carrier and/or excipient.

In one embodiment of the present invention, the pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier. For example, said carrier may be inert and may be selected from, but are not limited to, fillers such as sugar including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; starch including corn starch, wheat starch, rice starch and potato starch; cellulose family including cellulose, methyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl methylcellulose; gelatin, polyvinylpyrrolidone and the like. In addition, in some cases, a disintegrant such as crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added, but the disintegrant is not limited thereto.

For instance, the pharmaceutical composition may further include, but is not limited to, an anti-cohesive agent, a lubricant, a wetting agent, a flavor, an emulsifier and a preservative.

In addition, the compounds or pharmaceutical compositions of the present invention can be administered by any route as desired. The compounds or pharmaceutical compositions can be administered orally or parenterally, and examples of the parenteral administration route include, but are not limited to, various routes such as transdermal, nasal, peritoneal, muscular, subcutaneous, intravenous injection and the like. Specifically, the administration route of the compounds or pharmaceutical compositions of the present invention is preferably injection and oral administration.

The injectable preparations, for example, a sterilized injectable aqueous or oleaginous (oily) suspension, may be prepared using suitable dispersing agents, wetting agents or suspending agents according to the known technique. Solvents that may be used for this purpose include water, Ringer's solution and isotonic NaCl solution, and sterilized, fixed oils are also conventionally used as a solvent or suspending medium. Any non-irritating fixed oils, including mono- or di-glycerides, can be used for this purpose, and fatty acids such as oleic acid can also be used in injectable preparations.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules, and capsules and tablets are especially useful.

Tablets and pills are preferably prepared as enteric-coated ones. Solid dosage forms can be prepared by admixing a compound of Formula (1) according to the present invention with a carrier such as one or more inert diluents including sucrose, lactose, starch, etc.; lubricants such as magnesium stearate; disintegrants, binders and the like.

The compounds of Formula (I) or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions comprising the same according to the present invention, show an effect of inhibiting replication or transmission of an RNA virus such as coronavirus.

In one embodiment of the present invention, the compounds of Formula (I) or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions comprising the same, may be administered in combination with one or more additional agents having antiviral efficacy to prevent and treat respiratory viral infectious diseases.

Specifically, the pharmaceutical compositions may be administered in combination with one or more antiviral drugs such as antibiotics, Interferon, Ribavirin or Lopinavir/Ritonavir.

Therefore, in one embodiment, the present invention provides a combination for the treatment or prevention of coronavirus infection, comprising (a) the above compound of Formula (1), or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (b) one or more other therapeutic agents selected from the group consisting of antibiotics, Interferon, Ribavirin and Lopinavir/Ritonavir. Specifically, the coronavirus is Middle East respiratory syndrome-coronavirus (MERS-CoV). In one embodiment, the combination can be administered simultaneously, separately or sequentially.

Hereinafter, the present invention will be explained in more detail based on the following preparation examples and working examples. However, these preparation examples and working examples are provided only for the purpose of facilitating understanding of the present invention, and the scope of the present invention is not intended to be limited to these examples in any way.

Preparation Example 1: Synthesis of 2-( trifluoromethyl ) benzaldehyde oxime

2-(Trifluoromethyl)benzaldehyde (34.82 g, 200.0 mmol) was dissolved in ethanol (200 mL), and sodium hydroxide (12.00 g, 300.0 mmol) dissolved in purified water (50 mL) was added thereto. Hydroxylamine hydrochloride (16.68 g, 240 mmol) dissolved in purified water (50 mL) was added thereto, and the mixture was stirred for 3 hours. After the reaction was completed, ice was added to the reaction mixture, and the resulting solid was filtered, washed with purified water (600 mL) and dried to obtain the title compound as a white solid (32.15 g, 171 mmol, 85%).

¹H-NMR (400 MHz, CDCl₃, δ) = 7.23 (dd, 1H), 7.59 (dd, 1H), 7.76 (m, 2H), 8.39 (s, 1H), 11.63 (s, 1H)

Preparation Example 2: Synthesis of 2-( trifluoromethoxy ) benzaldehyde oxime

2-(Trifluoromethoxy)benzaldehyde (38.03 g, 200.0 mmol), sodium hydroxide (12.00 g, 300.0 mmol) and hydroxylamine hydrochloride (16.6 g, 240 mmol) were reacted in the same manner as in Preparation Example 1 to obtain the title compound in a white solid (38.67 g, 188 mmol, 94%).

¹H-NMR (400 MHz, CDCl₃, δ) = 7.43 (m, 2H), 7.54 (m, 1H), 7.89 (m, 1H), 8.24 (s, 1H), 11.76 (s, 1H)

Preparation Example 3: Synthesis of 3- fluorobenzaldehyde oxime

3-Fluorobenzaldehyde (24.82 g, 200.0 mmol), sodium hydroxide (12.00 g, 300.0 mmol) and hydroxylamine hydrochloride (16.68 g, 240 mmol) were reacted in the same manner as in Preparation Example 1 to obtain the title compound in a white solid (22.48 g, 162 mmol, 81%).

¹H-NMR (400 MHz, CDCl₃, δ) = 7.32 (m, 1H), 7.56 (m, 3H), 8.20 (s, 1H)

Preparation Example 4: Synthesis of N- hydroxy -2-( trifluoromethyl ) benzimidonyl chloride

2-(Trifluoromethyl)benzaldehyde oxime (30.0 g, 158.60 mmol) was dissolved in dimethylformamide (300 mL), and then N-chlorosuccinimide (23.31 g, 174.46 mmol) was added thereto and the mixture was stirred for 15 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and ethyl acetate (1,500 mL) was added thereto. The mixture was washed with saturated aqueous sodium chloride solution (1,000 mL) and purified water (1,000 mL), respectively, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the title compound in a pale yellow solid (32.81 g, 146.70 mmol, 93%).

¹H-NMR (400 MHz, CDCl₃, δ) = 7.73 (m, 2H), 7.80 (t, 1H), 7.86 (d, 1H), 12.61 (s, 1H)

Preparation Example 5: Synthesis of N- Hydroxy -2-(trifluoromethoxy)benzimidonyl chloride

Dimethylformamide (240 mL), 4-(trifluoromethoxy)benzaldehyde oxime (20.0 g, 97.50 mmol) and N-chlorosuccinimide (14.32 g, 107.26 mmol) were reacted in the same manner as in Preparation Example 4 to obtain the title compound in a pale yellow solid (19.48 g, 81.30 mmol, 83%).

¹H-NMR (400 MHz, CDCl₃, δ) = 7.49 (m, 1H), 7.62 (m, 1H), 7.68 (dd, 1H), 12.67 (s, 1H)

Preparation Example 6: Synthesis of 3- fluoro -N- hydroxybenzimidonyl chloride

Dimethylformamide (240 mL), 3-fluorobenzaldehyde oxime (20.0 g, 143.76 mmol) and N-chlorosuccinimide (21.12 g, 158.12 mmol) were reacted in the same manner as in Preparation Example 4 to obtain the title compound in a pale yellow solid (23.32 g, 134.40 mmol, 94%).

¹H-NMR (400 MHz, CDCl₃, δ) = 7.29 (m, 1H), 7.47 (m, 1H), 7.62 (m, 2H)

Preparation Example 7: Synthesis of methyl-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carboxylate

N-Hydroxy-2-(trifluoromethyl)benzimidonyl chloride (8.0 g, 35.78 mmol) and methyl acetoacetate (8.30 g, 71.56 mmol) were dissolved in methanol (160 mL). The temperature of the reactor was cooled to -10°C and stirred for 30 minutes before slowly adding sodium methoxide (5.80 g, 107.34 mmol). The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure to remove methanol, and ethyl acetate (200 mL) was added thereto. The reaction mixture was washed with purified water (200 mL) and saturated aqueous sodium chloride solution (200 mL), respectively, and dried over anhydrous sodium sulfate. Then, the reaction mixture was concentrated under reduced pressure and purified by column chromatography to obtain the title compound in a white solid (5.79 g, 20.31 mmol, 57%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.75 (s, 3H), 3.58 (s, 3H), 7.56 (m, 1H), 7.78 (m, 1H), 7.90 (m, 1H)

Preparation Example 8: Synthesis of methyl-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboxylate

Methanol (160 mL), N-hydroxy-4-(trifluoromethoxy)benzimidonyl chloride (8.00 g, 33.39 mmol), methyl acetoacetate (7.76 g, 66.78 mmol) and sodium methoxide (5.41 g, 100.17 mmol) were reacted in the same manner as in Preparation Example 7 to obtain the title compound in a white solid (7.04 g, 23.37 mmol, 70%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.73 (s, 3H), 3.64 (s, 3H), 7.53 (m, 1H), 7.61 (m, 1H), 7.69 (m, 1H)

Preparation Example 9: Synthesis of methyl-3-(3- fluorophenyl )-5-methylisoxazole-4-carboxylate

Methanol (160 mL), 3-fluoro-N-hydroxybenzimidonyl chloride (8.00 g, 46.09 mmol), methyl acetoacetate (10.07 g, 92.18 mmol) and sodium methoxide (7.47 g, 138.27 mmol) were reacted in the same manner as in Preparation Example 7 to obtain the title compound in a white solid (7.84 g, 33.33 mmol, 72%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.59 (s, 3H), 3.92 (s, 3H), 7.22 (m, 1H), 7.43 (m, 1H), 7.55 (m, 2H)

Preparation Example 10: Synthesis of (5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazol-4-yl)methanol

Diethyl ether (15 mL) was cooled to 0°C, and lithium aluminum hydride (0.23 g, 6.00 mmol) was gradually added dropwise. Methyl-5-methyl-3-(2-(trifluoro-methyl)phenyl)isoxazole-4-carboxylate (1.42 g, 5.00 mmol) dissolved in diethyl ether (15 mL) was gradually added dropwise thereto, and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was cooled again to 0°C, and distilled water (1.00 mL) and 10% sodium hydroxide (1 mL) were added thereto. After stirring for 30 minutes, the resulting solid was filtered and the filtrate was concentrated to obtain the title compound in a pale yellow liquid (0.92 g, 3.58 mmol, 72%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.54 (s, 3H), 4.35 (s, 2H), 7.48 (d, 1H), 7.64 (m, 2H), 7.83 (t, 1H)

Preparation Example 11: Synthesis of 5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methanol

Diethyl ether (28 mL), methyl-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboxylate (3.01 g, 10.00 mmol) and lithium aluminum hydride (0.57 g, 15.00 mmol) were reacted in the same manner as in Preparation Example 10 to obtain the title compound in a pale yellow liquid (1.82 g, 6.67 mmol, 67%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.54 (s, 3H), 4.35 (s, 2H), 7.52 (d, 1H), 7.74 (m, 2H), 7.81 (t, 1H)

Preparation Example 12: Synthesis of (3-(3- fluorophenyl )-5- methylisoxazol -4-yl)methanol

Diethyl ether (20 mL), 3-(3-fluorophenyl)-4-(methoxymethyl)-5-methylisoxazole (2.00 g, 8.50 mmol) and lithium aluminum hydride (0.37 g, 9.80 mmol) were reacted in the same manner as in Preparation Example 10 to obtain the title compound in a white solid (0.91 g, 4.39 mmol, 52%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.52 (s, 3H), 4.59 (d, 2H), 7.18 (t, 1H), 7.46 (q, 1H), 7.60 (d, 1H), 7.63 (t, 1H)

Preparation Example 13: Synthesis of 5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carboaldehyde

5-Methyl-3-(2-(trifluoromethyl)phenyl)isoxazol-4-yl)methanol (20.57 g, 80.00 mmol) and triethylamine (92.90 g, 800.00 mmol) were dissolved in dimethyl sulfoxide (200 mL). A solution of sulfur trioxide pyridine complex (38.20 g, 230.00 mmol) dissolved in dimethyl sulfoxide (200 mL) was slowly added to the reaction solution, followed by stirring for 4 hours. After the reaction solution was cooled to 0°C, a 36% hydrochloric acid solution was added and acidified to have a pH value of 4.5 to 5. To the reaction solution was added water (1500 mL), washed twice with ethyl acetate (1200 mL), and the respective organic layers were combined and washed twice with a 10% aqueous solution of sodium hydroxide (750 ml). Thereafter, the organic layer was dried over anhydrous sodium sulfate, concentrated, and then purified by column chromatography to obtain the title compound in a white solid (17.5 g, 68.58 mmol, 86%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.82 (s, 3H), 7.48 (m, 1H), 7.69 (m, 2H), 7.86 (m, 1H), 9.61 (s, 1H)

Preparation Example 14: Synthesis of 5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboaldehyde

Dimethyl sulfoxide (400 mL), 5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methanol (20.1 g, 73.57 mmol), triethylamine (85.46 g, 844.55 mmol) and sulfur trioxide pyridine complex (38.64 g, 242.77 mmol) were reacted in the same manner as in Preparation Example 13 to obtain the title compound in a white solid (14.83 g, 54.69 mmol, 74%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.83 (s, 3H), 7.58 (d, 1H), 7.72 (m, 2H), 7.80 (t, 1H), 9.69 (s, 1H)

Preparation Example 15: Synthesis of 3-(3- fluorophenyl )-5- methylisoxazole -4-carboaldehyde

Dimethyl sulfoxide (300 mL), (3-(3-fluorophenyl)-5-methylisoxazol-4-yl)methane (12.20 g, 58.88 mmol), triethylamine (65.80 g, 650.26 mmol), sulfur trioxide pyridine complex (31.00 g, 194.77 mmol) were reacted in the same manner as in Preparation Example 13 to obtain the title compound in a white solid (17.5 g, 68.58 mmol, 86%).

¹H-NMR (400 MHz, CDCl₃, δ) = 2.82 (s, 3H), 7.25 (m, 1H), 7.47 (d, 1H), 7.51 (m, 2H)

Example 1: Synthesis of 4-((4-(2- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole

5-Methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carboaldehyde (5.80 g, 22.73 mmol) and 1-(2-methoxyphenyl)piperizine (4.37 g, 22.73 mmol) were dissolved in 1,2-dichloroethane (70 mL), and sodium triacetoxyborohydride (6.74 g, 31.80 mmol) was added thereto, and the mixture was stirred at room temperature for 7 hours. The reaction solution was washed with a saturated aqueous sodium carbonate solution (80 mL). The aqueous layer was collected and washed with 1,2-dichloroethane (50 mL), and the respective organic layers were combined, dried over anhydrous sodium sulfate and concentrated. After concentration, the mixture was crystallized using n-hexane (30 mL), filtered and vacuum-dried at 50°C to obtain the title compound in a white solid (7.28 g, 16.87 mmol, 74%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.32 (brs, 4H), 2.78 (brs, 4H), 3.17 (s, 2H), 3.34 (s, 3H), 3.73 (s, 3H), 6.82 (m, 2H), 6.92 (m, 2H), 7.65 (d, 1H), 7.76 (m, 2H), 7.89 (d, 1H)

Example 2: Synthesis of 4-((4-(3- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole

1,2-Dichloroethane (36 mL), 5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carboaldehyde (3.0 g, 14.6 mmol), 1-(3-methoxyphenyl)piperazine (3.3 g, 17.1 mmol) and sodium triacetoxyborohydride (5.3 g, 20.5 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.1 g, 9.7 mmol, 66%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.39 (brs, 4H), 2.64 (brs, 4H), 3.17 (s, 2H), 3.32 (s, 3H), 3.84 (s, 3H), 6.88 (m, 2H), 7.66 (m, 2H), 7.82 (m, 2H), 7.89 (d, 1H)

Example 3: Synthesis of 4-((4-(3,4- dichlorophenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole

1,2-Dichloroethane (36 mL), 5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole-4-carboaldehyde (3.0 g, 14.6 mmol), 1-(3,4-dichlorophenyl)piperazine (3.4 g, 14.6 mmol) and sodium triacetoxyborohydride (5.3 g, 20.5 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.1 g, 9.7 mmol, 66%).

¹H-NMR (400 MHz, DMSO-d6, δ) =2.34 (brs, 4H), 2.82 (brs, 4H), 3.22 (s, 2H), 3.36 (s, 3H), 6.94 (dd, 1H), 7.02 (d, 1H), 7.28 (d, 1H), 7.58 (d, 1H), 7.69 (m, 2H), 7.91 (d, 1H)

Example 4: Synthesis of 4-((4-(2- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole

1,2-Dichloroethane (44 mL), 5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboaldehyde (3.62 g, 13.35 mmol), 1-(2-methoxyphenyl)piperazine (3.05 g, 13.35 mmol) and sodium triacetoxyborohydride (3.96 g, 18.69 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (5.02 g, 11.22 mmol, 84%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.32 (brs, 4H), 2.92 (brs, 4H), 3.28 (s, 2H), 3.34 (s, 4H), 3.68 (s, 3H), 6.34 (m, 2H), 6.44 (m, 1H), 7.06 (t, 1H), 7.51 (t, 2H), 7.64 (m, 1H), 7.72 (d, 1H)

Example 5: Synthesis of 4-((4-(3- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole

1,2-Dichloroethane (36 mL), 5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboaldehyde (3.0 g, 14.6 mmol), 1-(3-methoxyphenyl)piperazine (3.3 g, 17.1 mmol) and sodium triacetoxyborohydride (5.3 g, 20.5 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.1 g, 9.7 mmol, 66%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.34 (brs, 4H), 2.81 (brs, 4H), 3.27 (s, 2H), 3.34 (s, 3H), 3.72 (s, 3H), 6.81 (m, 2H), 6.91 (m, 2H), 7.52 (m, 2H), 7.64 (m, 1H), 7.75 (m, 1H)

Example 6: Synthesis of 4-((4-(3,4- dichlorophenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole

1,2-Dichloroethane (36 mL), 5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboaldehyde (3.0 g, 14.6 mmol), 1-(3,4-dichlorophenyl)piperazine (3.4 g, 14.6 mmol) and sodium triacetoxyborohydride (5.3 g, 20.5 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.1 g, 9.7 mmol, 66%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.31 (brs, 4H), 2.97 (brs, 4H), 3.28 (s, 2H), 3.33 (s, 3H), 6.84 (dd, 1H), 7.05 (d, 1H), 7.33 (d, 1H), 7.52 (m, 2H), 7.64 (m, 1H), 7.71 (d, 1H)

Example 7: Synthesis of 3-(3- fluorophenyl )-4-((4-(2- methoxyphenyl ) piperazin -1-yl)methyl)-5-methylisoxazole

1,2-Dichloroethane (60 mL), 3-(3-fluorophenyl)-5-methylisoxazole-4-carboaldehyde (3.50 g, 17.06 mmol), 1-(2-methoxyphenyl)piperazine (3.30 g, 17.16 mmol) and sodium triacetoxyborohydride (5.90 g, 27.84 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.62 g, 12.11 mmol, 71%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.47 (s, 3H), 2.56 (t, 4H), 2.95 (t, 4H), 3.34 (s, 2H), 3.77 (s, 3H), 6.89 (m, 4H), 7.34 (t, 1H), 7.56 (q, 1H), 7.78 (d, 1H), 7.95 (d, 1H)

Example 8: Synthesis of 3-(3- fluorophenyl )-4-((4-(3- methoxyphenyl ) piperazin -1-yl)methyl)-5-methylisoxazole

1,2-Dichloroethane (60 mL), 3-(3-fluorophenyl)-5-methylisoxazole-4-carboaldehyde (3.00 g, 14.62 mmol), 1-(3-methoxyphenyl)piperazine (3.30 g, 17.16 mmol) and sodium triacetoxyborohydride (5.90 g, 27.84 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.68 g, 12.27 mmol, 84%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.47 (s, 3H), 2.54 (t, 4H), 3.12 (t, 4H), 3.32 (s, 2H), 3.70 (s, 3H), 6.36 (d, 1H), 6.44 (s, 1H), 6.51 (d, 1H), 7.10 (t, 1H), 7.34 (t, 1H), 7.56 (q, 1H), 7.77 (d, 1H), 7.93 (d, 1H)

Example 9: Synthesis of 4-((4-(3,4- dichlorophenyl ) piperazin -1- yl )methyl)-3-(3-fluorophenyl)-5-methylisoxazole

1,2-Dichloroethane (60 mL), 3-(3-fluorophenyl)-5-methylisoxazole-4-carboaldehyde (3.00 g, 14.62 mmol), 1-(3,4-dichlorophenyl)piperazine (3.40 g, 14.71 mmol) and sodium triacetoxyborohydride (5.30 g, 25.01 mmol) were reacted in the same manner as in Example 1 to obtain the title compound in a white solid (4.11 g, 9.78 mmol, 67%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.46 (s, 3H), 2.52 (t, 4H), 3.18 (t, 4H), 3.35 (s, 2H), 6.92 (d, 1H), 7.11 (d, 1H), 7.35 (m, 2H), 7.55 (q, 1H), 7.76 (d, 1H), 7.90 (d, 1H)

Example 10: Synthesis of 4-((4-(2- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole hydrochloride

4-((4-(2-Methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole (0.95 g, 2.20 mmol) was dissolved in acetone (10 mL) and was cooled to 0°C, and then ethanolic hydrogen chloride (10%, 0.80 g, 2.20 mmol) was slowly added dropwise. The reaction mixture was stirred at room temperature for 8 hours, filtered and dried to obtain the title compound in a white solid (0.94 g, 2.01 mmol, 91%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.75 (s, 3H), 2.85 (m, 2H), 3.10 (t, 2H), 3.34 (m, 2H), 3.42 (m, 2H), 7.84 (m, 3H), 7.98 (m, 1H), 11.46 (s, 1H),

Example 11: Synthesis of 4-((4-(3- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole hydrochloride

Acetone (10 mL), 4-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole (0.98 g, 2.27 mmol), and ethanolic hydrogen chloride (11%, 0.74 g, 2.27 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (0.82 g, 1.75 mmol, 77%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.75 (s, 3H), 2.82 (m, 2H), 3.21 (t, 2H), 3.30 (m, 2H), 3.44 (m, 2H), 3.69 (s, 3H), 4.15 (s, 2H), 6.42 (m, 2H), 6.49 (d, 1H), 7.11 (t, 1H), 7.82 (m, 3H), 7.96 (s, 1H), 11.53 (s, 1H)

Example 12: Synthesis of 4-((4-(3,4- dichlorophenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole hydrochloride

Acetone (15 mL), 4-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole (1.50 g, 3.19 mmol), and ethanolic hydrogen chloride (11%, 1.06 g, 3.19 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (1.32 g, 2.61 mmol, 82%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.73 (s, 3H), 2.79 (m, 2H), 3.23 (t, 2H), 3.33 (m, 2H), 3.46 (m, 2H), 3.72 (s, 3H), 4.22 (s, 2H), 6.92 (d, 1H), 7.16 (d, 1H), 7.11 (m, 2H), 7.82 (m, 3H), 7.92 (s, 1H), 11.49 (s, 1H)

Example 13: Synthesis of 4-((4-(2- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole hydrochloride

Acetone (23 mL), 4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (3.37 g, 8.43 mmol), and ethanolic hydrogen chloride (11%, 2.79 g, 8.43 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (3.24 g, 6.70 mmol, 79%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.74 (s, 3H), 2.80 (brs, 2H), 3.20(m, 4H), 3.63 (m, 2H), 3.69 (s, 3H), 4.21 (s, 2H), 6.41 (m, 2H), 6.47 (d, 1H), 7.10 (t, 1H), 7.58 (m, 2H), 7.75 (m, 2H), 11.70 (s, 1H)

Example 14: Synthesis of 4-((4-(3- methoxyphenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole hydrochloride

Acetone (25 mL), 4-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (4.17 g, 9.32 mmol), and ethanolic hydrogen chloride (11%, 3.09 g, 9.32 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (3.93 g, 8.12 mmol, 87%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.74(s, 3H), 2.84 (brs, 2H), 3.12 (t, 2H), 3.25 (m, 4H), 3.71(s, 3H), 4.12 (s, 2H), 6.85 (m, 2H), 6.93 (d, 1H), 7.00 (m, 1H), 7.62 (m, 2H), 7.75 (m, 2H), 11.61 (s, 1H)

Example 15: Synthesis of 4-((4-(3,4- dichlorophenyl ) piperazin -1- yl )methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole hydrochloride

Acetone (33 mL), 4-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole (5.55 g, 11.41 mmol), and ethanolic hydrogen chloride (11%, 3.78 g, 11.41 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (5.32 g, 10.18 mmol, 89%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.73 (s, 3H), 2.81 (brs, 2H), 3.20 (m, 4H), 3.72 (t, 2H), 4.21(s, 2H), 6.90(dd, 1H), 7.14 (d, 1H), 7.41 (d, 1H), 7.85 (m, 2H), 7.74 (m, 2H), 11.47 (s, 1H)

Example 16: Synthesis of 3-(3- fluorophenyl )-4-((4-(2- methoxyphenyl ) piperazin -1-yl)methyl)-5-methylisoxazole hydrochloride

Acetone (2 mL), 3-(3-fluorophenyl)-4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-5-methylisoxazole (0.20 g, 0.52 mmol), and ethanolic hydrogen chloride (10%, 0.19 g, 0.52 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (0.13 g, 0.31 mmol, 60%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.69 (s, 3H), 2.97 (brs, 4H), 3.31 (brs, 4H), 4.42 (s, 2H), 6.98 (m, 4H), 7.43 (t, 1H), 7.60 (m, 3H), 10.75 (s, 1H)

Example 17: Synthesis of 3-(3- fluorophenyl )-4-((4-(3- methoxyphenyl ) piperazin -1-yl)methyl)-5-methylisoxazole hydrochloride

Acetone (2 mL), 3-(3-fluorophenyl)-4-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-5-methylisoxazole (0.20 g, 0.52 mmol), and ethanolic hydrogen chloride (10%, 0.19 g, 0.52 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (0.19 g, 0.46 mmol, 89%).

¹H-NMR (400 MHz, DMSO-d6, δ) = 2.68 (s, 3H), 2.98 (m, 4H), 3.69 (s, 3H), 4.43 (s, 2H), 6.45 (m, 3H), 7.42 (t, 1H), 7.59 (m, 3H), 10.69 (s, 1H)

Example 18: Synthesis of 4-((4-(3,4- dichlorophenyl ) piperazin -1- yl )methyl)-3-(3-fluorophenyl)-5-methylisoxazolehydrochloride

Acetone (2 mL), 4-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-3-(3-fluorophenyl)-5-methylisoxazole (0.20 g, 0.48 mmol), and ethanolic hydrogen chloride (10%, 0.18 g, 0.48 mmol) were reacted in the same manner as in Example 10 to obtain the title compound in a white solid (0.15 g, 0.33 mmol, 68%).

¹H-NMR(400 MHz, DMSO-d6, δ) = 2.67 (s, 3H), 2.90 (brs, 2H), 3.13 (t, 2H), 3.30 (brs, 2H), 3.76 (brs, 2H), 4.41 (s, 2H), 6.92 (d, 1H), 7.16 (d, 1H), 7.42 (m, 2H), 7.60 (m, 3H) 11.03 (s, 1H)

Experimental Example 1: Antiviral efficacy evaluation test

In this experiment, cells are infected with MERS-CoV (strain isolated in Korea, 002) at the same titer, and the subject compound is added to the overlay medium at each concentration, and the antiviral efficacy of the compound is evaluated by the plaque formation test method. The results of this experiment can be used to determine the efficacy of a compound against viruses by assessing the maximum dilution concentration of a compound that reduces by 50% or more the number of plaques formed in the negative control group that has been infected with only viruses, thereby measuring the EC₅₀ value.

1) Cell line

- Vero cells: 1 Х 10⁶/well (6-well plate)

2) Virus

- MERS-CoV, strain isolated in Korea (MERS-CoV/KOR/KNIH/002_05_2015)

- Virus titer: 7.75 Х 10⁶ PFU/ml

- Virus inoculation titer: 100 PFU/100μl

3) Plaque formation inhibitory test evaluation

- Basal medium: MEM F12, 5% NaHCO₃ 1ml, DEAE-Dextran

4) Test method

On the day before the test, the Vero cell line was inoculated on a 6-well plate at 1Х10⁶/well and cultured in a 5% CO₂ incubator at 37°C for 24 hours. The next day, the compounds were diluted in 2-fold serial dilution from 100 μM to 3.13 μM using the basal medium for plaque test, thereby preparing an overlay medium. Then, MERS-CoV was diluted in PBS to be 100 PFU/100 μl. The cells were washed with PBS, inoculated (infected) with MERS-CoV and allowed to react for 1 hour in a 5% CO₂ incubator at 37°C with shaking the cells well at 15-minute intervals so that they did not dry out. After suctioning the inoculated MERS-CoV, the overlay medium containing the compounds was mixed with oxoid agar and applied to the 6-well plate in 2 ml each from the low concentration. After incubation for 72 hours in a 5% CO₂ incubator at 37°C, cell staining was carried out using crystal violet, and plaque formation was observed by cell staining.

Experimental Example 2: Cytotoxicity evaluation

The compounds of Formula (1) are diluted stepwise by concentration and inoculated into the cell line (Vero) which is sensitive to MERS-CoV, and the maximum dilution concentration showing at least 50% of the survival difference compared with the negative control group (a test group with no treatment of compounds) is determined. In this experiment, it is evaluated whether or not the compound itself affects cell viability.

In Experimental Example 1, the compounds of Formula (1) were screened by in vitro efficacy against MERS-CoV (strain isolated in Korea, 002), and some compounds showing excellent effects were evaluated as to whether the compound itself affects cell viability in this experiment.

The results of this experiment are necessary to obtain selectivity index (SI) based on antiviral efficacy and cytotoxicity against MERS-CoV.

1) Cell line

- Vero cells: 4 Х 10⁴/well (96-well plate)

2) Cell viability evaluation

- WST-1 (Roche, Cat. No.: 11 644 807 001)

- Absorbance (440 nM to 600 nM)

3) Test method

On the day before the test, the Vero cell line was sub-cultured in a 96-well plate at 1Х10⁴/100 μl and cultured in a 5% CO₂ incubator at 37°C for 24 hours. After 24 hours, the compounds of Formula (1) to be evaluated were diluted in 2-fold serial dilution from 400 μM to 0.195 μM using a serum-free DMEM medium, and then inoculated into the Vero cell line prepared. The cell line should be washed once with PBS before inoculating the diluted active substance. After inoculation, the cell line was allowed to react for 72 hours in a 5% CO₂ incubator at 37°C. To measure cell viability, the cell line was treated with WST-1 in 10 μl per well (based on a total volume of 100 μl), and reacted for 2 hours in a 5% CO₂ incubator at 37°C. After 2 hours, the absorbance (440 nM, 600 nM) was measured to determine the CC₅₀ value relative to the negative control group which was not treated with the active substance.

From the results of Experimental Example 1, as shown in Figure 1, the concentration of the compound which reduces by 50% or more the number of plaques formed in the control group infected with only virus could be confirmed by counting the number of plaques. EC₅₀ values were measured using graphpad prism software as shown in [Table 1].

As a result of the experiment, it was found that the compound of Example 16 inhibited plaque formation at the lowest concentration of 1.15 μM; the compound of Example 10 inhibited plaque formation at the second lowest concentration of 17.50 μM; the compound of Example 11 inhibited plaque formation at the concentration of 22.26 μM; the compound of Example 18 inhibited plaque formation at the concentration of 67.68 μM; and the compound of Example 17 inhibited plaque formation at the concentration of 1215 μM.

In Experimental Example 2, compounds showing good efficacy were selected and inoculated into the cell line, followed by culturing for 72 hours. The reason for culturing the cell line for 72 hours was to be consistent with the reaction time of 72 hours between the compound and the cell line in the plaque formation inhibition test (plaque reduction assay). As a result, the viability of Vero cells could be evaluated according to the concentration of the compound showing good efficacy as shown in Figure 2. In addition, CC₅₀ values for compounds with good efficacy could be measured using graphpad prism software as shown in [Table 2].

As a result of this experiment, as shown in [Table 2], the three active substance compounds except for Examples 16 and 18 showed cell viability of 50% or more at a concentration of about 30 μM or less, and based on this, the optimum concentration of the active substance showing cytotoxicity could be analyzed. Based on the results, the selectivity index (SI) for the evaluation of antiviral efficacy against MERS-CoV could be measured.

Based on the results of Experimental Example 1 and Experimental Example 2, the selectivity index (SI) of the antiviral agent could be obtained using the CC₅₀ and EC₅₀ values as shown in [Table 3]. As shown in [Table 3], the compound of Example 10 exhibited the highest SI value of 2.89, the compound of Example 11 exhibited the SI value of 2.10, and the compound of Example 17 exhibited the SI value of 0.037. The antiviral efficacies of the compounds were in this order. In addition, the growth characteristics analysis results indicated that all of the compounds showing good efficacy showed such an efficacy in a dose-dependent manner.

In view of the results of the above experiments, it could be assumed from the plaque formation inhibition test that the compounds with good efficacy which had been selected through antiviral screening prevent the virus from re-infecting new cells after first infection into the cells. In addition, it could be confirmed that the inhibition of plaque formation is due to the antiviral efficacy of the active substance itself, rather than its cytotoxicity, in view of the fact that the EC₅₀ values are generally lower than the concentrations which reduce cell viability by 50% or more.

The antiviral efficacy test of the compounds prepared in the present invention was measured in the above Experimental Examples 1 and 2 using the compounds of the Examples in the present specification. As a result, it was confirmed that the compounds have chemical structures exhibiting antiviral ability.

The efficacy of the Example compounds was confirmed by the plaque formation inhibition test (plaque-reduction assay, 50% effect concentration, EC₅₀) in order to screen for compounds showing antiviral efficacy after virus infection in the Example compounds of the present invention, and compounds showing good efficacy could be identified.

The cytotoxicity concentration evaluation test (50% cytotoxic concentration, CC₅₀) was carried out for the compounds identified as effective among the Example compounds. From this test, the CC₅₀ and SI (Selectivity Index) values were measured, and the results are shown in [Table 1], [Table 2] and [Table 3].

1) EC₅₀ (Effective Concentration 50%): The minimum concentration of the compound that reduces by more than half the number of plaques in the control group

2) CC₅₀ (Cytotoxicity Concentration 50%): The maximum concentration of the compound that reduces by more than half the number of cells in the control group

3) SI (Selectivity Index): The value shown by CC₅₀/EC₅₀

[Table 1]

EC₅₀ values

[Table 2]

CC₅₀ values

[Table 3]

The present invention provides novel isoxazole derivative compounds having potent antiviral activity against coronaviruses, processes for preparing these compounds, and compositions comprising these compounds as active ingredients, which enable development of an antiviral agent for the prevention or treatment of viral respiratory diseases caused by coronavirus infection, particularly by Middle East respiratory syndrome-coronavirus (MERS-CoV) infection.

Claims

A compound of the following Formula (1), or a pharmaceutically acceptable salt, hydrate or solvate thereof:

[Formula 1]

wherein,

R₁ and R₂ each independently represent hydrogen; C₁-C₆alkyl optionally substituted with halogen; C₁-C₆alkoxy optionally substituted with halogen; or halogen; and

R₃, R₄ and R₅ each independently represent hydrogen, C₁-C₆alkoxy or halogen.
The compound of Claim 1, wherein

one of R₁ and R₂ represents hydrogen, and the other represents C₁-C₆alkyl optionally substituted with halogen, C₁-C₆alkoxy optionally substituted with halogen, or halogen; and

one or two of R₃, R₄ and R₅ represents each independently hydrogen, C₁-C₆alkoxy or halogen.
The compound of Claim 2, wherein

R₁ represents methyl substituted with halogen, or methoxy substituted with halogen, R₂ represents hydrogen, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, chloro or methoxy; or

R₁ represents hydrogen, R₂ represents halogen, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, chloro or methoxy.
The compound of Claim 1, wherein

one of R₁ and R₂ represents hydrogen, and the other represents trifluoromethyl, fluoro or trifluoromethoxy; and

one or two of R₃, R₄ and R₅ represents each independently hydrogen, methoxy or chloro.
The compound of Claim 4, wherein

R₁ represents trifluoromethyl or trifluoromethoxy, R₂ represents hydrogen, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, methoxy or chloro; or

R₁ represents hydrogen, R₂ represents fluoro, and one or two of R₃, R₄ and R₅ represents each independently hydrogen, methoxy or chloro.
The compound of Claim 1, wherein the compound is selected from the group consisting of the following compounds:

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole;

4-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole;

4-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethyl)phenyl)isoxazole;

4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole;

4-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole;

4-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-5-methyl-3-(2-(trifluoromethoxy)phenyl)isoxazole;

3-(3-fluorophenyl)-4-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-5-methylisoxazole;

3-(3-fluorophenyl)-4-((4-(3-methoxyphenyl)piperazin-1-yl)methyl)-5-methylisoxazole; and

4-((4-(3,4-dichlorophenyl)piperazin-1-yl)methyl)-3-(3-fluorophenyl)-5-methylisoxazole,

or a pharmaceutically acceptable salt, hydrate or salvate thereof.
The compound of Claim 6, wherein the pharmaceutically acceptable salt is hydrochloride salt.
A process for preparing a compound of Formula (1) as defined in Claim 1, comprising the following steps:

a) reacting a compound of the following Formula (2) with a hydroxylammonium chloride in the presence of a base to produce a compound of the following Formula (3);

b) chlorinating the compound of Formula (3) to produce a compound of the following Formula (4);

c) cyclizing the compound of Formula (4) to produce a compound of the following Formula (5), which is an isoxazole compound;

d) reducing the compound of Formula (5), which is an ester compound, with an alcohol to form a compound of the following Formula (6);

e) oxidizing the compound of Formula (6) with an aldehyde to form a compound of the following Formula (7); and

f) reacting the compound of Formula (7) with a compound of the following Formula (8) to form a compound of Formula (1):

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 1]

wherein R₁ to R₅ are as defined in Claim 1.
The process of Claim 8, wherein the preparation process of a compound of Formula (1) is carried out by using one or more solvents selected from the group consisting of tetrahydrofuran, 1,2-dichloroethane, methylene chloride, chloroform, methanol, ethanol, isopropyl alcohol, n-butanol, tert-butanol, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, ethyl acetate, diethyl ether, toluene and dioxane.
The process of Claim 8, wherein the preparation process of a compound of Formula (1) is carried out by using one or more bases selected from the group consisting of pyridine, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, lithium aluminum hydride, lithium borohydride, sodium nitrate and cesium carbonate.
The process of Claim 8, wherein the preparation process of a compound of Formula (1) is carried out by using one or more acids selected from the group consisting of trifluoroacetic acid, hydrochloric acid, nitric acid, sulfuric acid, bromic acid, zinc bromide and acetic acid.
A pharmaceutical composition for the treatment or prevention of coronavirus infection in a subject, comprising a compound of Formula (1), or a pharmaceutically acceptable salt, hydrate or solvate thereof as claimed in any one of Claims 1 to 7, and a pharmaceutically acceptable carrier or excipient
The pharmaceutical composition of Claim 12, wherein the coronavirus is Middle East respiratory syndrome-coronavirus (MERS-CoV).
The pharmaceutical composition of Claim 12, wherein the subject is an animal.
The pharmaceutical composition of Claim 14, wherein the animal is a mammal.
The pharmaceutical composition of Claim 15, wherein the mammal is a human.
A combination for the treatment or prevention of coronavirus infection, comprising

a) a compound of Formula (1), or a pharmaceutically acceptable salt, hydrate or solvate thereof as claimed in any one of Claims 1 to 7; and

b) one or more other therapeutic agents selected from the group consisting of antibiotics, Interferon, Ribavirin and Lopinavir/Ritonavir.
The combination of Claim 17, wherein the coronavirus is Middle East respiratory syndrome-coronavirus (MERS-CoV).