KR102234530B1 - Novel Toltrazuril Derivatives and Pharmaceutical Composition for Treating or Preventing Kudoa - Google Patents

Abstract

The present invention relates to a novel toltrazuril derivative and a pharmaceutical composition for the prevention and treatment of Kudoa septempunctata, comprising the derivative. The toltrazuril derivative according to the present invention lowers toxicity to fish while maintaining the exterminating effect of toltrazuril on Kudoa septempunctata, and thus can be broadly applicable to the development of aquatic medicine (anthelmintic) that can exterminate Myxozoa that causes death in aquaculture sites.

Description

Novel Toltrazuril Derivatives and Pharmaceutical Composition for Treating or Preventing Kudoa

The present invention relates to a novel toltrazuril derivative and a pharmaceutical composition for the prevention and treatment of Kudoae comprising the derivative.

Kudoa septempunctata was first identified in 2010 by Matsukane et al. (Matsukane, Y. et al., Parasitol Res 107:865, 2010) by detecting Kudoa septempunctata in the muscles of farmed flounder imported from Korea to Japan. In other words, it was first reported to academia as a new species different from the previously reported mucosa (Myxozoa) Kudoaworm through the shape of spores, SSU rDNA sequence, 7 polar vesicles, and distribution of body muscles. In addition, K. septempunctata is a causative agent that causes novel food poisoning after intake of flounder sashimi, which has been increasing since 2003, and was administered to suckling-mouse and house musk shrew. The human pathogenicity of this parasite was first reported by observing diarrhea and vomiting symptoms (Kawai, T. et al., Clin Infect Dis , 54:1046, 2012), and the Japanese Ministry of Health, Labor and Welfare reported a new type of kudoa septempunctata (Kawai, T. et al., Clin Infect Dis, 54:1046, 2012). Hereinafter referred to as'kudoworm') is defined as a causative agent of food poisoning that causes symptoms such as diarrhea and vomiting in humans.

Kudolarvae do not cause transient (within 1 to 9 hours) diarrhea and vomiting due to ingestion of the halibut muscle, which has a large number of parasitic halibut, which forms a pseudocyst in the halibut muscle part. However, the symptoms are mild and recover quickly, and the next day the prognosis is good without any sequelae. The method of prevention is to keep the infected fish at -20°C for more than 4 hours or heat treatment at a central temperature of 75°C for more than 5 minutes to kill the Kudoworm and inactivate the toxicity of the spores.

Kudolarvae are Myxozoa Subphylum, Myxosporea Class, Multivalvulida Order, Kudoa Family, and Kudoa Genus. More than 2,000 species of mucous spores are reported in the world, of which 97 species of Kudoas are, and more than 80 species are marine. Morphologically, it forms spores of the structure of a pole sac with a coil-shaped pole inside, and the spore is a multicellular body composed of spores including pole sacs and spore traits. The size of spores is about 10 μm, and the species are classified by the number of polar sacs or spores, shape of spores, and measurements (Yokoyama, H. Jpn J Food Microbiol 29:68, 2012; Jorge, CE et al. , Systematic Parasitology 87 :153, 2014). Kudolarvae have 5-7 polar cysts, mainly parasitic in the muscle cells of the halibut body muscle.

There are 9 types of Cudoa genus mucospore worms reported to be parasitic to flounder so far ( K. igami, K. lateolabracis , K. ogawai , K. paralichthys , K. sp., K. septempunctata, K. shiomitsui , K. thyrsites , K. yasunagi ) are known (Shin, SP et al. , Korean J Parasitology , 57:439, 2019), and are the only reported parasites that cause food poisoning. Previously reported K. lateolabracis , K. shiomitsui , K. thyrsites , K. Unlike yasunagi , kudolarvae do not have any external symptoms due to infection even if they are infected with live flounder, and clinical symptoms such as cyst formation or muscle fusion are not observed, making it impossible to distinguish them from uninfected fish in appearance. In addition, even if a large number of Kudoworms are infected, the flounder does not die, so there are many difficulties in preventing and diagnosing.

Most of the genus Cudois mucosporiasis are parasitic in fish and form visible cysts in organs such as body muscles, brain, kidneys, and gills. These do not affect the physiology and survival of infected fish, but in the case of species that are parasitic to the muscle fibers, after the fish dies, the muscles are dissolved by the parasite's proteolytic enzyme and become jelly, resulting in economic damage due to the decrease in the value of the product. .

They are said to have a two-host life cycle, typically with vertebrates and invertebrate annular animals as alternating hosts. It is known that fish are representative of vertebrates, and invertebrate annular animals are tamals in freshwater, and polychaetes are known in the case of seawater. In fish, it has the stage of mucous spores, and in annular animals it has the stage of actinosporangia. Of the about 2,000 species of mucilosporangiasis, only a few have revealed life history. In particular, the life history of the genus Kudoa mucilospores is not yet known, and it is estimated that they will have a life history similar to that of the marine mucospores known to date. In addition, it is only estimated that the infection route of the Kudoworm is similar to that of the mucous spores.

In the meantime, various studies have been attempted on diagnosing Kudolarvae, and qualitative-quantitative genetic testing, LAMP, and Immunochromatography assays have been developed. These various test methods have a detection sensitivity about 100 times higher than that of microscopic examination, and can detect not only spores, but also all stages of development, but they are only a screening method for Kudoworms. The spore must be confirmed to be final positive. This means that the target of the potential food poisoning is the spores of the Kudoworm, and it is the key to confirm the spores under a microscope to confirm that the Kudoworm is positive, and the detection limit of spores was ^{about 10 4 per gram of muscle.}

Despite the controversy over the human pathogenicity of Kudolarvae in Korea and Japan, recent cases of acute gastroenteritis such as vomiting, diarrhea, and abdominal pain after halibut intake have been steadily occurring in Korea. With regard to Kudolarvae disease, the number of reports of group outbreaks and the positive rate of diagnosis continue to increase after the first patient outbreak in 2015. In other words, as of '18, the ranking of the causes of group patients with water-borne and foodborne infectious diseases (11 cases in '15 → 68 cases in '18) and the cultivation rate of kudoworms (19% in '15 → 39% in '18) increase. It is a trend.

Since the fact that halibut has a large number of parasites and the possibility that Kudo larvae cause food poisoning can adversely affect consumption, we continue to investigate the actual condition of Kudo larvae infection in domestic farmed halibut and food poisoning through research on the path of infection. It is necessary to prepare measures to manage kudoworms in flounder farms to relieve distrust of concerns.

The in vitro deactivation effect on Kudo ahchung (K. septempunctata) of an exemplary anti-coccidiosis agent toll trad hungry (toltrazuril) with the aim of controlling the Kudo ahchung in halibut is first placed in the outside journals, possible administration as gujeje for Kudo ahchung (Ahn M. et al. , Parasite , 24:11, 2017).

The present inventors studied the safety of toltrazuril against flounder, and toltrazuril was observed to have various hematologic and histopathological degenerations (toxicity) in the liver and kidneys in a concentration-dependent manner, and a serious side effect of concern for safety was expected. It is difficult to accurately calculate the concentration of anthelmintic repellents used in aquaculture sites based on the weight of fish, and considering the reality that not only single administration but also repeated administration, it is necessary to develop a safe halibut kudoworm control agent.

Accordingly, the inventors of the present invention made diligent efforts to develop a safe remedy for kudoparatus that does not cause toxicity to fish while having an excellent effect of controlling kudoparasites, and as a result of synthesizing a derivative through structural change of toltrazuril, and the toltrazuril derivative It was confirmed that the toxicity to fish was reduced while maintaining the exterminating effect on, and the present invention was completed.

An object of the present invention is to provide a novel toltrazuril derivative compound.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating parasitic infections containing toltrazuril induction as an active ingredient.

In order to achieve the above object, the present invention provides a novel toltrazuryl derivative compound having the structure of the following formula (1):

, CF₃, F 및

로 구성된 군에서 선택되고,R1 is

, CF ₃ , F and

Is selected from the group consisting of,

,

,

및

으로 구성된 군에서 선택되고,R2 is NO ₂ , NH ₂ ,

,

,

And

Is selected from the group consisting of,

R3 is characterized in that H or a C1 ~ C3 alkyl group.

The present invention also provides a pharmaceutical composition for the prevention or treatment of parasitic infections containing as an active ingredient any one of toltrazuril derivatives having the structure of the following formula (1):

[Formula 1]

, CF₃, F 및

로 구성된 군에서 선택되고,R1 is

, CF ₃ , F and

Is selected from the group consisting of,

,

,

및

으로 구성된 군에서 선택되고,R2 is NO ₂ , NH ₂ ,

,

,

And

Is selected from the group consisting of,

R3 is characterized in that H or a C1 ~ C3 alkyl group.

The toltrazuril derivative according to the present invention maintains the control effect of toltrazuril against kudoae, while lowering the toxicity to fish, and is a pharmaceutical for fishery that can control mucospore parasites that cause mortality in aquaculture sites ( Insect repellent) development will also be able to be applied broadly.

1 shows the structure of toltrazuril and the structures of two metabolites.
FIG. 2 shows the toxicity response of toltrazuril to halibut colonization, and the dotted arrows indicate 50% cell viability.
3 shows the mortality results of halibut exposed to toltrazuril over time (maintaining water temperature of 23±0.5°C).
Figure 4 shows the cumulative mortality results for the flounder by toltrazuril concentration and administration route.
5 shows the results of GOT/GPT, ALP, and BUN analysis of flounder blood according to oral administration of toltrazuril concentration.
6 shows the results of GOT/GPT, ALP, and BUN analysis of flounder blood according to injection administration according to toltrazuril concentration.
Figure 7 shows the characteristics of the toltrazuril structure (left: 4-trifluoromethylsulfanyl phenyl, middle: methyl phenoxy, right: triazinetrione).
Figure 8 shows the synthetic structure design of toltrazuril derivatives.
9 shows the synthesis process (Scheme 1) of methylaniline.
10 shows the synthesis process (Scheme 2) of the toltrazuril derivative.
Figure 11 shows a fluorescence micrograph of Kudoa live spores and dead spores after HO&PI staining, general spores (A), blue-stained fresh spores after compound reaction (B), and red-stained dead spores (C. ).
Figure 12 shows the results of the halibut colonization cytotoxic reaction to toltrazuril derivative compounds (7 types).

Until now, no drugs have been developed that can prevent infection in flounder or inhibit or inactivate the survival of Kudoae. Among animal anticoccidial agents, the in vitro inactivation efficacy of toltrazuril against Kudoae was first reported in foreign journals (Ahn M. et al. , Parasite , 24:11, 2017). However, with the exception of this report, no anthelmintic activity test of toltrazuril against kudoworm and efficacy and safety (side effects) against flounder were published at all. In the present invention, 7 kinds of toltrazuril derivative compounds with reduced toxicity to fish are synthesized while maintaining the effect of exterminating Kudoa insects of toltrazuril, and while the compound exhibits in vitro inactivation effect against Kudoa larvae, flounder coinage. It was confirmed that it exhibits a low toxic reaction to cells.

Accordingly, in one aspect, the present invention relates to a novel toltrazuryl derivative compound having the structure of Formula 1:

[Formula 1]

, CF₃, F 및

로 구성된 군에서 선택되고,R1 is

, CF ₃ , F and

Is selected from the group consisting of,

,

,

및

으로 구성된 군에서 선택되고,R2 is NO ₂ , NH ₂ ,

,

,

And

Is selected from the group consisting of,

R3 is characterized in that H or a C1 ~ C3 alkyl group.

In the present invention, the toltrazuryl derivative may be characterized in that it is a compound having a structure represented by the following Chemical Formulas 2 to 15.

[Formula 2]

,

,

[Formula 3]

,

,

[Formula 4]

,

,

[Formula 5]

,

,

[Formula 6]

,

,

[Formula 7]

,

,

[Formula 8]

,

,

[Formula 9]

,

,

[Formula 10]

,

,

[Formula 11]

,

,

[Formula 12]

,

,

[Formula 13]

,

,

[Formula 14]

및

And

[Formula 15]

.

.

Toltrazuril is an anticoccidium drug that is widely administered to various livestock including cattle, pigs, and chickens, and its mechanism of action has not been accurately identified. The administered toltrazuril is rapidly metabolized into oxidized toltrazuril sulfoxide and toltrazuril sulfone (FIG. 1). Since oxidized sulfone is more stable, its half-life is relatively long (pig: 5-7 days, chicken: 3-4 days, cattle: 2-3 days, goat: 3-8 days).

Chicken to toll Trad is known to be hungry distributed throughout the tissue and after gradually removed after administration, when the 11, 12, 18 and 19 days of age the chicks was administered a Toll trad hungry ^{(Baycox ⓡ) 7 mg / kg} , suspended administration 24 Toltrazuril metabolites were detected in chicken breasts even after days (Vertommen, M. et al. , Veterinary Quarterly , 12:183, 1990). On the other hand, in the investigation of the residual degree of toltrazuril in ^{the tissues, when toltrazuril (Baycox ⓡ} ) 7 mg/kg was administered to young chicks (30 days of age) for 2 days and a biopsy was performed, it was detected at the highest level in the kidney. Next, liver, lung, heart, skin, and fat were detected in order. It was not detected in tissues and plasma 7 days after discontinuation of administration (Soliman, A. et al, Inter J Basic & Applied Sci , 4:310, 2015).

Toltrazuril is not marketed as an unlicensed drug in the United States, but there are still cases of death from side effects of horses prescribed toltrazuril as a combination drug from a veterinarian (FDA, https://www.fda.gov/) (FDA, https://www.fda.gov/ animal-veterinary/cvm-updates/ compounded-unapproved-animal-drugs-rapid-equine-solutions-linked-three-horse-deaths., 2019). However, in the case of diclazuril and ponazuril (toltrazuril sulfone), which is also the main metabolite of toltrazuril, administration is permitted for protozoal myeloencephalitis (EPM) caused by horse protozoa. .

According to the report of EMEA (Toltrazuril. Summary report (1). EMEA/MRL/314/97-FINAL, 1998), with respect to the efficacy and effect of toltrazuril, changes in the microstructure at the development stage of coccidium protozoa, mainly It is said to cause swelling of the endoplasmic reticulum, swelling of the Golgi apparatus, and abnormalities of the nuclear membrane cavity, inhibiting nuclear fission, and inducing a decrease in the activity of parasite respiratory enzymes, but the mechanism of biochemical action is so far unclear. Once absorbed into the animal body, it is detected as two metabolites and used as a residual marker (FIG. 1), and it is known to be highly detectable in muscles and fats because it is hardly soluble in water.

The anticoccidial effect of toltrazuril is widely known, but no studies have yet been reported on the efficacy and safety of flounder infected with Kudolarvae. The use of toltrazuril in red snapper infected with Myxobolus sp. has been reported to be effective in the early spore stage of Myxobolus sp. (Schmahl, G. et al. , Archiv fㆌr Protistenkunde , 140:83, 1991). When used alone, it has been reported that it reduces the infection rate of red snapper infected by mucosporiasis from 80% to 33%, of which salinomycin and amprolium combination formulations are more effective than alone (Karagouni, E. et al., Vet). Parasitol, 34:215, 2005). However, although it is effective both alone and in combination to control mucosal spores, pathologic problems such as abdominal distension, inflammatory necrosis, and hematopoietic tissue abnormalities occurred (Athanassopoulou, F. et al., Dis Aquat Organ , 62:217, 2004). ).

Accordingly, in the present invention, a derivative for toltrazuril was synthesized in order to ensure excellent anthelmintic effect against parasites, absorption, distribution, embryonicity and persistence of the administered drug and safety (toxicity) for the target animal. .

Accordingly, the present invention relates to a pharmaceutical composition for the prevention or treatment of parasitic infections containing as an active ingredient any one of toltrazuril derivatives having the structure of the following formula (1), from another viewpoint:

[Formula 1]

, CF₃, F 및

로 구성된 군에서 선택되고,R1 is

, CF ₃ , F and

Is selected from the group consisting of,

,

,

및

으로 구성된 군에서 선택되고,R2 is NO ₂ , NH ₂ ,

,

,

And

Is selected from the group consisting of,

R3 is characterized in that H or a C1 ~ C3 alkyl group.

In the present invention, the toltrazuryl derivative may be characterized in that it is a compound having a structure represented by the following Chemical Formulas 2 to 15.

[Formula 2]

,

,

[Formula 3]

,

,

[Formula 4]

,

,

[Formula 5]

,

,

[Formula 6]

,

,

[Formula 7]

,

,

[Formula 8]

,

,

[Formula 9]

,

,

[Formula 10]

,

,

[Formula 11]

,

,

[Formula 12]

,

,

[Formula 13]

,

,

[Formula 14]

및

And

[Formula 15]

.

.

In the present invention, the parasite may be characterized in that it is selected from the group consisting of mucous spores, ciliated worms, monositic flukes, microspores, parasitic protozoa, flatworms, roundworms and hookworms, and the mucous spores Kudoa septempunctata , Myxobolus spp. , Myxosoma spp., Henneguya spp., and the ciliated worms may be Ichthyophthirius multifiliis , Trichodina spp., Apiosoma spp., and the monosynthetic flukes are Dactylogylus vastator , Dactylogylus extensus , Dactylogylus cornu , Pserodilla, arcodlus , arcodactyus , Diplozoon paradoxum, Diplozoon homoion , remind US gondii may be a Glugea anomala, the parasitic protozoa is Cystoisospora suis, Eimeria spp., Eimeria ahsata, Eimeria acervulina, Eimeria bovis, Eimeria crandallis, Eimeria columbarum, Eimeria falciformis, Eimeria flavescens, Eimeria intestinalis, Eimeria labbeana, Eimeria magna, Eimeria maxima, Eimeria mulardi, Eimeria perforans, Eimeria stiedai, Eimeria tenella, Eimeria zuerinii, Hepatozoon canis, Isopora spp., be a Isopora felis, Isopora rivolta, Isopora suis, Neospora caninum, Sarcocystis neurona, Sarcocystis calchasi, Toxoplasma gondii, and the The flatworm may be Trichuris vulpis , the roundworm may be Toxocara canis , and the hookworm may be Ancylostoma caninum , Uncinaria stenocephala , but is not limited thereto.

In one embodiment of the present invention, 16 novel toltrazuril derivative compounds were synthesized to develop a remedy for halibut kudoparasitica, and in vitro inactivation effects and toxic reactions to halibut colonization cells were investigated. In addition, 7 kinds of toltrazuril derivative compounds (PK05, PK06, PK07, PK08, PK09, PK11, PK12), which are closest to their development potential, were developed through a study on the efficacy and safety of Kudolarvae in halibut in the future.

Accordingly, the compound of the present invention is N -(3-Methyl-4-(4-(trifluoromethoxy)phenoxy)phenyl)acetamide, N -(3-Methyl-4-(4-(trifluoromethyl)phenoxy)phenyl)acetamide, N -(4-(4-Methoxyphenoxy)-3-methylphenyl)acetamide, N -(4-(4-Fluorophenoxy)-3-methylphenyl)acetamide, N -Methyl- N -(3-methyl-4-(4- Consisting of (trifluoromethoxy)phenoxy)phenyl)acetamide, N -(4-(4-Methoxyphenoxy)-3-methylphenyl)- N -methylacetamide and N -(4-(4-Fluorophenoxy)-3-methylphenyl)- N -methylacetamide It may be characterized in that it is a derivative selected from the group.

The derivative of the present invention is a synthetic set of four basic structures that have changed the left structure of toltrazuril, and these are various structural changes to the right from PK01 to PK04, PK05 to PK08, PK09 to PK12, and PK13 to PK16, respectively. have. In other words, as a result of introducing variously to obtain insecticidal effect on the right (triazine trione) centering on the left basic structure, PK05, PK06, PK07, PK08> PK09, PK11, PK12 achieved excellent results in the inactivation efficacy investigation in the order of PK05, PK06, PK07, PK08> PK09, PK11, PK12. All.

The carrier used in the pharmaceutical composition of the present invention includes pharmaceutically acceptable carriers, adjuvants and vehicles, and is collectively referred to as "pharmaceutically acceptable carrier". Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal Silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene-polyoxypropylene-blocking polymer, polyethylene glycol and wool paper, and the like.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal.

Oral and parenteral administration is preferred. The term “parenteral” as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical composition may be in the form of a sterile injectable preparation as an aqueous or oily suspension for sterile injection. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (eg, a solution in 1,3-butanediol). Vehicles and solvents that can be used permissively include mannitol, water, Ringel's solution and isotonic sodium chloride solution. In addition, sterile nonvolatile oils are commonly used as solvents or suspending media. For this purpose, any less irritating nonvolatile oil can be used, including synthetic mono or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in injection formulations as are pharmaceutically acceptable natural oils (eg olive oil or castor oil), especially their polyoxyethylated.

The pharmaceutical composition of the present invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If necessary, sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of the present invention with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycol.

The term “therapeutically effective amount” means for fish, the therapeutically effective amount used in the composition for treating infections caused by pathogens is 5 to 500 mg. In the case of livestock, the therapeutically effective amount used in the composition for treating infectious diseases caused by pathogens is 2.5 to 200 mg.

When the pharmaceutical composition according to the present invention is injected into the subcutaneous cells of fish, it can be administered to the sac or the digestive tract. Injection can be injected into muscle cells or other cells in muscle tissue, and can be injected into visceral cells in the abdominal cavity.

In a preferred embodiment, the pharmaceutical composition for oral administration may be prepared by mixing the active ingredient with a solid excipient, and may be prepared in the form of granules for preparing in the form of tablets or dragees. Suitable excipients include sugar forms such as lactose, sucrose, mannitol and sorbitol, or starch from corn, wheat flour, rice, potatoes or other plants, cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose or sodium carboxymethylcellulose. , Arabic gum, gums, such as gums, such as gums, or protein fillers such as gelatin and collagen may be used. If necessary, disintegrants or solubilizing agents in the form of respective salts such as crosslinked polyvinylpyrrolidone, agar and alginic acid or sodium alginic acid may be added.

As a preferred embodiment, for parenteral administration, the pharmaceutical composition of the present invention can be prepared as an aqueous solution. Preferably, a physically appropriate buffer solution such as Hank's solution, Ringer's solution, or physically buffered saline may be used. Aqueous injection suspensions may be added with a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. In addition, suspensions of the active ingredient can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension can use suitable stabilizing agents or agents to increase the solubility of the compound and to prepare high concentration solutions.

Hereinafter, the present invention will be described in more detail by examples. These examples are merely for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the technical scope of the present invention is not limited to these examples.

Example 1: Toltrazuril in vitro Confirmation of cytotoxic reaction

The cytotoxicity of the drug was measured using fish-coated cells (flounder embryo-derived cells, HINAE). Cells of 1×10 ⁵ /well were cultured at 20° C. for 24 h to check 80% density, and then toltrazuril was treated at different concentrations (0, 1, 2, 5, 10, 20, 40, 100 ppm). . After incubation for 96 hours, a cytotoxic drug (CCK-8, Dojindo) was treated and absorbance was measured at 450 nm.

HINAE cells showed a decrease in viability in a concentration-dependent manner, and when toltrazuril was added at a concentration of 20 ppm, the cell survival rate was at a level of 54% compared to the normal control group (FIG. 2). As the concentration of toltrazuril increased, the number of cells decreased, and in addition, it was confirmed that the shape of the cells was condensed compared to the control group.

Example 2: Half-lethal concentration of toltrazuril for flounder (LC ₅₀ )

All experimental fish were tested for aquatic biological pathogens (parasites, bacteria, viruses) and then tested for 2 weeks through acclimatization. _{The half-lethal concentration (LC 50} ) of 70 halibut (17.2±0.37 cm, 45.1±3.97 g) was investigated. Dissolve commercially available toltrazuril (No. 34000, Sigma) in DMSO (dimethyl sulfoxide) and maintain a 5L water bath (water temperature 23±0.5℃) by concentration (0, 1, 2, 5, 10, 20, 40, 100 ppm) ) Was immersed for 96 hours, and the mortality and clinical symptoms inside and outside the fish body and histopathologic investigation were conducted by time.

To investigate the acute toxicity concentration of toltrazuril, it was divided into the first and second phases. In the first investigation, the control, 1, 5, 10, 20, and 50 ppm were investigated in 6 sections, and all died above 5 ppm. , In particular, at a high concentration (more than 20 ppm), excessive mucus secretion and internal bleeding were observed in dead subjects. Trichodina and Icthio complement were detected in fish and breeding water. Subsequently, as a result of irradiation with 8 sections of control, 1, 0.1, 0.2, 0.5, 1, 2, 5, and 10 ppm in the second irradiation, 80% death occurred at 10 ppm (Fig. 3). As a result of the acute toxicity investigation, 96h-LC ₅₀ was found to be 9.2 ppm. In particular, the dead subjects had excessive secretion of mucus and internal bleeding. As a result of these results, it was estimated that acute toxicity was very high when a fish was infected with a pathogen.

As a result of histopathologic examination of surviving subjects, toltrazuril showed a significant difference in a concentration-dependent manner. Compared with the control group, it was confirmed that degenerative changes were observed in the liver, spleen, gills, stomach, intestines, and kidneys. Hepatic congestion and microvesicular type fatty changes occurred in the liver, and glomerulonephritis (RES, Reticuloendothelial system) activity, epithelial cell necrosis and melano macrophage center (MMC) production in the gills, vacuolization of gastric gland epithelium, intestinal epithelial necrosis, and inflammation in the digestive tract occurred multiple times. In the kidney, tubular necrosis and degeneration, and RES activity were found significantly.

The toxicity of toltrazuril to aquatic organisms was varied. In rainbow trout, 96h-LC ₅₀ was 0.44 mg/L. In algae ( Selenastrum capricornutum ), EC ₅₀ (half-effect concentration) was 3.16 mg/L. In addition, Daphnia magna showed weak toxicity (1.0<LC/EC ₅₀ <10 mg/L) (Rojickova et al., 1998). The EC ₅₀ of zebrafish embryos was reported to be 1.1 mg/L (Carlsson G. et al., Aquat. Toxicol , 126:30, 2013).

Example 3: Half-lethal dose (LD ₅₀ ) Confirm

On the basis of the cytotoxic response and LC ₅₀ , a single (one time) oral administration and injection of toltrazuril to 120 flounder (19.5±0.74 cm, 72.8±7.60 g) resulted in the death of 50% of the test group. LD ₅₀ ) was investigated. Commercially available toltrazuril (No. 34000, Sigma) was dissolved in DMSO and administered orally or by injection at different concentrations (0, 5, 10, 20, 50, 100, 200 mg/kg body weight).

Death began to occur from the high concentration of 100 mg/kg BW in both the oral administration and injection administration, and the LD ₅₀ of the oral administration was 44.75 mg/kg, and the LD ₅₀ of the injection administration was 26.28 ppm (FIG. 4).

In the acute toxicity test in which toltrazuril was administered orally to Wistar female rats, the half-lethal dose (LD ₅₀ ) was 2,000 mg/kg or more, and in the acute toxicity test in which toltrazuril sulfone was administered orally to male and female rats, LD ₅₀ was 5,000 mg. /kg or more (Japan Food Safety Committee, 2007, 2008, 2016).

Example 4: Blood biochemical investigation of toltrazuril flounder

Experimental instruments that were orally administered and injected to 300 flounder (21.5±1.4 cm, 96.0±18.6 g) were prepared with 0, 5, 10, 15 mg/kg BW of toltrazuril and 1, 3, 7, 14, after administration. Blood was collected on the 28th day, serum was separated, and stored at -80°C until analysis. Analysis items were transaminase GOT(AST)/GPT(ALT), glucose (GLU), total protein (TP), Total cholesterol (TCHO), alkaline phosphatase (ALP), and urea nitrogen (BUN) were measured. For blood biochemical analysis, a Fuji-Dri Chem 4500 (Fuji, Japan) analyzer was used. The result is SPSS program for Windows ver. One-way ANOVA and Duncan's multiple range test by 23.0 (SPSS Inc. USA) were performed to test the significance (p<0.05) between means.

GPT and GOT, which are indicators of hepatotoxicity, which are indicators of liver function when oral administration by the concentration of toltrazuril, showed a tendency to increase significantly on the 1st day of oral administration, and the highest concentrations of GPT and GOT on the 1st day of oral administration. After showing, it decreased as time passed. In the case of ALP, a significant increase was maintained from the 7th day to the 14th day of administration and then decreased on the 28th day. In the case of BUN, which can be said to be an index of renal function, a tendency to decrease significantly from the 3rd day of oral administration was shown, and significant differences were shown for each dose concentration (Table 1 and FIG. 5). When absorption disorders occurred in the digestive tract, GLU, TP, and TCHO, which are used as indicators of nutritional status, showed a significant increase by oral administration concentration, but GLU increased from day 1 and maintained until day 28, showing the highest value for a long time. TP increased from the 1st day, but maintained gently until the 7th day, and the TCHO increased slightly from the 7th day and continued until the 28th.

When administered by injection according to the concentration of toltrazuril showed an overall similar trend to that of oral administration (Tables 2 and 6).

Blood biochemical analysis of flounder according to oral administration of toltrazuril concentration term density
(ppm) Body
weight(g) Length
(cm) GOT
(U/L) GPT
(U/L) GLU
(mg/dL) BUN
(mg/dL) ALP
(mg/dL) TCHO
(mg/dL) TP
(g/dL) One
First 0 92.2±7.0 21.4±0.4 14±1.4 ^a 7.2±0.4 ^a 8.8±1.4 ^a 1.6±0.1 ^a 99.0±6.4 ^a 158.6±15.8 ^a 2.7±0.2 ^ab 5 84.5±11.3 21.8±1.0 19.8±2.1 ^b 9.6±0.7 ^b 11.4±1.2 ^ab 2.6±0.3 ^c 99.0±0.9 ^a 151.4±14.8 ^a 3.2±0.3 ^b 10 87.7±4.3 21.0±0.2 22.4±2.5 ^c 12.0±1.9 ^c 13.8±1.7 ^b 3.0±0.3 ^cd 108.6±6.2 ^a 164.6±16.5 ^a 3.9±0.2 ^c 15 100.0±8.0 21.9±0.6 29.6±2.6 ^d 14.2±1.0 ^d 13.8±1.4 ^b 3.3±0.2 ^d 85.4±10.9 ^a 167.6±14.2 ^a 4.3±0.4 ^d 3
First 0 93.3±3.4 21.3±0.3 13.6±0.7 ^a 7.0±0.3 ^a 9.8±1.7 ^a 1.5±0.0 ^a 102.0±10.3 ^a 167.6±17.1 ^a 2.9±0.3 ^ab 5 94.6±8.8 21.6±0.5 16.4±2.2 ^ab 7.8±0.4 ^a 12.4±1.6 ^ab 2.5±0.2 ^bc 112.4±10.8 ^a 157.0±14.3 ^a 3.1±0.3 ^b 10 90.0±6.6 21.1±0.7 21.2±2.6 ^c 8.2±0.9 ^ab 14.6±1.3 ^b 2.8±0.1 ^c 135.8±17.0 ^b 158.6±15.7 ^a 3.1±0.3 ^b 15 87.5±9.1 20.4±1.0 20.3±2.2 ^c 9.2±1.1 ^b 14.6±1.4 ^b 2.8±0.2 ^c 101.8±18.0 ^a 182.0±18.7 ^ab 3.6±0.2 ^bc 7
First 0 90.0±5.9 21.5±0.4 13.4±1.0 ^a 6.0±0.3 ^a 9.6±1.4 ^a 1.7±0.1 ^a 107.0±10.6 ^a 162.0±14.3 ^a 2.8±0.3 ^ab 5 103.3±8.8 22.2±0.6 15.6±2.2 ^ab 6.6±0.9 ^a 14.2±1.1 ^b 2.2±0.2 ^b 143.2±13.9 ^b 178.6±18.5 ^ab 2.9±0.3 ^ab 10 104.3±7.8 22.4±0.6 17.2±1.03 ^b 7.2±0.5 ^ab 16.2±1.3 ^b 2.6±0.2 ^c 223.0±19.2 ^d 201.6±22.2 ^b 3.2±0.3 ^b 15 101.6±6.5 22.0±0.7 18.8±2.0 ^b 8.2±0.8 ^b 15.4±2.3 ^b 2.6±0.2 ^c 160.8±11.6 ^bc 213.4±21.4 ^b 3.3±0.4 ^bc 14
First 0 23.5±0.6 117.9±8.7 12.6±1.4 ^a 5.8±0.4 ^a 9.2±1.4 ^a 1.7±0.2 ^a 109.8±6.6 ^a 167.2±16.4 ^a 2.9±0.2 ^ab 5 21.4±0.5 91.1±4.4 15.6±0.2 ^ab 5.8±0.4 ^a 13.2±1.0 ^ab 2.0±0.0 ^ab 164.2±17.0 ^bc 247.4±23.6 ^c 2.5±0.2 ^a 10 22.6±0.8 107.3±10.0 17.4±1.7 ^b 7.0±0.7 ^ab 16.2±1.6 ^b 2.2±0.2 ^b 200.8±17.3 ^c 235.4±20.8 ^c 3.1±0.3 ^b 15 23.4±0.7 115.4±8.4 18.2±2.0 ^b 7.2±0.7 ^ab 18.4±1.2 ^bc 2.3±0.2 ^bc 222.8±22.6 ^d 237.4±23.9 ^c 3.2±0.4 ^b 28
First 0 23.5±0.8 115.7±11.3 12.8±0.7 ^a 5.6±0.2 ^a 10.3±0.9 ^a 1.6±0.1 ^a 105.0±10.3 ^a 170.0±10.7 ^a 2.3±0.2 ^a 5 21.8±0.4 97.8±5.3 13.8±0.6 ^a 5.6±0.2 ^a 17.8±1.9 ^bc 1.6±0.1 ^a 157.2±7.0 ^bc 270.6±27.6 ^cd 2.3±0.2 ^a 10 23.6±0.7 119.4±13.2 14.0±1.3 ^a 5.4±0.9 ^a 21.2±2.1 ^c 1.5±0.1 ^a 186.4±11.5 ^c 280.0±25.8 ^cd 2.5±0.3 ^a 15 22.3±0.7 100.2±9.4 15.0±1.9 ^ab 6.0±0.5 ^a 24.2±1.2 ^d 1.7±0.1 ^a 178.0±5.8 ^c 298.0±30.0 ^d 2.7±0.3 ^ab * GOT; glutamic oxaloacetic transaminase, GPT; glutamic pyruvic transaminase, ALP; alkaline phosphatase, GLU; glucose, BUN; blood urea nitrogen, TCHO; total cholesterol, TP; total protein

term density
(ppm) Body
weight(g) Length
(cm) GOT
(U/L) GPT
(U/L) GLU
(mg/dL) BUN
(mg/dL) ALP
(mg/dL) TCHO
(mg/dL) TP
(g/dL) One
First 0 97.2±9.3 21.2±0.8 25.0±1.6 ^a 8.1±0.5 ^a 9.6±0.6 ^a 1.6±0.1 ^a 93.6±4.8 ^a 149.4±15.4 ^a 2.7±0.3 ^ab 5 91.3±4.0 21.6±0.4 54.2±3.8 ^b 10.4±1.0 ^b 11.8±1.0 ^a 2.6±0.2 ^b 135.2±13.4 ^ab 155.2±16.5 ^a 2.8±0.3 ^ab 10 102.4±93.3 22.0±0.3 76.2±7.9 ^c 11.2±1.6 ^c 12.8±1.0 ^a 3.1±0.3 ^c 161.8±11.9 ^b 170.5±16.3 ^a 2.9±0.1 ^ab 15 93.3±5.2 21.4±0.5 82.5±9.2 ^d 12.6±1.8 ^d 15.5±1.4 ^ab 3.3±0.3 ^d 119.3±11.7 ^ab 191.4±17.2 ^ab 2.9±0.3 ^ab 3
First 0 104.4±6.9 22.3±0.7 23.4±2.2 ^a 7.6±0.2 ^a 9.3±2.3 ^a 1.6±0.1 ^a 98.0±10.2 ^a 155.4±18.0 ^a 2.9±0.3 ^ab 5 96.3±6.4 21.8±0.8 30.0±2.0 ^a 7.0±0.5 ^a 16.2±0.9 ^ab 2.5±0.3 ^b 144.3±8.1 ^b 161.0±16.4 ^a 3.0±0.3 ^ab 10 110.0±8.0 22.2±0.6 39.8±3.8 ^ab 7.6±0.6 ^a 18.0±1.3 ^ab 2.8±0.3 ^bc 181.0±17.7 ^c 171.4±18.8 ^a 3.3±0.2 ^b 15 99.8±9.0 21.9±0.4 42.5±3.48 ^ab 8.3±0.2 ^a 19.0±0.7 ^ab 3.0±0.3 ^c 135.2±11.6 ^b 178.1±16.4 ^a 3.5±0.3 ^c 7
First 0 107.9±12.0 22.3±0.8 25±0.8 ^a 7.3±1.0 ^a 8.7±0.9 ^a 1.6±0.1 ^a 100.0±10.6 ^a 155.2±19.0 ^a 2.8±0.3 ^ab 5 111.5±12.9 22.9±1.0 19.6±1.24 ^a 7.0±0.3 ^a 16.8±0.7 ^ab 2.1±0.1 ^ab 153.0±10.8 ^b 168.2±18.2 ^a 2.9±0.3 ^ab 10 107.3±9.6 22.5±0.6 19.4±2.9 ^a 6.4±0.2 ^a 20.2±1.5 ^b 2.2±0.1 ^ab 185.5±8.5 ^c 178.8±16.3 ^a 2.8±0.3 ^ab 15 105.2±7.8 22.3±0.6 21.5±2.1 ^a 6.8±0.9 ^a 25.6±2.5 ^c 3.1±0.4 ^c 170.4±16.3 ^bc 221.4±20.2 ^b 3.2±0.3 ^b 14
First 0 117.8±4.3 23.3±0.5 21.5±0.5 ^a 6.6±1.0 ^a 9.6±1.1 ^a 1.8±0.1 ^a 99.0±4.8 ^a 160.0±16.3 ^a 2.6±0.3 ^a 5 123.0±18.3 23.5±1.3 20.7±3.6 ^a 6.6±0.2 ^a 22.8±2.9 ^c 2.1±0.2 ^ab 161.8±14.5 ^bc 199.0±20.8 ^ab 2.7±0.3 ^ab 10 116.2±4.4 23.6±0.6 20±2.9 ^a 6.6±0.2 ^a 24.5±2.2 ^c 2.2±0.3 ^ab 190.2±18.3 ^c 229.2±23.9 ^b 2.8±0.2 ^ab 15 111.4±10.3 22.6±0.8 18.8±1.4 ^a 6.6±0.7 ^a 25.0±2.4 ^c 2.4±0.1 ^ab 262.0±22.3 ^d 240.2±23.2 ^b 3.0±0.2 ^ab 28
First 0 97.7±7.5 22.2±0.7 22.8±1.7 ^a 6.0±1.1 ^a 9.6±0.7 ^a 1.7±0.1 ^a 95.0±4.8 ^a 163.0±16.3 ^a 2.4±0.2 ^a 5 93.1±7.1 22.2±0.6 18±1.0 ^a 5.6±0.5 ^a 16.5±1.4 ^ab 1.8±0.1 ^a 170.2±13.3 ^bc 256.2±28.8 ^bc 2.4±0.2 ^a 10 119.4±13.2 23.6±0.7 18.8±1.4 ^a 5.6±0.4 ^a 19.0±1.1 ^b 1.8±0.2 ^a 199.0±9.4 ^c 271.8±23.6 ^bc 2.5±0.3 ^a 15 100.2±9.4 22.3±0.7 18.6±0.4 ^a 5.3±0.4 ^a 19.6±3.9 ^b 1.9±0.1 ^a 145.0±14.1 ^b 282.0±27.5 ^c 2.4±0.2 ^a * GOT; glutamic oxaloacetic transaminase, GPT; glutamic pyruvic transaminase, ALP; alkaline phosphatase, GLU; glucose, BUN; blood urea nitrogen, TCHO; total cholesterol, TP; total protein

Example 5: Histopathological investigation of toltrazuril flounder

After the blood biochemical investigation, the flounder from the same sample was dissected and the gills, liver, spleen, kidney, stomach, intestine, and heart were excised and fixed in neutral buffered formalin fixative for 24 hours. Each fixed organ is shredded again and fixed in the same fixative for a second time (12 hours), washed with water, subjected to dehydration, clarification, and paraffin permeation, and then a paraffin specimen is made, and a slide is cut to a thickness of 4-5 μM using a microtome. Attached to the glass and dried. Hematoxyline and Eosin (H&E) staining, which is used for general tissue observation, was performed to make a tissue sample and the degree of degeneration was observed under a microscope.

In addition to the major organs (gills, liver, kidneys) for which toxicity can be evaluated, a total of 7 organs including the stomach, intestine, heart, and spleen were examined. The pathologic toxicity assessment was based on the method of Bernet et al . (Bernet, D. et al., J Fish Dis 22:25, 1999), based on circulatory disorders (bleeding, congestion), degenerative degeneration (cell necrosis, atrophy), and progressive degeneration. Significant changes were investigated for (hypertrophy, proliferation) and inflammatory degeneration.

Compared to the control, toltrazuril 5 mg/kg oral administration of congestion, atrophy and hepatitis in the liver, infiltration and activity of lymphocytic inflammatory cells in the spleen, and inflammation in the kidney (reticuloendothelial system activity, glomerulonephritis) in the kidney, and Hematopoietic activity was enhanced. In particular, among the tissues subject to oral administration of 10 mg/kg and 15 mg/kg, the liver showed symptoms of congestion and severe fatty liver and atrophy due to circulatory disorders, while the kidney showed significant inflammatory changes in hematopoietic and glomerulonephritis. Trazuril showed histopathologic degeneration in liver and kidney in a concentration-dependent manner.

Similar to the oral administration group, the toltrazuril injection group showed a significant change in the pathologic histology mainly in the immune tissue in a concentration-dependent manner. Compared to the control, the liver showed mild congestion and atrophy in the liver, mild hematopoietic activity in the kidney, and symptoms of glomerulonephritis, whereas the spleen, an inflammatory lesion, showed additional lymphocyte infiltration. At 10 ppm, the liver showed mild congestion, fatty liver and hyaline droplet formation, and showed significant changes in severe hematopoietic activity and tubular atrophy. At a high concentration of 15 ppm, hepatocellular hypertrophy, lymphocyte infiltration, and multiple granulomatous formation in the kidney were observed.

Example 6: Synthesis of toltrazuryl derivative compound

Toltrazuril can be divided into large subdivisions as shown in FIG. 7, 4-trifluoromethylsulfanyl phenyl on the left, methyl phenoxy on the center, and triazinetrione on the right, and characteristically contains fluorine (F), sulfur (S), and triazinetrione ( Fig. 7).

Fluorine plays a very important role in the field of new drug development, as approximately 25% of commercially available drugs have more than one. When introduced into the structure, metaboilc stability, binding to target organs, and In addition, as pharmacological properties such as membrane permeability are improved, bioavailability and medicinal efficacy may increase. This is useful not only in the development of small molecular drugs, but also in the development of drugs containing peptides with low bioavailability.

Sulfur is easily oxidized or metabolized to sulfoxide and sulfone in the body as well as in the air. And since oxidized sulfone is more stable as described above, its half-life is relatively long.

Triazine is also known to exhibit strong biological activities such as anti-inflammatory, anti-mycobacterial, anti-viral, and anti-cancer, and is limited to anticancer drugs (Altretamine and triethylenemelamine), herbicides (atrazine), and animal repellents (cyromazine). It is commercially available.

These factors are expected to significantly affect the retention of drugs in the body as well as the strong insecticidal effect of toltrazuril, and the strong insecticidal effect may act as a side effect in the flounder. Therefore, by dividing the toltrazuril structure into subdivisions, it is intended to synthesize compounds that can maintain drug efficacy while securing safety by making appropriate changes such as introducing various moieties similar to triazine, adding or subtracting fluorine, and introducing oxygen instead of sulfur. .

The structure-activity relationship between coccidiosis and toltrazuril has not been clearly identified, but toltrazuril contains triazine, which is used as an insecticide, in the form of triazine trione, and fluorin, which has an effect such as retention in the body, When oxidized in the body, sulfur becomes more stable as it forms sulfon. This leads to problems such as strong insecticidal effect and long half-life of toltrazuril, resulting in toxicity and retention in vivo.

Thus, as shown in FIG. 8, oxygen was introduced instead of sulfur in the left part or changed to (-OCF ₃ ), (-CF ₃ ), (-OCH ₃ ), (-F), and the triazine trione part on the right In order to check the minimum part for obtaining the insecticidal effect and the size of the space of the bonding part, dimethyl, acetyl, acetylamide, and oxobutanoic acid were introduced into aniline to obtain 16 structures (FIG. 8).

To synthesize this, first, four kinds of phenol (1, 2, 3, 4) were reacted with 2-Fluoro-5-nitrotoluene and nucleophilic aromatic substitution to obtain methylnitrobenzene (5, 6, 7, 8), and then Fe and NH ₄ Cl was used to reduce the nitro group to obtain methylaniline (9, 10, 11, 12) (Scheme 1, Fig. 9).

The obtained methylaniline was _{reductive methylation using paraformaldehyde and NaBH 3} CN to introduce a dimethyl group to obtain the final compounds PK01, PK02, PK03, PK04, and methylaniline was acetylated using acetylchloride and NaH, PK05, PK06, PK07, PK08 was obtained, and PK05, PK06, PK07, and PK08 thus obtained were methylated using methyliodide and NaH, respectively, to obtain PK09, PK10, PK11, and PK12. In addition, by reacting methylaniline and succinic anhydride, acid forms of PK13, PK14, PK15, and PK16 were obtained (Scheme 2, Fig. 10).

Among the 16 final compounds, four acid compounds (PK13, PK14, PK15, PK16) synthesized considering the very low solubility of toltrazuril were included. These are compounds designed to increase dissolution in water by making PK13, PK14, PK15, and PK16, which have poor solubility in water, with Na salt. As can be observed in the example of PK13 below, the acid form has solubility in water. Is very low, but it was confirmed that it dissolves well in water when it becomes Na salt by dissolving it in the same amount of NaOH aqueous solution.

The structure of the 16 compounds synthesized in this way ^{was confirmed by 1} H NMR and mass spectrometry. Among them, ¹ H NMR data of PK01, PK05, PK09, and PK16 and mass data of PK05 are shown below.

(1) Synthesis of methylnitrophenoxybenzene (5, 6, 7, 8)

(A) 2-Methyl-4-nitro-1-(4-(trifluoromethoxy)phenoxy)benzene (5)

2-Fluoro-5-nitrotoluene (776 mg, 5.0 mmol, 1.0 eq) and K ₂ CO ₃ (1.38 g, 10.0 mmol, 2.0 eq) were added to a 15 mL one-arm flask, substituted with argon gas, and distilled. It was dissolved by adding DMF (5 mL). Trifluoromethoxyphenol (0.78 mL, 6.0 mmol, 1.2 equivalent) was added thereto, and then ^{stirred at 108 o} C for 22 hours. After the reaction was completed and cooled to room temperature, it was diluted with EtOAc (ethyl acetate, 50 mL) and washed with distilled water (50 mL). After separating the two layers, the aqueous layer was extracted 3 times with EtOAc (100 mL). The extracted organic layers were collected _{, moisture was removed with anhydrous MgSO 4} , filtered, and then concentrated. Compound 5 was synthesized by separating and purifying by column chromatography using silica gel, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 1.57 g, 50 mmol, 100%, colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.17 (dd, J = 2.8, 1.2 Hz, 1H), 8.02 (dd, J = 8.8, 2.8 Hz, 1H), 7.28 (m, 1H), 7.25 (m , 1H), 7.08-7.02 (m, 2H), 6.81 (d, J = 8.8 Hz, 1H), 2.40 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 160.7, 154.0, 145.7, 143.2, 130.0, 127.0, 123.3, 123.1, 120.8, 120.5 (q, J _CF = 258.3 Hz), 116.5, 16.4.

HRMS (ESI) m/z: calculated for C ₁₄ H ₁₀ F ₃ NO ₄ 313.0562, found 313.0564.

(B) 2-Methyl-4-nitro-1-(4-(trifluoromethyl)phenoxy)benzene (6)

DMF (5 mL) obtained by distilling 2-Fluoro-5-nitrotoluene (776 mg, 5.0 mmol, 1.0 eq) and K ₂ CO ₃ (1.38 g, 10.0 mmol, 2.0 eq) in the same manner as the synthesis method of compound 5 And melted it. Compound 6 was synthesized by adding 4-hydroxybenzotrifluoride (973 mg, 6.0 mmol, 1.2 equiv) and ^{stirring at 106 o} C for 19 hours and 118 ^o C for 5 hours, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 1.22 g, 4.10 mmol, 82%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.19 (dd, J = 2.8, 0.4 Hz, 1H), 8.06 (ddd, J = 8.8, 2.8, 0.4 Hz, 1H), 7.66 (d, J = 8.4 Hz , 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.8 Hz, 1H), 2.38 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 159.6, 158.6, 143.8, 130.7, 127.7 (d, J _CF = 2.5 Hz), 127.1, 126.6 (q, J _CF = 32.8 Hz), 124.0 (q, J _CF = 272.2 Hz), 123.3, 118.9, 118.0, 16.4.

HRMS (ESI) m/z: calculated for C ₁₄ H ₁₀ F ₃ NO ₃ 297.0613, found 297.0614.

(C) 1-(4-Methoxyphenoxy)-2-methyl-4-nitrobenzene (7)

DMF (5 mL) obtained by distilling 2-Fluoro-5-nitrotoluene (776 mg, 5.0 mmol, 1.0 eq) and K ₂ CO ₃ (1.38 g, 10.0 mmol, 2.0 eq) in the same manner as the synthesis method of compound 5 And melted it. Compound 7 was synthesized by adding 4-methoxyphenol (745 mg, 6.0 mmol, 1.2 equiv) and ^{stirring at 110 o} C for 22 hours, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 1.30 g, 5.0 mmol, 100%, pale yellow solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.13 (dd, J = 2.8, 0.8 Hz, 1H), 7.96 (dd, J = 9.2, 2.8 Hz, 1H), 7.01 -6.91 (m, 4H), 6.67 (d, J = 9.2 Hz, 1H), 3.83 (s, 3H), 2.42 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 162.3, 156.9, 148.4, 142.1, 128.7, 126.7, 123.2, 121.5, 115.3, 114.5, 55.7, 16.4.

HRMS (ESI) m/z: calculated for C ₁₄ H ₁₃ NO ₄ 259.0845, found 259.0846.

(la) 1-(4-Fluorophenoxy)-2-methyl-4-nitrobenzene (8)

DMF (5 mL) obtained by distilling 2-Fluoro-5-nitrotoluene (800 mg, 5.16 mmol, 1.0 eq) and K ₂ CO ₃ (1.38 g, 10.0 mmol, 1.94 eq) in the same manner as for the synthesis of compound 5 And melted it. Compound 8 was synthesized by adding 4-fluorophenol (0.56 mL, 6.09 mmol, 1.18 eq) and ^{stirring at 110 o} C for 19 hours, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 1.27 g, 5.14 mmol, 100%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.15 (dd, J = 2.8, 0.8 Hz, 1H), 7.99 (ddd, J = 9.2, 2.8, 0.4 Hz, 1H), 7.15-7.07 (m, 2H) , 7.05-6.99 (m, 2H), 6.72 (d, J = 9.2 Hz, 1H), 2.41 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 161.5, 159.7 (d, J _CF = 244.4 Hz), 151.1, 142.6, 129.3, 126.8, 123.2, 121.6 (d, J _CF = 8.8), 116.9 (d, J _CF = 23.9), 115.3, 16.4.

HRMS (ESI) m/z: calculated for C ₁₃ H ₁₀ FNO ₃ 247.0645, found 247.0647.

(2) Synthesis of methylphenoxyaniline (9, 10, 11, 12)

(end) 3-Methyl-4-(4-(trifluoromethoxy)phenoxy)aniline (9)

Fe (894 mg, 16 mmol, 5.0 eq) and NH ₄ Cl (171 mg, 3.2 mmol, 1.0 eq) were added to a 50 mL round bottom flask and dissolved in distilled water (8 mL). Methylnitrobenzene (5, 1.0 g, 3.2 mmol, 1 eq) was dissolved in EtOH (16 mL), added to the flask, and ^{stirred at 90 o} C for 1 hour. After the reaction was completed and cooled to room temperature, excess Fe was removed using a celite pad, and washed with EtOH (10 mL) and EtOAc (100 mL). After the filtrate was concentrated, it was further diluted with EtOAc (10 mL) and washed with distilled water (10 mL). After separating the two layers, the aqueous layer was extracted twice with EtOAc (25 mL). The extracted organic layers were collected _{, moisture was removed with anhydrous MgSO 4} , filtered, and then concentrated. Separation and purification using silica gel column chromatography to obtain compound 9, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties:844 mg, 2.98 mmol, 93%, brown oil.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 7.28 (d, J = 9.2 Hz, 2H), 6.88-6.81 (m, 2H), 6.70 (d, J = 8.4 Hz, 1H), 6.50 (dd , J = 2.8, 0.8 Hz, 1H), 6.44 (dd, J = 8.4, 2.8 Hz, 1H), 4.97 (s, 2H), 1.96 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 157.9, 146.2, 142.8, 142.2, 130.1, 122.7, 121.8, 120.2 (q, J _CF = 255.8 Hz), 116.4, 116.3, 112.8, 15.8.

HRMS (ESI) m/z: calculated for C ₁₄ H ₁₃ F ₃ NO ₂ ⁺ [M+H] ⁺ 284.0893, found 284.0894.

(B) 3-Methyl-4-(4-(trifluoromethyl)phenoxy)aniline (10)

In the same manner as for the synthesis of compound 9, Fe (894 mg, 16 mmol, 5.0 eq) and NH ₄ Cl (171 mg, 3.2 mmol, 1.0 eq) were dissolved in distilled water (8 mL). Methylnitrobenzene ( 6 , 951 mg, 3.2 mmol, 1.0 eq) was dissolved in EtOH (16 mL), added to the flask, and ^{stirred at 90 o} C for 1 hour to synthesize compound 10, and mass spectometry (mass spectometry, MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 830 mg, 3.11 mmol, 97%, brown oil.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 7.65 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 2.8 Hz, 1H), 6.45 (dd, J = 8.4, 2.8 Hz, 1H), 5.01 (s, 2H), 1.94 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 162.0, 146.5, 142.2, 130.1, 127.3, 124.5 (q, J _CF = 270.9 Hz), 121.8 (q, J _CF = 32.8 Hz), 121.9, 116.3, 115.6, 112.8, 15.8.

HRMS (ESI) m/z: calculated for C ₁₄ H ₁₃ F ₃ NO ⁺ [M+H] ⁺ 268.0949, found 268.0949.

(C) 4-(4-Methoxyphenoxy)-3-methylaniline (11)

In the same manner as for the synthesis of compound 9, Fe (894 mg, 16 mmol, 5.0 eq) and NH ₄ Cl (171 mg, 3.2 mmol, 1.0 eq) were dissolved in distilled water (8 mL). Methylnitrobenzene ( 7 , 830 mg, 3.2 mmol, 1.0 eq) was dissolved in EtOH (16 mL), added to the flask, and ^{stirred at 90 o} C for 1 hour to synthesize compound 11, mass spectometry, MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 702 mg, 3.06 mmol, 96%, brown solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 6.89-6.80 (m, 2H), 6.75-6.67 (m, 2H), 6.61 (d, J = 8.4 Hz, 1H), 6.46 (dd, J = 2.8, 0.8 Hz, 1H), 6.39 (ddd, J = 8.4, 2.8, 0.8 Hz, 1H), 4.85 (s, 2H), 3.69 (s, 3H), 1.98 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 153.9, 152.6, 145.4, 144.2, 129.7, 121.1, 116.6, 116.3, 114.7, 112.6, 55.4, 16.0.

HRMS (ESI) m/z: calculated for C ₁₄ H ₁₆ NO ₂ ⁺ [M+H] ⁺ 230.1176, found 230.1180.

(D) 4-(4-Fluorophenoxy)-3-methylaniline (12)

In the same manner as for the synthesis of compound 9, Fe (894 mg, 16 mmol, 5.0 eq) and NH ₄ Cl (171 mg, 3.2 mmol, 1.0 eq) were dissolved in distilled water (8 mL). Methylnitrobenzene ( 8 , 791 mg, 3.2 mmol, 1.0 eq) was dissolved in EtOH (16 mL), added to the flask, and ^{stirred at 90 o} C for 1 hour to synthesize compound 12, and mass spectometry (mass spectometry, MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 678 mg, 3.12 mmol, 98%, brown oil.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 7.13-7.07 (m, 2H), 6.80-6.74 (m, 2H), 6.66 (d, J = 8.4 Hz, 1H), 6.48 (dd, J = 2.8, 0.8 Hz, 1H), 6.42 (dd, J = 8.4, 2.8 Hz, 1H), 4.92 (s, 2H), 1.96 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 156.8 (d, J _CF = 236.9 Hz), 155.2, 145.8, 143.4, 129.9, 121.5, 116.7 (d, J _CF = 8.8 Hz), 116.3, 116.0 ( d, J _CF = 22.7 Hz), 112.6, 15.9.

HRMS (ESI) m/z: calculated for C ₁₃ H ₁₃ FNO ⁺ [M+H] ⁺ 218.0976, found 218.0977.

(3) N,N Synthesis of ,3-trimethyl phenoxyaniline (PK01, PK02, PK03, PK04)

(end) N,N ,3-Trimethyl-4-(4-(trifluoromethoxy)phenoxy)aniline (PK01)

Methylaniline (10, 107 mg, 0.40 mmol, 1.0 eq) was added to a 5 mL one-arm flask, replaced with argon gas, and dissolved with glacial acetic acid (2.1 mL). Paraformaldehyde (120 mg, 4.0 mmol, 10.0 eq) and NaBH ₃ CN (118 mg, 1.88 mmol, 4.7 eq) were ^{added thereto at 0 o} C, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, a 2N NaOH aqueous solution ^{was added at 0 o} C to bring the pH to 14. The aqueous layer was extracted three times each with EtOAc (20 mL) and CH ₂ Cl ₂ (20 mL), and the extracted organic layers were collected _{to remove moisture with anhydrous Na 2} SO ₄ , filtered, and concentrated. Compound PK01 was synthesized by separating and purifying by column chromatography using silica gel, and mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 126 mg, 405 μmol, 83%, colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.09 (dd, J = 5.2, 0.8 Hz, 2H), 6.87 (d, J = 8.8 Hz, 1H), 6.84-6.80 (m, 2H), 6.62 (d , J = 2.8 Hz, 1H), 6.58 (dd, J = 8.8, 2.8 Hz, 1H), 2.94 (s, 6H), 2.14 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 157.8, 148.3, 144.5, 143.2, 131.0, 122.4, 121.8, 120.6 (q, J _CF = 255.8 Hz), 116.6, 115.5, 111.6, 41.1, 16.6.

HRMS (ESI) m/z: calculated for C ₁₆ H ₁₇ F ₃ NO ₂ ⁺ [M+H] ⁺ 312.1206, found 312.1212.

(I) N,N ,3-Trimethyl-4-(4-(trifluoromethyl)phenoxy)aniline (PK02)

In the same manner as for the synthesis of compound PK01, methylaniline (10, 107 mg, 0.40 mmol, 1.0 equivalent) was dissolved by adding glacial acetic acid (2.1 mL). To this, paraformaldehyde (120 mg, 4.0 mmol, 10.0 eq) and NaBH ₃ CN (118 mg, 1.88 mmol, 4.7 eq) were ^{added at 0 o} C, and the mixture was stirred at room temperature for 16 hours to synthesize compound PK02. , Mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 112 mg, 379 μmol, 95%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.50 (dd, J = 8.4, 0.8 Hz, 2H), 6.90-6.87 (m, 3H), 6.63-6.58 (m, 2H), 2.95 (s, 6H) , 2.12 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 162.0, 148.5, 143.8, 131.1, 127.0 (q, J _CF = 3.8 Hz), 124.4 (q, J _CF = 272.2 Hz), 123.4 (q, J _CF F = 32.8 Hz), 122.0, 115.6, 115.4, 111.6, 41.0, 16.6.

HRMS (ESI) m/z: calculated for C ₁₆ H ₁₇ F ₃ NO ⁺ [M+H] ⁺ 296.1257, found 296.1260.

(C) 4-(4-Methoxyphenoxy)-N,N,3-trimethylaniline (PK03)

In the same manner as for the synthesis of compound PK01, methylaniline ( 11 , 91.7 mg, 0.40 mmol, 1.0 equivalent) was dissolved by adding glacial acetic acid (2.1 mL). To this, paraformaldehyde (120 mg, 4.0 mmol, 10.0 eq) and NaBH ₃ CN (118 mg, 1.88 mmol, 4.7 eq) were ^{added at 0 o} C, and the mixture was stirred at room temperature for 16 hours to synthesize compound PK03. , Mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and Formation: 74.6 mg, 290 μmol, 73%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 6.85-6.75 (m, 5H), 6.62 (d, J = 3.2 Hz, 1H), 6.56 (dd, J = 8.8, 3.2 Hz, 1H), 3.77 (s , 3H), 2.91 (s, 6H), 2.18 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 154.4, 152.9, 147.6, 146.1, 130.6, 120.9, 117.4, 115.8, 114.7, 111.7, 55.7, 41.3, 16.8.

HRMS (ESI) m/z: calculated for C ₁₆ H ₂₀ NO ₂ ⁺ [M+H] ⁺ 258.1489, found 258.1491.

(C) 4-(4-Fluorophenoxy)- N,N ,3-trimethylaniline (PK04)

In the same manner as for the synthesis of compound PK01, methylaniline ( 12 , 87 mg, 0.40 mmol, 1.0 equivalent) was dissolved by adding glacial acetic acid (2.1 mL). To this, paraformaldehyde (120 mg, 4.0 mmol, 10.0 eq) and NaBH ₃ CN (118 mg, 1.88 mmol, 4.7 eq) were ^{added at 0 o} C, and the mixture was stirred at room temperature for 16 hours to synthesize compound PK04. , Mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 80.5 mg, 328 μmol, 82%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 6.96-6.90 (m, 2H), 6.84 (d, J = 8.8 Hz, 1H), 6.82 -6.77 (m, 2H), 6.62 (d, J = 3.2 Hz , 1H), 6.57 (dd, J = 8.8, 3.2 Hz, 1H), 2.93 (s, 6H), 2.16 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 157.7 (d, J _CF = 239.4 Hz), 155.2, 148.0, 145.3, 130.8, 121.4, 117.1 (d, J _CF = 2.5 Hz), 115.9 (d, J _CF = 22.3 Hz), 115.6, 111.6, 41.2, 16.6.

HRMS (ESI) m/z: calculated for C ₁₅ H ₁₇ FNO ⁺ [M+H] ⁺ 246.1289, found 246.1291.

(4) Synthesis of (Methylphenoxyphenyl)acetamide (PK05, PK06, PK07, PK08)

(end) N -(3-Methyl-4-(4-(trifluoromethoxy)phenoxy)phenyl)acetamide (PK05)

NaH (60% dispersion in mineral oil, 15.4 mg, 385 μmol, 1.1 equivalent) was added to a 5 mL one-arm flask, replaced with argon gas, and the synthesized methylaniline 9 (99 mg, 350 μmol, 1.0 equivalent) was distilled. It was dissolved in acetonitrile (1.75 mL), and this solution was added to the flask at ^{0 o C.} The mixture was stirred at room temperature for 1 hour. The mixture ^{was cooled to 0 o} C and acetyl chloride (27 μL, 385 μmol, 1.1 equivalent) was added. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction was diluted with EtOAc (5 mL) and washed with distilled water (5 mL). After separating the two layers, the aqueous layer was extracted twice with EtOAc (10 mL). The extracted organic layers were collected _{, moisture was removed with anhydrous MgSO 4} , filtered, and then concentrated. Compound PK05 was synthesized by separation and purification using silica gel column chromatography, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 93.8 mg, 288 μmol, 82%, pale yellow solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 9.93 (s, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.8, 2.4 Hz, 1H), 7.32 (d , J = 8.4 Hz, 2H), 6.95-6.90 (m, 3H), 2.10 (s, 3H), 2.03 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 168.1, 156.8, 148.3, 142.7, 136.3, 129.8, 122.9, 121.9, 120.8, 120.1 (q, J _CF = 255.8 Hz), 118.3, 117.3, 23.9, 15.9 .

HRMS (ESI) m/z: calculated for C ₁₆ H ₁₅ F ₃ NO ₃ ⁺ [M+H] ⁺ 326.0999, found 326.1002.

(I) N -(3-Methyl-4-(4-(trifluoromethyl)phenoxy)phenyl)acetamide (PK06)

In the same manner as for the synthesis of compound PK05, in NaH (60% dispersion in mineral oil, 17.6 mg, 440 μmol, 1.1 equivalent), methylaniline 10 (107 mg, 400 μmol, 1.0 equivalent) was distilled in acetonitrile (2.0 mL). The dissolved solution was added at 0 ^o C. The mixture was stirred at room temperature for 1 hour. The mixture ^{was cooled to 0 o} C, and acetyl chloride (31 μL, 440 μmol, 1.1 equivalent) was added to synthesize compound PK06, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 109 mg, 352 μmol, 88%, pale yellow solid.

Compound PK06 (Pale yellow solid, 109 mg, 352 μmol, 88%) in the same way

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 9.96 (s, 1H), 7.68 (dd, J = 9.2, 0.8 Hz, 2H), 7.58 (dd, J = 2.8, 2.0 Hz, 1H), 7.47 (dd, J = 8.8, 2.8 Hz, 1H), 7.04-6.95 (m, 3H), 2.08 (s, 3H), 2.04 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 168.2, 161.0, 147.4, 136.8, 130.1, 127.4 (q, J _CF = 5.0 Hz), 124.4 (q, J _CF = 277.2 Hz), 122.4 (q, J _CF = 32.1 Hz), 121.9, 121.4, 118.4, 116.1, 23.9, 15.9.

HRMS (ESI) m/z: calculated for C ₁₆ H ₁₅ F ₃ NO ₂ ⁺ [M+H] ⁺ 310.1049, found 326.1052.

(All) N -(4-(4-Methoxyphenoxy)-3-methylphenyl)acetamide (PK07)

In the same method as the synthesis of compound PK05, in NaH (60% dispersion in mineral oil, 18 mg, 451 μmol, 1.1 equiv) and methylaniline 11 (94.7 mg, 410 μmol, 1.0 equiv) distilled acetonitrile (2.0 mL). The dissolved solution was added at 0 ^o C. The mixture was stirred at room temperature for 1 hour. The mixture ^{was cooled to 0 o} C and acetyl chloride (32 μL, 451 μmol, 1.1 eq) was added to synthesize compound PK07, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 95.7 mg, 353 μmol, 86%, pale yellow solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): 9.85 (s, 1H), 7.49 (dd, J = 2.4, 0.8 Hz, 1H), 7.35 (ddd, J = 8.8, 2.4, 0.8 Hz, 1H), 6.94-6.86 (m, 2H), 6.86-6.69 (m, 2H), 6.75 (d, J = 8.8 Hz, 1H), 3.71 (s, 3H), 2.14 (s, 3H), 2.02 (s, 3H) .

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 168.0, 154.7, 151.1, 150.2, 135.1, 128.8, 121.9, 119.0, 118.2, 118.1, 114.9, 55.4, 23.9, 16.1.

HRMS (ESI) m/z: calculated for C ₁₆ H ₁₈ NO ₃ ⁺ [M+H] ⁺ 272.1281, found 272.1283.

(la) N -(4-(4-Fluorophenoxy)-3-methylphenyl)acetamide (PK08)

In the same method as the synthesis of compound PK05, in NaH (60% dispersion in mineral oil, 19.8 mg, 495 μmol, 1.1 equiv), methylaniline 12 (97.8 mg, 450 μmol, 1.0 equiv) was distilled in acetonitrile (2.3 mL). The dissolved solution was added at 0 ^o C. The mixture was stirred at room temperature for 1 hour. The mixture ^{was cooled to 0 o} C and acetyl chloride (35 μL, 495 μmol, 1.1 equivalent) was added to synthesize compound PK08, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 110 mg, 424 μmol, 94%, pale yellow solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 9.89 (s, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.40 (dd, J = 8.4, 2.4 Hz, 1H), 7.21-7.10 (m, 2H), 6.92-6.81 (m, 3H), 2.11 (s, 3H), 2.02 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 168.1, 157.4 (d, J _CF = 238.1 Hz), 154.0, 149.2, 135.8, 129.4, 121.9, 120.0, 118.2, 118.0 (d, J _CF = 8.8 Hz ), 116.3 (d, J _CF = 22.7), 23.9, 16.0.

HRMS (ESI) m/z: calculated for C ₁₅ H ₁₅ FNO ₂ ⁺ [M+H] ⁺ 260.1081, found 260.1084.

(5) Synthesis of N-Methyl-N-(methylphenoxyphenyl)acetamide (PK09, PK10, PK11, PK12)

(end) N -Methyl- N -(3-methyl-4-(4-(trifluoromethoxy)phenoxy)phenyl)acetamide (PK09)

The synthesized Acetamide (PK07, 92.5 mg, 341 μmol, 1.0 eq) was placed in a 1 mL one-arm flask, replaced with argon gas, and distilled tetrahydrofuran (0.51 mL) was added thereto to dissolve. NaH (60% dispersion in paraffin liquid, 40.8 mg, 1.02 mmol, 3.0 eq) was added at ^{0 °C.} Subsequently, methyliodide (0.21 mL, 3.41 mmol, 10.0 eq) was added, and the mixture ^{was stirred at 80 o} C for 10 hours. After the reaction was completed, the reaction was diluted with EtOAc (5 mL) and washed with saturated aqueoud NH ₄ Cl (3 mL). After separating the two layers, the aqueous layer was extracted twice with EtOAc (10 mL). The extracted organic layers were collected _{, moisture was removed with anhydrous MgSO 4} , filtered, and then concentrated. Compound PK09 was synthesized by separation and purification using silica gel column chromatography, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 91.1 mg, 268 μmol, 89%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.19 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 2.0 Hz, 1H), 7.03-6.86 (m, 4H), 3.26 (s, 3H) ), 2.26 (s, 3H), 1.90 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 170.6, 155.8, 153.7, 144.4, 140.6, 131.4, 130.1, 125.9, 122.8, 120.5 (q, J _CF = 257.0 Hz), 120.1, 118.6, 37.3, 22.4, 16.2 .

HRMS (ESI) m/z: calculated for C ₁₇ H ₁₇ F ₃ NO ₃ ⁺ [M+H] ⁺ 340.1155, found 340.1160.

(I) N -Methyl- N -(3-methyl-4-(4-(trifluoromethyl)phenoxy)phenyl)acetamide (PK10)

In the same manner as for the synthesis of compound PK09, the synthesized Acetamide (PK06, 112 mg, 362 μmol, 1.0 equivalent) was dissolved by adding distilled tetrahydrofuran (0.54 mL). NaH (60% dispersion in paraffin liquid, 43.6 mg, 1.09 mmol, 3.0 eq) was added at ^{0 °C.} Then, methyliodide (0.23 mL, 3.62 mmol, 10.0 eq) was added and the mixture ^{was stirred at 80 o} C for 10 hours to synthesize compound PK10, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 102 mg, 315 μmol, 87%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.58 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 1.6 Hz, 1H), 7.07 -6.94 (m, 4H), 3.27 (s, 3H) ), 2.23 (s, 3H), 1.91 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 170.5, 160.2, 152.6, 141.3, 132.0, 131.7, 130.2, 127.2 (q, J _CF = 3.8 Hz), 126.1, 124.2 (q, J _CF = 340.2 Hz), 121.2, 116.9, 37.3, 22.5, 16.2.

HRMS (ESI) m/z: calculated for C ₁₇ H ₁₇ F ₃ NO ₂ ⁺ [M+H] ⁺ 324.1206, found 324.1210.

(All) N -(4-(4-Methoxyphenoxy)-3-methylphenyl)- N -methylacetamide (PK11)

In the same manner as the synthesis of compound PK09, the synthesized Acetamide (PK07, 92.5 mg, 341 μmol, 1.0 equivalent) was dissolved by adding distilled tetrahydrofuran (0.51 mL). NaH (60% dispersion in paraffin liquid, 40.8 mg, 1.02 mmol, 3.0 eq) was added at ^{0 °C.} Then, methyliodide (0.21 mL, 3.41 mmol, 10.0 eq) was added and the mixture ^{was stirred at 80 o} C for 10 hours to synthesize compound PK11, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 94.4 mg, 331 μmol, 97%, white solid

¹ H NMR (400 MHz, CDCl ₃ ): 7.04 (d, J = 2.8 Hz, 1H), 6.95-6.87 (m, 5H), 6.74 (d, J = 8.4 Hz, 1H), 3.81 (s, 3H) , 3.23 (s, 3H), 2.31 (s, 3H), 1.88 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 170.8, 155.9, 155.6, 150.2, 139.2, 130.1, 129.7, 125.5, 120.1, 117.8, 115.0, 55.7, 37.3, 22.4, 16.3.

HRMS (ESI) m/z: calculated for C ₁₇ H ₂₀ NO ₃ ⁺ [M+H] ⁺ 286.1438, found 286.1442.

(la) N -(4-(4-Fluorophenoxy)-3-methylphenyl)- N -methylacetamide (PK12)

In the same manner as the synthesis of compound PK09, the synthesized Acetamide (PK08, 108 mg, 416 μmol, 1.0 equivalent) was dissolved by adding distilled tetrahydrofuran (0.62 mL). NaH (60% dispersion in paraffin liquid, 50 mg, 1.25 mmol, 3.0 eq) was added at ^{0 °C.} Then, methyliodide (0.26 mL, 4.16 mmol, 10.0 eq) was added and the mixture ^{was stirred at 80 o} C for 10 hours to synthesize compound PK12, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 91.3 mg, 334 μmol, 80%, white solid.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.09-6.99 (m, 3H), 6.97-6.8 (m, 3H), 6.80 (dd, J = 8.4, 0.4 Hz, 1H), 3.24 (s, 3H) , 2.28 (s, 3H), 1.89 (s, 3H).

¹³ C NMR (126 MHz, CDCl ₃ ): δ 170.7, 158.7 (d, J _CF = 241.9 Hz), 154.6, 152.9, 139.9, 130.7, 130.0, 125.7, 119.6 (d, J _CF = 8.3 Hz), 118.9, 116.4 (d, J _CF = 23.4 Hz), 37.3, 22.4, 16.3.

HRMS (ESI) m/z: calculated for C ₁₆ H ₁₇ FNO ₂ ⁺ [M+H] ⁺ 274.1238, found 274.1241.

(6) Synthesis of ((Methylphenoxyphenyl)amino)-4-oxobutanoic acid (PK13, PK14, PK15, PK16)

(A) 4-((3-Methyl-4-(4-(trifluoromethoxy)phenoxy)phenyl)amino)-4-oxobutanoic acid (PK13)

Succinic anhydride (44 mg, 440 μmol, 1.1 eq) was added to a 10 mL one-arm flask and replaced with argon gas. The synthesized methylaniline (11, 91.7 mg, 400 μmol, 1.0 equivalent) was dissolved in distilled toluene (4 mL), and this was added to the flask. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction was diluted with EtOAc (10 mL) and washed with distilled water (5 mL). After separating the two layers, the aqueous layer was extracted 3 times with EtOAc (10 mL). The extracted organic layers were collected _{, moisture was removed with anhydrous MgSO 4} , filtered, and then concentrated. The remaining residue was washed with hexane (2 mL) to synthesize PK13, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 152 mg, 397 μmol, 99%, white solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 12.15 (s, 1H), 9.97 (s, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.8, 2.4 Hz , 1H), 7.35-7.29 (m, 2H), 6.96-6.90 (m, 3H), 2.58-2.51 (m, 4H), 2.10 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 173.8, 170.0, 156.8, 148.2, 142.7, 136.3, 129.9, 122.9, 121.9, 120.9, 120.1 (q, J _CF = 256.3 Hz), 118.2, 117.3, 31.0 , 28.8, 15.9.

HRMS (ESI) m/z: calculated for C ₁₈ H ₁₇ F ₃ NO ₅ ⁺ [M+H] ⁺ 384.1053, found 384.1057.

(I) 4-((3-Methyl-4-(4-(trifluoromethyl)phenoxy)phenyl)amino)-4-oxobutanoic acid (PK14)

In the same manner as the synthesis of compound PK13, a solution in Succinic anhydride (44 mg, 440 μmol, 1.1 eq) and synthesized methylaniline (10 , 107 mg, 400 µmol, 1.0 eq.) was dissolved in toluene (4 mL). Added. The mixture was stirred at room temperature for 16 hours to synthesize compound PK14, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 137 mg, 373 μmol, 93%, white solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 12.16 (bs, 1H), 10.03 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 2.4 Hz, 1H ), 7.46 (dd, J _CF = 8.4, 2.4 Hz, 1H), 7.04 -6.96 (m, 3H), 2.60 -2.51 (m, 4H), 2.08 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 173.8, 170.1, 161.0, 147.4, 136.7, 130.1, 127.4 (q, J _CF = 5.0 Hz), 124.4 (q, J _CF = 272.2 Hz), 122.4 ( q, J _CF = 31.5 Hz), 121.9, 121.4, 118.3, 116.1, 31.0, 28.9, 15.9.

HRMS (ESI) m/z: calculated for C ₁₈ H ₁₇ F ₃ NO ₄ ⁺ [M+H] ⁺ 368.1104, found 368.1108.

(C) 4-((4-(4-Methoxyphenoxy)-3-methylphenyl)amino)-4-oxobutanoic acid (PK15)

In the same manner as for the synthesis of compound PK13, a solution in Succinic anhydride (44 mg, 440 μmol, 1.1 eq) and synthesized methylaniline ( 11, 91.7 mg, 400 μmol, 1.0 eq) was dissolved in distilled toluene (4 mL). Added. The mixture was stirred at room temperature for 16 hours to synthesize compound PK15, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 129 mg, 392 μmol, 98%, white solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 12.16 (brs, 1H), 9.90 (s, 1H), 7.52 (d, J = 2.4 Hz, 1H), 7.35 (dd, J = 8.8, 2.4 Hz , 1H), 6.93-6.86 (m, 2H), 6.85-6.69 (m, 2H), 6.75 (d, J = 8.4 Hz, 1H), 3.71 (s, 3H), 2.57-2.51 (m, 4H), 2.13 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 173.9, 169.8, 154.7, 151.2, 150.1, 135.1, 128.9, 121.9, 119.1, 118.2, 118.0, 114.9, 55.4, 31.0, 28.9, 16.1.

HRMS (ESI) m/z: calculated for C ₁₈ H ₁₉ NO ₅ Na ⁺ [M+Na] ⁺ 352.1155, found 352.1160.

(D) 4-((4-(4-Fluorophenoxy)-3-methylphenyl)amino)-4-oxobutanoic acid (PK16)

In the same manner as for the synthesis of compound PK13, a solution in Succinic anhydride (44 mg, 440 μmol, 1.1 eq) and synthesized methylaniline ( 11, 91.7 mg, 400 μmol, 1.0 eq) was dissolved in distilled toluene (4 mL). Added. The mixture was stirred at room temperature for 16 hours to synthesize compound PK16, mass spectometry (MS), ¹ H The resulting compound was confirmed using NMR and ^{13 C NMR.}

Yield and properties: 124 mg, 391 μmol, 98%, white solid.

¹ H NMR (400 MHz, DMSO- d ₆ ): δ 12.14 (s, 1H), 9.93 (s, 1H), 7.55 (d, J = 4.0 Hz, 1H), 7.40 (dd, J = 8.0, 4.0 Hz , 1H), 7.19-7.12 (m, 2H), 6.91-6.82 (m, 3H), 2.57-2.51 (m, 4H), 2.11 (s, 3H).

¹³ C NMR (126 MHz, DMSO- d ₆ ): δ 173.8, 169.9, 157.4 (d, J _CF = 239.4 Hz), 154.0, 149.1, 135.8, 129.4, 121.9, 120.1, 118.1, 118.0 (d, J _CF = 8.8 Hz), 116.3 (d, Jcf = 23.9 Hz), 31.0, 28.8, 16.0.

HRMS (ESI) m/z: calculated for C ₁₇ H ₁₇ FNO ₄ ⁺ [M+H] ⁺ 318.1136, found 318.1141.

Thereby, 8 intermediates and 16 new compounds, a total of 24 compounds, are shown in Table 3.

Structure and physical characteristics of 8 synthesized intermediates and 16 novel compounds Code No. Name Structure Physical Property 5 2-Methyl-4-nitro-1-(4-
(trifluoromethoxy)phenoxy)
benzene

colorless oil 6 2-Methyl-4-nitro-1-(4-
(trifluoromethyl)phenoxy)
benzene

white solid 7 1-(4-Methoxyphenoxy)-2-
methyl-4-nitrobenzene

pale yellow solid 8 1-(4-Fluorophenoxy)-2-methyl-4-nitrobenzene

white solid 9 3-Methyl-4-(4-(trifluoromethoxy) phenoxy)aniline

brown oil 10 3-Methyl-4-(4-(trifluoromethyl) phenoxy)aniline

brown oil 11 4-(4-Methoxyphenoxy)-3-
methylaniline

brown solid 12 4-(4-Fluorophenoxy)-3-
methylaniline

brown oil PK01 N,N ,3-Trimethyl-4-(4-
(trifluoromethoxy)phenoxy)
aniline

colorless oil PK02 N,N ,3-Trimethyl-4-(4-
(trifluoromethyl)phenoxy)
aniline

white solid PK03 4-(4-Methoxyphenoxy)- N,N ,3- trimethylaniline

white solid PK04 4-(4-Fluorophenoxy)- N,N ,3- trimethylaniline

white solid PK05 N -(3-Methyl-4-(4-
(trifluoromethoxy)phenoxy)
phenyl)acetamide

pale yellow
solid PK06 N -(3-Methyl-4-(4-
(trifluoromethyl)phenoxy)phenyl) acetamide

pale yellow
solid PK07 N -(4-(4-Methoxyphenoxy)-3-
methylphenyl)acetamide

pale yellow
solid PK08 N -(4-(4-Fluorophenoxy)-3-
methylphenyl)acetamide

pale yellow
solid PK09 N -Methyl- N -(3-methyl-4-(4-
(trifluoromethoxy)phenoxy)
phenyl)acetamide

white solid PK10 N -Methyl- N -(3-methyl-4-(4-
(trifluoromethyl)phenoxy)
phenyl)acetamide

white solid PK11 N -(4-(4-Methoxyphenoxy)-3-
methylphenyl)- N-
methylacetamide

white solid PK12 N -(4-(4-Fluorophenoxy)-3-
methylphenyl)- N-
methylacetamide

white solid PK13 4-((3-Methyl-4-(4-
(trifluoromethoxy)phenoxy)
phenyl)amino)-4-oxobutanoic acid

white solid PK14 4-((3-Methyl-4-(4-
(trifluoromethyl)phenoxy)
phenyl)amino)-4-oxobutanoic acid

white solid PK15 4-((4-(4-Methoxyphenoxy)-3-
methylphenyl)amino)-4-
oxobutanoic acid

white solid PK16 4-((4-(4-Fluorophenoxy)-3-
methylphenyl)amino)-4-
oxobutanoic acid

white solid

Example 7: Toltrazuril Derivative Compounds Against Kudoae in vitro Inactivation efficacy investigation

The inactivating efficacy of the toltrazuril derivative synthesized in Example 6 against Kudoae was confirmed.

In flounder farms, the process of selecting positives for separating Kudo larvae should be performed quickly and asepticly to prevent cross-contamination. First of all, in order to easily screen positive for Kudoworm, a commercially available rapid diagnostic kit reagent (POCT Rapido kit, Solpotu) was used. That is, the back muscle (5 cm width 5 cm length 5 cm depth 1 cm) of the flounder (around 500 g) collected at the farm was cut and peeled, and then the sample was treated according to the method described in the instruction manual of the product. Kudolar spores were confirmed by microscopic examination from the flounder confirmed as the final positive body, and Kudolarvae were quickly recovered from the positive body infected at a high concentration according to the method of Ohnishi et al. (Ohnishi T. et al., Parasitology Research . 115:2519, 2016). Separation and purification were performed to investigate the inactivation efficacy.

The isolated Kudoae spores were ^{purified to a final concentration of 1x10 6} /mL, and the diluted solution for each concentration of the compound was used for inactivation efficacy investigation. In other words, 90 μL of Kudoaeum spore solution diluted in HBSS (Hank's Balanced Salt solution) and 10 μL of synthetic compound were mixed, and the control was mixed with spore solution (90 μL) and HBSS (10 μL) for 24 hours in an e-tube. After the reaction, each deactivation efficacy was confirmed. This was all repeated twice.

The staining method for determining the inactivation of Kudoae is by Yokoyama et al . (Yokoyama, H. et al., Foodbornw Pathogens and Disease , 13:21) by HO&PI (Hoechst 33342 and propidium iodide), a fluorescent dye used as a method for determining the survival of cells. , 2016) and Do et al . (Do, JW et al., J Fish Mar Sci Edu , 28:1822, 2016). That is, when observed with an optical microscope (a in Fig. 11), the isolated and purified Kudoaworm shows 9 (1 to 9) spores (5 to 7 polar sacs), but it is not known whether or not they survive. However, when HO&PI staining was performed and then observed with a fluorescence microscope, all 9 spores were shown in blue (B in FIG. 11) and 4 of the 9 spores (3, 6, 7, 8) were all red, 1 In dogs (5), 50% of the polar sac appeared red, and the remaining four (1, 2, 4, 9) were not observed red at all (FIG. 11C). Therefore, spores that only show blue and do not appear red are alive, and spores that respond only to red are judged dead.

In order to investigate the inactivation of Kudoae spores in the control group and the concentration-specific compound treatment group, 100 spores stained with a fluorescence microscope were counted 1 hour after HO&PI staining. In this process, only the cases in which all of the spores (5~7) were blue were reflected in the survival rate (%).

The spore survival rate (%) of the control group was set to 100, and the spore survival rate of the synthetic compound-treated group was converted to a percentage to determine the inactivation effect. In other words, the survival rate range of spores is divided into 5 stages: 0~20%, 20~40%, 40~60%, 60~80%, and 80% or more, and the range of 0~20% is that the inactivation effect of spores is (++ ++), in the range of 20 to 40%, the deactivation effect is (+++), in the range of 40 to 60%, the deactivation effect is (++), and in the range of 60 to 80%, the deactivation effect is (+), 80 In% or more, the inactivation effect was classified as (-). In the order of (++++)> (+++)> (++)> (+), the inactivation effect is high, and the test section showing (-) is considered to have no inactivation effect. However, when the cells of the spores are all destroyed and the inactivation cannot be confirmed with a microscope, "N" is indicated.

Table 4 shows the in vitro deactivation effect of 16 compounds of toltrazuril derivatives against Kudoae isolated and purified from flounder in high concentration tests (1000, 5000, 10000 ppm).

And, based on 1000 ppm in Table 4, the deactivation efficacy was classified and summarized in Table 5. Compounds with an inactivation effect of (++++) showed the best results in the order of PK12 (0%), PK08 (2.6%), PK05 (7.9%), PK07 (15.8%), and PK06 (18.4%). Compounds with an inactivation effect of (+++) were PK11 (21.1%) and PK09 (34.2%), and the compound with (++) was PK14 (49.5%). Compounds with a weak (+) inactivation effect were PK13 (65.8%) and PK16 (78.9%), and the rest were (-), indicating that they had no inactivation effect.

Derivative name Concentration (ppm) Kudolar spore survival rate (%) PK01 1000 100 5000 13.2 10000 N PK02 1000 86.8 5000 0 10000 0 PK03 1000 92.1 5000 7.9 10000 N PK04 1000 100 5000 0 10000 N PK05 1000 7.9 5000 0 10000 0 PK06 1000 18.4 5000 0 10000 0 PK07 1000 15.8 5000 0 10000 0 PK08 1000 2.6 5000 0 10000 0 PK09 1000 34.2 5000 0 10000 0 PK10 1000 100 5000 0 10000 0 PK11 1000 21.1 5000 0 10000 0 PK12 1000 0 5000 0 10000 0 PK13 1000 65.8 5000 0 10000 0 PK14 1000 49.5 5000 29.8 10000 0 PK15 1000 87.3 5000 46.4 10000 0 PK16 1000 78.9 5000 0 10000 0 * N: All spore cells are destroyed, making it impossible to confirm survival with a microscope.

In vitro deactivation effect of Cudoaeum when treated with 1000 ppm of toltrazuril derivative compounds (16 types) compound The range and inactivation effect of the survival rate (%) of Kudolarvae spores 0-20%
(++++) 20-40%
(+++) 40-60%
(++) 60～80%
(+) 80% or more
(-) PK01 - PK02 - PK03 - PK04 - PK05 ++++ PK06 ++++ PK07 ++++ PK08 ++++ PK09 +++ PK10 - PK11 +++ PK12 ++++ PK13 + PK14 ++ PK15 - PK16 +

In the 1000 ppm standard, 0~20% (++++) and 20~40% (+++) were classified as groups with high inactivation effect. That is, with respect to seven species of PK05, PK06, PK07, PK08, PK09, PK11, and PK12, this time, they were diluted to a low concentration (10, 100, 500 ppm), and the spore survival rate was calculated by the same method (Table 6).

As a result, the concentration expected to be administered to farmed flounder was set from the lowest 10 ppm to the highest 100 ppm and examined. At 10 ppm, the spore survival rate was the lowest in PK07 at 12%, followed by PK05 at 20%, the rest (PK06, PK08, PK09, PK11) at 33.3 to 45%, and PK12 at 89.3%. The inactivation effect of subworms was highest in the order of PK07> PK05, and the rest were similar in inactivation efficacy, and PK12 was the lowest.

At 100 ppm, the spore survival rate was the lowest in PK06 and PK05 at 4-5%, followed by PK07 at 13.7%, the rest (PK08, PK09, PK11) at 31.2 to 46.9%, and PK12 at 78.9%. The inactivation effect of kudoworms was highest in the order of PK06, PK05> PK07, the rest had similar inactivation efficacy, and PK12 was the lowest.

Inactivation effect in vitro when treated with toltrazuril derivative compounds (7 types) derivative Concentration (ppm) Kudoa spore survival rate (%) PK05 10 20.0 100 5.0 500 0.9 PK06 10 37.2 100 4.0 500 0 PK07 10 12.0 100 13.7 500 2.7 PK08 10 40.0 100 46.9 500 0.9 PK09 10 45.0 100 41.1 500 0.9 PK11 10 33.3 100 31.2 500 19.0 PK12 10 89.3 100 78.9 500 0

Example 8: In vitro cytotoxic reaction of toltrazuril derivative compounds

Confirmation of the in vitro cytotoxic reaction of this example was carried out in the same manner as in Example 2.

The safety (toxicity) of the synthetic compound in the body of flounder was evaluated through cytotoxic reaction. The effect of the selected seven compounds (PK05, PK06, PK07, PK08, PK09, PK11, PK12) on the proliferation of HINAE cells is shown in FIG. 12.

As a result, when the concentration expected to be administered to farmed flounder is considered to be from the lowest 10 ppm to the highest 100 ppm, the cell viability is more than 90% for PK06 and PK11, more than 80% for PK07, and more than 70% for PK08, PK09, and PK12. And PK05 was the lowest at 22.1%.

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

A novel toltrazuryl derivative compound, characterized in that it has the structure of any one of the following Formulas 2 to 15;
[Formula 2]

,
[Formula 3]

,
[Formula 4]

,
[Formula 5]

,
[Formula 6]

,
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

,
[Formula 11]

,
[Formula 12]

,
[Formula 13]

,
[Formula 14]

And
[Formula 15]

.
delete A pharmaceutical composition for preventing or treating parasitic infections containing any one of the toltrazuril derivative derivatives having the structure of the following formula (1) as an active ingredient:
[Formula 1]

R1 is

, CF ₃ , F and

Is selected from the group consisting of,
R2 is NO ₂ , NH ₂ ,

,

,

And

Is selected from the group consisting of,
R3 is characterized in that H or a C1 ~ C3 alkyl group.
The pharmaceutical composition of claim 3, wherein the toltrazuril derivative has a structure of any one of the following formulas 2 to 15:
[Formula 2]

,
[Formula 3]

,
[Formula 4]

,
[Formula 5]

,
[Formula 6]

,
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

,
[Formula 11]

,
[Formula 12]

,
[Formula 13]

,
[Formula 14]

And
[Formula 15]

.
[4] The pharmaceutical composition of claim 3, wherein the parasite is selected from the group consisting of mucous spores, ciliated worms, monosporangial fluke, microspore worms, parasitic protozoa, flatworms, roundworms and hookworms.
The method of claim 4, wherein N -(3-Methyl-4-(4-(trifluoromethoxy)phenoxy) phenyl)acetamide, N -(3-Methyl-4-(4-(trifluoromethyl)phenoxy)phenyl)acetamide, N- (4-(4-Methoxyphenoxy)-3-methylphenyl) acetamide, N -(4-(4-Fluorophenoxy)-3-methylphenyl)acetamide, N -Methyl- N -(3-methyl-4-(4-(trifluoromethoxy) )phenoxy) phenyl)acetamide, N -(4-(4-Methoxyphenoxy)-3-methylphenyl)- N -methylacetamide and N -(4-(4-Fluorophenoxy)-3-methylphenyl)- N -methylacetamide A pharmaceutical composition comprising a selected derivative.

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