KR20200023034A - Isoxazole derivatives and preparation process thereof - Google Patents

Abstract

The present invention relates to: an isoxazole derivative compound of chemical formula 1, useful as a substance for treating s respiratory viral infection disease coronavirus, in particular, Middle East respiratory syndrome coronavirus (MERS-CoV); a pharmaceutically acceptable derivative thereof; a manufacturing method thereof; and a pharmaceutical composition for treating coronavirus infection, which contains the compound as an active component.

Description

Isoxazole derivatives and preparation method thereof

The present invention relates to novel isoxazole derivatives which have antiviral activity against coronaviruses and are particularly useful for the prevention and treatment of respiratory diseases caused by Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The present invention also provides methods, uses, pharmaceutical compositions comprising these compounds, methods of preparing these compounds, and methods of using these compounds for the treatment or prevention of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection. It relates to synthetic intermediates.

Coronaviruses are enveloped, single-stranded, positive RNA viruses, with genome sizes ranging from 25-32 kb, which are relatively large among RNA viruses known to date. Spike protein, a club-like protrusion on the outer shell, has a flame or crown-specific structure, and the virus name is derived from the Latin Corona. Since it was first discovered in chicken in 1937, it has been found in a variety of birds and animals, including bats, birds, cats, dogs, cows, pigs, and rats, with four groups (α-, β-, γ-, and δ-coronavirus). Divided by. The alpha and beta coronavirus groups are primarily infected with mammals and the gamma and delta coronavirus groups are found in birds. Coronaviruses are known to cause a variety of diseases in animals, such as gastrointestinal and respiratory diseases.

Human coronaviruses infected with humans include HCoV-229E and HCoV-OC43, which were discovered in the 1960s, and HCoV-NL63 (2004) and HCoV-HKU1 (2005), which were discovered after the SARS pandemic. It is known to cause severe lung disease in patients with immunodeficiency. It is reported that the infection rate of coronavirus increases mainly in winter or early spring, and it is known that the proportion of coronaviruses among adult cold patients is the causative agent. In 2003, the first SARS-CoV was discovered, causing Severe Acute Respiratory Syndrome (SARS), and the World Health Organization (WHO) reported that 8,273 patients worldwide from 2002 to 2003 And 775 deaths (approximately 10% mortality rate) occurred, and by 2004 additional cases and deaths were reported.

In September 2012, a patient with severe respiratory illness, which showed respiratory symptoms such as high fever, cough, and shortness of breath, was identified as a new coronavirus (HCoV-EMC).

The new coronavirus was classified as "Middle East respiratory syndrome-coronavirus (MERS-CoV)" by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses in May 2013. Genetic sequencing is similar to the Pipistrellus Bat CoV-HKU5 and HKU4 found in bats, suggesting that bats are the most likely source of infection, but in a recent paper published in the journal Lancet Infectious Diseases, Oman Analysis of blood samples from 50 dromedary camels revealed all traces of MERS-CoV antibody, but in the Canary Islands, 15 out of 105 tested positive for the antibody, although the virus itself was not found. At which point they were infected with MERS or similar virus And the possibility that the virus host camels showed very high.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection was officially reported to WHO worldwide in June 2012 after the first infection was confirmed in Saudi Arabia in September 2012, with 808 deaths and 313 deaths worldwide. (34.5%).

There was a concern that the patient's clinical symptoms were similar to SARS at the time of the initial confirmation of MERS-CoV. However, the molecular genetics of the respiratory syndrome coronavirus (MERS-CoV) were significantly correlated with SARS coronavirus. It turns out that it is a new virus that is quite different from SARS.

First, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) showed 55% lower nucleotide homology with SARS Coronavirus, and in the systematic analysis, the two viruses showed different evolutionary differences. Middle Respiratory Syndrome Coronavirus (MERS-CoV) was in the C group of beta coronavirus. Belonging to the beta coronavirus group are SARS coronavirus and human coronaviruses HCoV-HKU1 and HCoV-OC43.

SARS and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) also show different pathways to enter the cell. SARS virus has an ACE2 receptor that is distributed in the ciliary cells of human airways, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is non-islet. Intracellular DDP4 (also called CD26) is used. Both receptors are distributed more in the lower airways than in the upper respiratory tract of humans, and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and SARS virus have a high mortality rate, but since they progress to pneumonia, they must spread through large droplets of influenza It suggests that infectivity can be as low as compared to diseases that spread to aerosols, as in the case of measles. However, in case of SARS, infectious risk is still high because the propagation power can be considerably higher in the case of close exposure to SARS or in aerosolization of respiratory droplets.

It is not yet known what causes the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) to be transmitted and caused by humans. Two of the newly confirmed cases of new coronavirus were identified as farm visits and animal contact. This shows the potential for concomitant transmission of new coronaviruses.

The main clinical symptoms are pneumonia with fever (87%), cough (89%) and shortness of breath. Some patients also have vomiting and diarrhea (35%). Renal failure also occurs in patients with reduced immune function, with a very high mortality rate of 34.5%. It is estimated to occur in the Middle East (Saudi Arabia, Qatar, etc.), and these areas are considered infected areas, but the exact path of infection is not known. However, there has been no evidence of widespread infection among humans, but it has been confirmed that it spreads in close contact with family and medical staff. The incubation period is estimated to be 9 to 12 days, and there are many differences in incubation periods for each person. In the MEJM (2008.02) report, it was 1.9 to 14.7 days (average 5.2 days).

So far, no drug or preventive vaccine has been developed that has been shown to have a therapeutic effect on the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Therefore, to date, there is no special treatment and prevention method except avoiding contact with patients suspected of viral infection and thoroughly sanitation. Currently, antibiotics, ribavirin, interferon, and serum of patients have been used to treat Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infections, but these drugs do not have specific effects.

Therefore, the development of alternative drugs for the treatment and prevention of diseases for the Middle East respiratory syndrome coronavirus (MERS-CoV) is very urgent.

The present invention provides a novel isoxazole derivative having potent antiviral activity against coronaviruses, particularly Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

In another aspect, the present invention is to provide a pharmaceutical composition for the prevention or treatment of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection comprising the compound as an active ingredient and a method for producing these compounds.

The present inventors have studied to solve the above problems, and as a result, have found a compound of formula 1 having a completely different structure from the compound structure that has been developed so far, and completed the present invention.

Accordingly, an object of the present invention is to provide an isoxazole derivative of Formula 1, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof.

<Formula 1>

In the above formula

R ₁ and R ₂ are each independently hydrogen; Lower alkyl optionally substituted with halogen; Lower alkoxy optionally substituted with halogen; Or halogen;

R ₃ , R ₄ and R ₅ each independently represent hydrogen, lower alkoxy, or halogen.

The present invention also comprises a compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, prodrug or complex thereof, and a pharmaceutically acceptable carrier or excipient, a medicament for preventing or treating coronavirus infection To a pharmaceutical composition.

The present invention also relates to the use of a compound of formula 1, a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof, for preventing or treating a coronavirus infection.

The invention also provides a therapeutically effective amount of a compound of Formula 1, a pharmaceutically acceptable salt, hydrate, solvate, prodrug, or complex thereof, in a mammal, including a person in need of treatment or prevention of a coronavirus infection. A method of preventing or treating a coronavirus infection in a mammal comprising administering.

The present invention provides compounds of formula (1) having a antiviral activity against coronaviruses, particularly useful for the prevention and treatment of respiratory diseases caused by Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and methods for their preparation.

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

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

Figure 1 is first infected with MERS-CoV (National isolate, 002) of the same titer and then treated with the compound of the present invention at each concentration, and then the antiviral of the compound through a plaque reduction assay (plaque reduction assay) The result of measuring the efficacy evaluation is a photograph. This gives an EC ₅₀ value.
FIG. 2 is a graph obtained by evaluating whether the compound itself affects cell viability according to the concentration of the active substance of the selected compound through an antiviral efficacy screening test for MERS-CoV (Domestic isolate, 002) to be. The CC ₅₀ value can be obtained.

The present inventors have discovered isoxazole derivatives, which are compounds of formula (1) exhibiting high antiviral activity against Middle East Respiratory Syndrome coronavirus, in which no therapeutic agent currently exists.

Thus, as a particularly preferred aspect, the present invention provides a compound of formula 1 and pharmaceutically acceptable derivatives thereof.

<Formula 1>

In the above formula

R ₁ and R ₂ are each independently hydrogen; Lower alkyl optionally substituted with halogen; Lower alkoxy optionally substituted with halogen; Or halogen,

R ₃ , R ₄ and R ₅ each independently represent hydrogen, lower alkoxy, or halogen.

In one embodiment of the invention, R ₁ and R ₂ are each independently hydrogen; C ₁ -C ₆ alkyl optionally substituted with halogen; C ₁ -C ₆ alkoxy optionally substituted with halogen; Or halogen;

R ₃ , R ₄ and R ₅ each independently represent hydrogen, C ₁ -C ₆ alkoxy, or halogen.

In the present invention, reference to "compound of formula 1" (or "compound of the present invention", etc.), unless otherwise specified, pharmaceutically acceptable salts, hydrates, solvates, prodrugs, complexes, moieties thereof It is understood to include pharmaceutically acceptable derivatives of the compounds of formula (I) including stereoisomers or enantiomers.

In the present invention, the term "lower alkyl" preferably refers to straight or branched chain saturated aliphatic hydrocarbon radicals comprising 1 to 12, alternatively 1 to 8 carbon atoms, alternatively 1 to 6 carbon atoms. Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secondary-butyl, tert-butyl, pentyl, isoamyl, n-hexyl, and the like. It is not limited to this. The alkyl group of the present invention may be optionally substituted.

The term "alkoxy" also means an oxygen moiety that additionally has an alkyl substituent. The alkoxy group of the present invention may be optionally substituted.

The term "lower halo alkyl" is also preferably a straight or branched chain saturated aliphatic radical containing 1 to 12, alternatively 1 to 8, alternatively 1 to 6 carbon atoms, wherein halogen is substituted for hydrogen. Means that.

In addition, the term "halogen" denotes a fluoro, chlorine, bromine or iodine atom, preferably fluoro, chlorine. The halogen group of the present invention may be optionally substituted.

The compounds of the present invention also include salts within the scope of the present invention. Compounds of the present invention, for example compounds of Formula 1, are understood to mean salts thereof unless otherwise indicated.

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

Examples of such salts include, but are not limited to: acetic acid, adipic acid, benzenesulfonic acid, benzoic acid, kempperic acid, camphorsulfonic acid, citric acid, citric acid, ethane-1,2-disulfonic acid, ethanesulfuric acid Phonic acid, 2-hydroxy ethanesulfonic 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-toluene sulfonic acid, propionic acid, glycolic acid, succinic acid, tartaric acid, oxalic acid, amino acids (eg lysine), salicylic acid, 2,2- Chloroacetic acid, L-aspartic acid, (+)-(1S) -camphor-10-sulfonic acid, 4-acetaamido benzoic acid, caproic acid, cinnamic acid, gentisic acid, glutaric acid, malonic acid, mandelic acid Acid addition salts can be formed with ortic acid, pamoic acid, aminosalicylic acid and the like. Specifically, the pharmaceutically acceptable salt of the compound of formula 1 of the present invention is a hydrochloride salt. When multiple basic groups are present, it is possible to form mono or poly acid addition salts.

As used herein, the term “pharmaceutically acceptable derivative” refers to a pharmaceutically acceptable salt of a compound of the invention that retains the desired biological activity of the compound and exhibits minimal or no unwanted toxicological effects. , Hydrate, solvate, prodrug, complex, diastereomer or enantiomer.

The invention also includes prodrugs of the compounds of the invention. The term “prodrug” is intended to denote a compound covalently bound to a carrier, which prodrug may release the active ingredient when administered to a mammalian subject. Release of the active ingredient may occur in vivo and prodrugs may be prepared by techniques known to those skilled in the art. These techniques modify suitable functional groups in certain compounds. However, these modified functional groups regenerate the original functional groups in routine manipulation or in vivo. Examples of prodrugs include, but are not limited to, esters (eg, acetate, formate and benzoate derivatives) and the like.

The compound of the present invention has a strong antiviral activity against coronaviruses, and shows an excellent effect in the treatment and prevention of infection of the Middle East Respiratory Syndrome (MERS-CoV) coronavirus and its similar virus.

In one embodiment of the invention, one of R ₁ and R ₂ represents hydrogen and the other is lower alkyl optionally substituted with halogen, lower alkoxy optionally substituted with halogen, or halogen, preferably fluoro or chlorine Represent; One or two of R ₃ , R ₄ and R ₅ each independently represent hydrogen, lower alkoxy, or halogen.

In one embodiment of the invention, R ₁ represents methyl substituted with halogen, or methoxy substituted with halogen, R ₂ represents hydrogen, one or two of R ₃ , R ₄ and R ₅ are each Independently represent a compound that is hydrogen, chlorine, methoxy; R ₁ represents hydrogen, R ₂ represents halogen, preferably fluoro, and one or two of R ₃ , R ₄ and R ₅ each independently represent a compound which is hydrogen, chlorine, methoxy.

In another embodiment of the invention, more preferred compounds are those in which one of R ₁ and R ₂ represents hydrogen and the other represents trifluoromethyl, fluoro, or trifluoromethoxy, and R ₃ , R ₄ and One or two of R ₅ each independently represent hydrogen, methoxy, or chlorine.

In one embodiment of the invention, R ₁ represents trifluoromethyl, or trifluoromethoxy, R ₂ represents hydrogen, one or two of R ₃ , R ₄ and R ₅ are each independently hydrogen, Methoxy, or chlorine; R ₁ represents hydrogen, R ₂ represents fluoro, and one or two of R ₃ , R ₄ and R ₅ each independently represent hydrogen, methoxy, or chlorine.

According to the present invention, the compound of Formula 1 may be prepared by the following synthesis process:

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

b) producing a compound of formula 4 through chlorolation of the resulting compound of formula 3;

c) producing a compound of Formula 5, which is an isoxazole compound, through a cyclization reaction of the compound of Formula 4;

d) reducing the compound of formula 5, which is an ester compound, to an alcohol to produce a compound of formula 6;

e) oxidizing the resulting compound of Formula 6 to aldehyde to produce a compound of Formula 7; And

f) reacting the resultant compound of formula 7 with a compound of formula 8 to produce a compound of formula 1:

<Formula 2>

<Formula 3>

　　　　　　　　　　　　　　　　　　　　　　

　　　　　　　　　　　　　　　　　　　　　　

<Formula 4>

<Formula 5>

<Formula 6>

<Formula 7>

<Formula 8>

<Formula 1>

In the above formula

R ₁ to R ₅ are as defined above.

In one embodiment of the present invention, the compound of Formula 1 may be prepared by the following Scheme 1:

<Scheme 1>

Specifically, the phenylaldehyde compound of <Formula 2> in which R ₁ and R ₂ are substituted in the benzene ring as a starting material was purchased from a commercially available one. Benzaldehyde oxime of <Chemical Formula 3> is synthesized by reacting the phenylaldehyde compound of <Chemical Formula 2> in the presence of a base, preferably with hydroxylammonium chloride or an equivalent thereof, and through the chlorolation reaction, benzimidonyl To produce a chloride compound. Subsequently, the compound of Formula 5 may be obtained through a cyclization reaction, which is a commonly used synthetic method using alkyl acetoacetate or alkylcyanoacetate. The ester compound of <Formula 5> is reduced to obtain an alcohol compound of <Formula 6>, and then oxidized to obtain an aldehyde compound of <Formula 7>. The synthesized aldehyde compound of Formula 7 may be reacted with a compound of Formula 8 to prepare an isoxazole derivative of Formula 1, which is a final compound.

Isomers and solvates (eg hydrates) of the compounds of Formula 1 are also within the scope of the present invention. Methods of solvation are commonly known in the art. Thus, the compounds of the present invention may be in free hydrate or salt form.

The preparation method according to the invention may preferably be carried out in the presence of a base or an acid in a solvent. At this time, the solvent, acid and base may use any conventional solvent, base or acid catalyst that does not adversely affect the reaction, for example, as a solvent, tetrahydrofuran, 1,2-dichloroethane, methylene chloride, chloroform, Group consisting of methanol, ethanol, isopropyl alcohol, n-butanol, tert-butanol, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, ethyl acetate, diethyl ether, toluene and dioxane One or more selected from can be used. For example, as a base, pyridine, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, sodium methoxide, sodium ethoxide, sodium tert One or more selected from the group consisting of butoxide, potassium tert-butoxide, lithium aluminum hydride, lithium borohydride, sodium nitrate and cesium carbonate can be used. Further, for example, as the 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 may be mentioned.

The starting materials used in the preparation of the compounds according to the invention according to the above methods are all commercially available or are readily available due to their convenient purchase. The reaction may usually be carried out under cooling to warming, and after completion of the reaction, the final compound may be purified through conventional post-treatment processes such as column chromatography, recrystallization, and the like.

On the other hand, the present invention relates to a pharmaceutical composition, treatment method and use for the treatment or prevention of coronavirus infection and the like, comprising an effective amount of the compound of Formula 1 or a pharmaceutically acceptable derivative thereof, in particular Middle East respiratory coronavirus It is effective in treating (MERS-CoV) infections and can therefore be used effectively in the treatment of such diseases.

Typical suitable dosages in single or separate doses required for the treatment of the compounds of the present invention as such have been shown to be effective range from about 0.01 to 750 mg / kg body weight per day, preferably 0.1 to 100 mg, most preferred Preferably, the dosage ranges from 0.5 to 25 mg, but specific dosage levels for individual patients vary depending on the specific compound to be used, the weight of the patient, sex, diet, time of administration of the drug, method of administration, excretion rate, drug mixture, and the condition and age of the patient. Can be.

The compound of the present invention may be administered as it is during processing, but is preferably provided as a pharmaceutical formulation.

Accordingly, the present invention provides a pharmaceutical formulation (pharmaceutical composition, etc.) obtained by mixing a compound of Formula 1 or a pharmaceutically acceptable derivative thereof with a pharmaceutically acceptable carrier and / or excipient.

According to one embodiment of the invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For example, the carrier may be inert and may include sugars, including corn lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, corn starch, wheat starch, rice starch and potato starch. Celluloses including starch, cellulose, methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like, and fillers such as gelatin, polyvinylpyrrolidone, etc., but may not be limited thereto. . In addition, in some cases, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, but may not be limited thereto.

For example, the pharmaceutical composition may further include, but may not be limited to, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.

In addition, the compounds or pharmaceutical compositions of the invention may be administered by any route as desired. It may be administered orally or parenterally, and the parenteral route of administration may include, but is not limited to, various routes such as, for example, transdermal, nasal, abdominal, intramuscular, subcutaneous, intravenous, and the like. Specifically, the route of administration of the compound or pharmaceutical composition of the present invention is preferably injection and oral administration.

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, can be prepared using suitable dispersing agents, wetting agents or suspending agents according to known techniques. Solvents that can be used for this are water, Ringer's solution and isotonic NaCl solution, and sterile fixed oils are also commonly used as solvents or suspending media. Any non-irritating fixed oil may be used for this purpose, including mono- and diglycerides, and fatty acids such as oleic acid may also be used in the preparation of injectables.

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

Tablets and pills are preferably prepared with enteric agents. Solid dosage forms can be prepared by mixing the compound of formula 1 according to the present invention with a carrier such as one or more inert diluents such as sucrose, lactose, starch and the like, lubricants such as magnesium stearate, disintegrants, binders and the like.

The compound of formula 1 of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, has an effect of inhibiting the replication or transmission of an RNA virus such as Coronavirus.

In one embodiment of the invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, further comprises one or more additional agents having antiviral efficacy for preventing and treating a respiratory viral infection disease. It can be administered in combination with the drug.

Specifically, the pharmaceutical composition may be administered with one or more of antiviral effects such as antibiotics, Interferon, Ribavirin, or Lopinavir / Ritonavir. have.

Thus, in one embodiment, the present invention provides a composition comprising: a) a compound of Formula 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof; And b) at least one other therapeutic agent selected from the group consisting of antibiotics, Interferon, Ribavirin and Lopinavir / Ritonavir. Prophylactic combinations are provided. Specifically, the coronavirus is Middle East respiratory coronavirus (MERS-CoV). In one embodiment, the combination may be administered simultaneously, separately or sequentially.

Hereinafter, the present invention will be described in more detail based on the following Preparation Examples and Examples. However, these preparation examples and examples are only intended to help the understanding of the present invention, it is not intended that the scope of the present invention is limited to these in any sense.

Production Example 1: 2 -( Trifluoromethyl ) Benzaldehyde oxime  synthesis

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

1 H-NMR (400 MHz, CDCl 3, δ) = 7.23 (dd, 1H), 7.59 (dd, 1H), 7.76 (m, 2H), 8.39 (s, 1H), 11.63 (s, 1H)

Production Example 2: 2 -( Trifluoromethoxy ) Benzaldehyde oxime  synthesis

Similar to Preparation Example 1, white solid using 2- (trifluoromethoxy) benzaldehyde (38.03g, 200.0mmol), sodium hydroxide (12.00g, 300.0mmol), hydroxylamine hydrochloride (16.6g, 240mmol) Compound (38.67 g, 188 mmol, 94%) was obtained.

1 H-NMR (400 MHz, CDCl 3, δ) = 7.43 (m, 2H), 7.54 (m, 1H), 7.89 (m, 1H), 8.24 (s, 1H), 11.76 (s, 1H)

Production Example 3: 3 - Fluorobenzaldehyde oxime  synthesis

Similar to Preparation Example 1, a white solid target compound (22.48 g) was prepared using 3-fluorobenzaldehyde (24.82 g, 200.0 mmol), sodium hydroxide (12.00 g, 300.0 mmol), and hydroxylamine hydrochloride (16.68 g, 240 mmol). , 162 mmol, 81%).

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

Production Example  4: N-hydroxy-2- ( Trifluoromethyl ) Of benzimidonylchloride  synthesis

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

1 H-NMR (400 MHz, CDCl 3, δ) = 7.73 (m, 2H), 7.80 (t, 1H), 7.86 (d, 1H), 12.61 (s, 1H)

Production Example  5: N-hydroxy-2- ( Trifluoromethoxy ) Of benzimidonylchloride  synthesis

Similar to Preparation Example 4, light yellow color was obtained using dimethylformamide (240 mL), 4- (trifluoromethoxy) benzaldehyde oxime (20.0 g, 97.50 mmol) and N-chlorosuccinimide (14.32 g, 107.26 mmol). A solid target compound (19.48 g, 81.30 mmol, 83%) was obtained.

1 H-NMR (400 MHz, CDCl 3, δ) = 7.49 (m, 1H), 7.62 (m, 1H), 7.68 (dd, 1H), 12.67 (s, 1H)

Production Example 6: 3 - Fluoro -N- Of hydroxybenzimidonylchloride  synthesis

Similar to Preparation Example 4, a pale yellow solid target compound was obtained using dimethylformamide (240 mL), 3-fluorobenzaldehyde oxime (20.0 g, 143.76 mmol), and N-chlorosuccinimide (21.12 g, 158.12 mmol). 23.32 g, 134.40 mmol, 94%).

1 H-NMR (400 MHz, CDCl 3, δ) = 7.29 (m, 1H), 7.47 (m, 1H), 7.62 (m, 2H)

Production Example  7: methyl -5- methyl -3- (2- ( Trifluoromethyl Phenyl) Isoxazole -4- Carboxyle Synthesis of sites

N-hydroxy-2- (trifluoromethyl) benzimidonylchloride (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, followed by the slow addition of sodium methoxide (5.80 g, 107.34 mmol). The reaction mixture was heated to room temperature, stirred for 3 hours, concentrated under reduced pressure to remove methanol, ethyl acetate (200 mL) was added, washed with purified water (200 mL) and saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, and decompressed. Concentrated. Purified by column chromatography to obtain a white solid target compound (5.79g, 20.31mmol, 57%).

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

Production Example  8: methyl -5- methyl -3- (2- ( Trifluoromethoxy Phenyl) Isoxazole -4- Carboxyl Rate Synthesis

Methanol (160 mL), N-hydroxy-4- (trifluoromethoxy) benzimidonylchloride (8.00 g, 33.39 mmol), methyl acetoacetate (7.76 g, 66.78 mmol) and sodium methoxide were similarly prepared in Preparation Example 7. (5.41 g, 100.17 mmol) was used to obtain a white solid target compound (7.04 g, 23.37 mmol, 70%).

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

Production Example  9: methyl -3- (3- Fluorophenyl ) -5- Methylisoxazole -4- Carboxylate  synthesis

Similar to Preparation 7, methanol (160 mL), 3-fluoro-N-hydroxybenzimidonyl chloride (8.00 g, 46.09 mmol), methyl acetoacetate (10.07 g, 92.18 mmol), sodium methoxide (7.47 g, 138.27 mmol) was obtained as a white solid target compound (7.84 g, 33.33 mmol, 72%).

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

Production Example  10: (5- methyl -3- (2- ( Trifluoromethyl Phenyl) Isoxazole Synthesis of -4-yl) methanol

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

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

Production Example 11: 5 - methyl -3- (2- ( Trifluoromethoxy Phenyl) Isoxazole Synthesis of -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 hydroxide similarly to Preparation Example 10 A pale yellow liquid target compound (1.82 g, 6.67 mmol, 67%) was obtained using lead (0.57 g, 15.00 mmol).

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

Production Example  12: (3- (3- Fluorophenyl ) -5- Methylisoxazole Synthesis of -4-yl) methanol

Similar to Preparation Example 10, 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) was used to obtain a white solid target compound (0.91 g, 4.39 mmol, 52%).

1 H-NMR (400 MHz, CDCl 3, 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)

Production Example 13: 5 - methyl -3- (2- ( Trifluoromethyl Phenyl) Isoxazole -4- Caboaldehyde Synthesis of

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 dimethyl sulfoxide (200 mL) The solution dissolved in sulfur trioxide pyridine complex (38.20 g, 230.00 mmol) in dimethyl sulfoxide (200 mL) was slowly added to the reaction solution, which was then stirred for 4 hours. After the reaction solution was cooled to 0 ° C., 36% hydrochloric acid solution was added and acidified to have a pH value of 4.5-5. The reaction solution was added with water (1500 mL), washed twice with ethyl acetate (1200 mL), and each organic layer. The combined solution was washed twice with 10% aqueous sodium hydroxide solution (750 ml). The organic layer was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to obtain a white solid target compound (17.5g, 68.58mmol, 86%).

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

Production Example 14: 5 - methyl -3- (2- ( Trifluoromethoxy Phenyl) Isoxazole -4- Cabo Aldehy Synthesis of de

Similar to Preparation 13, dimethyl sulfoxide (400 mL), 5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazol-4-yl) methanol (20.1 g, 73.57 mmol), triethylamine ( 85.46g, 844.55mmol) and sulfur trioxide pyridine complex (38.64g, 242.77mmol) were used to obtain a white solid compound (14.83g, 54.69mmol, 74%).

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

Production Example 15: 3 -(3- Fluorophenyl ) -5- Methylisoxazole -4- Carboaldehyde  synthesis

Similar to Preparation 13, dimethyl sulfoxide (300 mL), (3- (3-fluorophenyl) -5-methylisoxazol-4-yl) methane (12.20, 58.88 mmol), triethylamine (65.80 g, 650.26 mmol) and a sulfur trioxide pyridine complex (31.00 g, 194.77 mmol) gave a white solid compound (17.5 g, 68.58 mmol, 86%).

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

Example 1: 4 -((4- (2- Methoxyphenyl Synthesis of Piperazine-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), 1- (2-methoxyphenyl) piperazine (4.37 g, 22.73 mmol) The solution was dissolved in 1,2-dichloroethane (70 mL), and sodium triacetoxyborohydride (6.74 g, 31.80 mmol) was added thereto, followed by stirring at room temperature for 7 hours. The reaction solution is washed with saturated aqueous sodium carbonate solution (80 mL). The aqueous layer was collected, washed with 1,2-dichloroethane (50 mL), and each organic layer was collected, dried over anhydrous sodium sulfate, and concentrated. After concentration, crystallized with n-hexane (30 mL), filtered and dried under vacuum at 50 ℃ to give a white solid target compound (7.28g, 16.87mmol, 74%).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3- Methoxyphenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethyl) phenyl) isoxazole

Similar to Example 1 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 used to obtain a white solid target compound (4.1 g, 9.7 mmol, 66%). Got it.

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3,4- Dichlorophenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethyl) phenyl) isoxazole

Similar to Example 1 1,2-dichloroethane (36 mL), 5-methyl-3- (2- (trifluoromethyl) phenyl) isoxazole-4-carboaldehyde (3.0 g, 14.6 mmol), 1- White solid target compound (4.1 g, 9.7 mmol, 66%) using (3,4-dichlorophenyl) piperazine (3.4 g, 14.6 mmol) and sodium triacetoxyborohydride (5.3 g, 20.5 mmol) Got.

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (2- Methoxyphenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole

Similar to Example 1 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 used to obtain a white solid target compound (5.02 g, 11.22 mmol, 84%). Got it.

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3- Methoxyphenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole

Similar to Example 1 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 used to obtain a white solid target compound (4.1 g, 9.7 mmol, 66%). Got it.

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3,4- Dichlorophenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole

Similar to Example 1 1,2-dichloroethane (36 mL), 5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole-4-carboaldehyde (3.0 g, 14.6 mmol), 1- White solid target compound (4.1 g, 9.7 mmol, 66%) using (3,4-dichlorophenyl) piperazine (3.4 g, 14.6 mmol) and sodium triacetoxyborohydride (5.3 g, 20.5 mmol) Got.

1 H-NMR (400 MHz, DMSO-d 6, 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: 3 -(3- Fluorophenyl ) -4-((4- (2- Methoxyphenyl Synthesis of) Piperazine-1-yl) methyl) -5-methylisoxazole

Similar to Example 1 1,2-dichloroethane (60 mL), 3- (3-fluorophenyl) -5-methylisoxazole-4-carboaldehyde (3.50 g, 17.06 mmol), 1- (2- Using methoxyphenyl) piperazine (3.30 g, 17.16 mmol) and sodium triacetoxyborohydride (5.90 g, 27.84 mmol), a white solid target compound (4.62 g, 12.11 mmol, 71%) was obtained.

1 H-NMR (400 MHz, DMSO-d 6, 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: 3 -(3- Fluorophenyl ) -4-((4- (3- Methoxyphenyl Synthesis of) Piperazine-1-yl) methyl) -5-methylisoxazole

Similar to Example 1 1,2-dichloroethane (60 mL), 3- (3-fluorophenyl) -5-methylisoxazole-4-carboaldehyde (3.00 g, 14.62 mmol), 1- (3- Using methoxyphenyl) piperazine (3.30 g, 17.16 mmol) and sodium triacetoxyborohydride (5.90 g, 27.84 mmol), a white solid target compound (4.68 g, 12.27 mmol, 84%) was obtained.

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3,4- Dichlorophenyl Synthesis of Piperazine-1-yl) methyl) -3- (3-fluorophenyl) -5-methylisoxazole

Similar to Example 1 1,2-dichloroethane (60 mL), 3- (3-fluorophenyl) -5-methylisoxazole-4-carboaldehyde (3.00 g, 14.62 mmol), 1- (3, Using 4-dichlorophenyl) piperazine (3.40 g, 14.71 mmol) and sodium triacetoxyborohydride (5.30 g, 25.01 mmol), a white solid target compound (4.11 g, 9.78 mmol, 67%) was obtained.

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (2- Methoxyphenyl Synthesis of 1) 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) Dissolve in acetone (10 mL), cool to 0 ° C, and add hydrochloric ethanol (10%, 0.80 g, 2.20 mmol) dropwise. The reaction was stirred at room temperature for 8 hours, filtered and dried to give a white solid target compound (0.94 g, 2.01 mmol, 91%).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3- Methoxyphenyl Synthesis of 1) piperazin-1-yl) methyl) -5-methyl-3- (2- (trifluoromethyl) phenyl) isoxazole hydrochloride

Similar to Example 10, acetone (10 mL), 4-((4- (3-methoxyphenyl) piperazin-1-yl) methyl) -5-methyl-3- (2- (trifluoromethyl) phenyl A white solid target compound (0.82 g, 1.75 mmol, 77%) was obtained using isoxazole (0.98 g, 2.27 mmol) and hydrogen ethanol (11%, 0.74 g, 2.27 mmol).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3,4- Dichlorophenyl Synthesis of 1) piperazin-1-yl) methyl) -5-methyl-3- (2- (trifluoromethyl) phenyl) isoxazole hydrochloride

Similar to Example 10, 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 hydrogen ethanol (11%, 1.06 g, 3.19 mmol) were used to obtain a white solid target compound (1.32 g, 2.61 mmol, 82%).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (2- Methoxyphenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole hydrochloride

Similar to Example 10, acetone (23 mL), 4-((4- (2-methoxyphenyl) piperazin-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl A white solid target compound (3.24 g, 6.70 mmol, 79%) was obtained using isoxazole (3.37 g, 8.43 mmol) and hydrochloric ethanol (11%, 2.79 g, 8.43 mmol).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3- Methoxyphenyl Synthesis of Piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole hydrochloride

Similar to Example 10, acetone (25 mL), 4-((4- (3-methoxyphenyl) piperazin-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl A white solid target compound (3.93 g, 8.12 mmol, 87%) was obtained using isoxazole (4.17 g, 9.32 mmol) and hydrochloric ethanol (11%, 3.09 g, 9.32 mmol).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3,4- Dichlorophenyl Synthesis of) piperazine-1-yl) methyl) -5-methyl-3- (2- (trifluoromethoxy) phenyl) isoxazole hydrochloride

Similar to Example 10, 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 hydrochloric ethanol (11%, 3.78 g, 11.41 mmol) were used to obtain a white solid target compound (5.32 g, 10.18 mmol, 89%).

1 H-NMR (400 MHz, DMSO-d 6, 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: 3 -(3- Fluorophenyl ) -4-((4- (2- Methoxyphenyl Synthesis of) Piperazine-1-yl) methyl) -5-methylisoxazole Hydrochloride

Similar to Example 10, acetone (2 mL), 3- (3-fluorophenyl) -4-((4- (2-methoxyphenyl) piperazin-1-yl) methyl) -5-methylisoxazole (0.20 g, 0.52 mmol) and hydrogen chloride ethanol (10%, 0.19 g, 0.52 mmol) were used to obtain a white solid target compound (0.13 g, 0.31 mmol, 60%).

1 H-NMR (400 MHz, DMSO-d 6, 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: 3 -(3- Fluorophenyl ) -4-((4- (3- Methoxyphenyl Synthesis of) Piperazine-1-yl) methyl) -5-methylisoxazole Hydrochloride

Similar to Example 10, acetone (2 mL), 3- (3-fluorophenyl) -4-((4- (3-methoxyphenyl) piperazin-1-yl) methyl) -5-methylisoxazole (0.20 g, 0.52 mmol) and hydrogen chloride ethanol (10%, 0.19 g, 0.52 mmol) were used to obtain a white solid target compound (0.19 g, 0.46 mmol, 89%).

1 H-NMR (400 MHz, DMSO-d 6, 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: 4 -((4- (3,4- Dichlorophenyl Synthesis of Piperazine-1-yl) methyl) -3- (3-fluorophenyl) -5-methylisoxazole hydrochloride

Similar to Example 10, acetone (2 mL), 4-((4- (3,4-dichlorophenyl) piperazin-1-yl) methyl) -3- (3-fluorophenyl) -5-methylisox White solid target compound (0.15 g, 0.33 mmol, 68%) was obtained by using azole (0.20 g, 0.48 mmol) and hydrochloric ethanol (10%, 0.18 g, 0.48 mmol).

1 H-NMR (400 MHz, DMSO-d 6, 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 test

In this experiment, MERS-CoV (Korean isolate, 002) of the same titer was first infected with cells, and then each compound was added to the overlay medium for each concentration. The results of this experiment can determine the efficacy evaluation of the compound against the antiviral by measuring the EC ₅₀ value by evaluating the maximum dilution concentration of the compound that is reduced by 50% or more than the number of plaques formed in the virus-only negative control.

1) cell line

Vero cell: 1 x 10 ⁶ / well (6 well plate)

2) virus

-MERS-CoV Korean securities (MERS-CoV / KOR / KNIH / 002_05_2015)

Virus titer: 7.75 X 10 ⁶ PFU / ml

Virus inoculation titer: 100 PFU / 100μl

3) Evaluation of plaque formation inhibition test

Basal medium: MEM F12, 5% NaHCO ₃ 1ml, DEAE-Dextran

4) Test method

The day before the test, Vero cell lines were incubated in 6-well plates at 1 × 10 ⁶ / well and then incubated for 24 hours in a 5% CO ₂ , 37 ° C. incubator. The next day, the compound is diluted in two steps from 100μM to 3.13μM using a plaque test basal medium to prepare overlay medium. After diluting MERS-CoV to 100 PFU / 100μl in PBS, the cells were washed with PBS and infected, and then shaken well at 15% intervals in a 5% CO ₂ , 37 ° C incubator and reacted for 1 hour. After inoculation, inoculate the inoculated MERS-CoV, mix oxoid agar in overlay medium containing the compound, and apply 2 ml of each to a 6-well plate from low concentration. After incubation for 72 hours in a 5% CO ₂ , 37 ℃ incubator, proceed with cell staining using crystal violet, it can be observed whether the plaque formation through the cell staining.

Experimental Example  2: Cytotoxicity Assessment

After diluting the compound of Formula 1 by concentration in a cell line (Vero) susceptible to MERS-CoV, the maximum dilution concentration showing a difference in survival rate of 50% or more compared to the negative control group (the experimental group not treated with the compound) was evaluated. In other words, it is an experiment evaluating whether the compound itself affects cell viability.

In Experiment 1, in vitro efficacy evaluation of the compound of Formula 1 against MERS-CoV (Korean isolate, 002) was performed to evaluate whether the compound itself affects the cell viability of the compound having excellent effects through screening. .

The test results are essential for obtaining selectivity index (SI) values based on antiviral efficacy and cytotoxicity against MERS-CoV.

1) cell line

Vero cell: 4 x 10 ⁴ / well (96 well plate)

2) Cell viability

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

Absorbance (440nM-600nM)

3) Test method

The day before performing this test, Vero cell lines are sub-cultured in 96-wells at 1 × ¹⁰ 4/100 μl and incubated in a 37 ° C. 5% CO ₂ incubator for 24 hours. After 24 hours, the compound of formula 1 to be evaluated is subjected to stepwise dilution from 400 μM to 0.195 μM in serum-free DMEM medium, and then treated to the prepared Vero cell line. At this time, the cell line should be washed once with PBS and treated with diluted active substance. After the treatment was reacted for 72 hours in a 37 ℃ 5% CO ₂ incubator. To measure cell viability, WST-1 was treated with 10 μl per well (based on a total volume of 100 μl) and reacted for 2 hours in a 37 ° C. 5% CO ₂ incubator. After 2 hours, the absorbance (440 nM, 600 nM) was measured to determine the CC ₅₀ value compared to the negative control untreated active material.

Through the results of Experimental Example 1, as shown in [FIG. 1], the concentration of the compound which reduced by 50% or more than the number of plaques formed in the virus-infected control group was confirmed by checking the plaque number, and using the graphpad prism software. EC ₅₀ values were measured as shown in [Table 1].

Experimental results showed that the compound of Example 16 inhibited plaque formation at the lowest 1.15 μM concentration, and the compound of Example 10 inhibited the formation of plaque at the next low concentration of 17.50 μM, the compound of Example 11 It was found that at 22.26 μM concentration of silver, the compound of Example 18 inhibited plaque formation at 67.68 μM concentration, and the compound of Example 17 in the order of 1215 μM concentration.

In the results of Experimental Example 2, compounds showing effective efficacy were selected and cultured for 72 hours after treatment in cell lines. The reason is that the reaction time between the compound and the cell line in the plaque reduction assay (72 hours) was the same. As a result, it was possible to evaluate the survival rate of Vero cells in accordance with the concentration of the compound showing the effective efficacy as shown in FIG. In addition, the graphpad prism software was able to evaluate the CC ₅₀ values for compounds exhibiting effective efficacy as shown in Table 2.

As shown in [Table 2], the three active substance compounds except for Examples 16 and 18 showed more than 50% cell viability at concentrations of about 30 μM or less, and based on this, the optimal concentration of the active substance exhibiting cytotoxicity. Could be analyzed. Therefore, based on the present results, it was possible to measure the selective index (SI) for antiviral efficacy evaluation for MERS-CoV.

Therefore, on the basis of the results of Experimental Example 1 and Experimental Example 2, the selection index (selective index, SI) of the antiviral agent was obtained as shown in [Table 3] using CC ₅₀ and EC ₅₀ values. As shown in Table 3, the compound of Example 10 showed the highest SI value of 2.89, the compound of Example 11 showed the antiviral efficacy in the order of 2.10 and Example 17 of the compound having an SI value of 0.037. In addition, growth characteristics analysis showed that the efficacy is shown to be dose dependent (dose dependent) in all compounds showing effective efficacy.

Based on the results of this test, plaque formation suppression test could be inferred that compounds showing effective efficacy selected through antiviral screening inhibit the virus from re-infection into new cells. Formation inhibition was confirmed that the EC ₅₀ value is lower than the concentration that reduces the overall cell survival rate of more than 50%, it was confirmed that the antiviral efficacy of the effective substance itself, not due to cytotoxicity.

Efficacy test for the antiviral of the compound prepared in the present invention was measured in the compounds of the Examples of the present specification using Experimental Example 1 to Experimental Example 2 as a result confirmed that the chemical structure having antiviral capacity It was.

The efficacy of the example compounds was determined by plaque-reduction assay (50% effect concentration, EC ₅₀ ) to screen for compounds that showed antiviral efficacy after viral infection in the cells of the example compounds herein. It was confirmed, it was confirmed that the compound showing an effective efficacy.

Cytotoxicity concentration assay (50% cytotoxic concentration, CC ₅₀ ) was carried out mainly on the compounds identified as effective substances in these compounds to determine CC ₅₀ , and SI (Selective index) values. 1], [Table 2] and [Table 3].

1) EC ₅₀ (Effective Concentration 50%): Minimum concentration of compound that reduces by more than half the number of plaques in the control group.

2) CC ₅₀ (Cytotoxicity concentration 50%): the maximum concentration of the compound that is reduced by more than half the number of cells in the control group

3) SI (Selective Index): Value represented by CC ₅₀ / EC ₅₀

The present invention provides a novel isoxazole derivative compound having potent antiviral activity against coronaviruses, a method for preparing these compounds, and a composition comprising these compounds as an active ingredient, thereby providing a coronavirus infection, particularly Middle East Respiratory Syndrome coronavirus ( It is possible to develop antiviral agents for the prevention and treatment of viral respiratory diseases caused by MERS-CoV) infection.

Claims (18)

A compound of Formula 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof:
<Formula 1>

In the above formula
R ₁ and R ₂ are each independently hydrogen; C ₁ -C ₆ alkyl optionally substituted with halogen; C ₁ -C ₆ alkoxy optionally substituted with halogen; Or halogen;
R ₃ , R ₄ and R ₅ each independently represent hydrogen, C ₁ -C ₆ alkoxy, or halogen. The method of claim 1,
One of R ₁ and R ₂ represents hydrogen, the other represents C ₁ -C ₆ alkyl optionally substituted by halogen, C ₁ -C ₆ alkoxy optionally substituted by halogen, or halogen, R ₃ , R ₄ and Wherein one or two of R ₅ each independently represent hydrogen, C ₁ -C ₆ alkoxy, or halogen. The method of claim 2,
R ₁ represents methyl substituted with halogen, or methoxy substituted with halogen, R ₂ represents hydrogen, and one or two of R ₃ , R ₄ and R ₅ each independently represents hydrogen, chlorine, methoxy Represent; or
R ₁ represents hydrogen, R ₂ represents halogen, and one or two of R ₃ , R ₄ and R ₅ each independently represent hydrogen, chlorine, methoxy. The method of claim 1,
One of R ₁ and R ₂ represents hydrogen and the other represents trifluoromethyl, fluoro, or trifluoromethoxy and one or two of R ₃ , R ₄ and R ₅ are each independently hydrogen, meth Toxin or chlorine. The method of claim 4, wherein
R ₁ represents trifluoromethyl or trifluoromethoxy, R ₂ represents hydrogen and one or two of R ₃ , R ₄ and R ₅ each independently represent hydrogen, methoxy, or chlorine; or
R ₁ represents hydrogen, R ₂ represents fluoro, and one or two of R ₃ , R ₄ and R ₅ each independently represent hydrogen, methoxy, or chlorine. The method of claim 1,
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
Compound selected from the group consisting of 4-((4- (3,4-dichlorophenyl) piperazin-1-yl) methyl) -3- (3-fluorophenyl) -5-methylisoxazole Or pharmaceutically acceptable salts, hydrates or solvates thereof. The compound of claim 6, wherein the pharmaceutically acceptable salt is a hydrochloride salt. a) reacting a compound of Formula 2 with hydroxylammonium chloride under a base to produce a compound of Formula 3;
b) producing a compound of formula 4 through chlorolation of the resulting compound of formula 3;
c) producing a compound of Formula 5, which is an isoxazole compound, through a cyclization reaction of the compound of Formula 4;
d) reducing the compound of formula 5, which is an ester compound, to an alcohol to produce a compound of formula 6;
e) oxidizing the resulting compound of Formula 6 to aldehyde to produce a compound of Formula 7; And
f) preparing a compound of formula 1 as defined in claim 1, comprising reacting the resulting compound of formula 7 with a compound of formula 8 to produce a compound of formula 1
<Formula 2>

<Formula 3>

<Formula 4>

<Formula 5>

<Formula 6>

<Formula 7>

<Formula 8>

<Formula 1>

In the above formula
R ₁ to R ₅ are as defined in claim 1. The method of claim 8, wherein the method for preparing the compound of Formula 1 is tetrahydrofuran, 1,2-dichloroethane, methylene chloride, chloroform, methanol, ethanol, isopropyl alcohol, n-butanol, tert-butanol, dimethyl sulfoxide, A process characterized in that it is carried out using at least one solvent selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide, ethyl acetate, diethyl ether, toluene and dioxane. The method of claim 8, wherein the method for preparing the compound of formula 1 is pyridine, triethylamine, diethylamine, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, sodium methoxide, sodium Toxide, sodium tert-butoxide, potassium tert-butoxide, lithium aluminum hydride, lithium borohydride, sodium nitrate and cesium carbonate characterized in that it is carried out using at least one base selected from the group consisting of Way.  The method of claim 8, wherein the method for preparing the compound of Formula 1 is carried out using at least one acid selected from the group consisting of trifluoroacetic acid, hydrochloric acid, nitric acid, sulfuric acid, bromic acid, zinc bromide and acetic acid. Way. A coronavirus infection in a subject containing a compound of formula 1 according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions for the treatment or prophylaxis. 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) a compound of formula (1) according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, hydrate or solvate thereof; And
b) treatment or prevention of coronavirus infection further comprising at least one other therapeutic agent selected from the group consisting of antibiotics, Interferon, Ribavirin and Lopinavir / Ritonavir Combination. 18. The combination of claim 17, wherein the coronavirus is Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

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