KR100982661B1 - Pharmaceutical composition for the prevention and treatment of malaria containing a compound that inhibits plasmincin II activity as an active ingredient and a method for treating malaria using the same - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing and treating malaria, and comprising administering to the mammal an effective amount of malaria, characterized in that it comprises a compound that binds to the active site of Plascexin II and inhibits activity as an active ingredient. The present invention relates to a prophylactic and therapeutic method, and more particularly, to a composition of the present invention, an N-alkoxy amidine derivative, a guanidine derivative, an amide derivative, a urea or a thiourea derivative, and an N- (2-{[3- (1,3- At least one compound selected from the group consisting of benzodioxol-5-yl) -3-oxo-1-propen-1-yl] amino} phenyl) -4-nitrobenzene sulfonamide.

The compound included in the pharmaceutical composition of the present invention is used for the prevention and treatment of malaria by binding to the active site of Plascexin II and inhibiting its activity, and has an effective effect against malaria that is resistant to existing known malaria therapeutics. Can be.

Malaria, Plaschexin, Plasmodium Palmiparum, Protease, Red Blood Cells

Description

Pharmaceutical composition for preventing and treating malaria comprising compounds that inhibit Plasmepsin II activity and the method of treating malaria using about}

The present invention relates to a pharmaceutical composition effective for the treatment of malaria comprising a compound that binds to and inhibits the activity of Plascinsin II. More specifically, the compositions of the present invention include N-alkoxyamidine derivatives, guanidine derivatives, amide derivatives, urea or thiourea derivatives, and N- (2-{[3- (1,3-benzodioxol-5-yl) At least one compound selected from the group consisting of -3-oxo-1-propen-1-yl] amino} phenyl) -4-nitrobenzene sulfonamide. The compound included as an active ingredient in the pharmaceutical composition of the present invention is bound to the active site of Plascecin II, the tropical fever malaria ( Plasmodium falciparum ), which is used as an inhibitor of protease plasmincin II. Furthermore, the present invention relates to a method for preventing and treating malaria comprising administering the pharmaceutical composition to a mammal in an effective amount.

Malaria is a very serious and complex disease that threatens human health in the 21st century. Approximately 3 million people are infected with malaria worldwide, and 1.5 million die every year. Malaria is an infectious disease caused by four types of malaria protoplasm, Plasmodium , which is a tropical malaria, Plasmodium. falciparum ) is the most serious. To date, attempts to develop a vaccine against tropical malaria have failed, and the treatment and prevention of malaria is limited to drugs. However, resistance to many anti-malarial drugs that are widely used is rapidly spreading and new drugs are needed.

Plasmodium falciparum enters the human body through wounds of bites of female gelatinous mosquitoes. Malaria protozoa initially stay in the liver and then reproduce in red blood cells during the infection cycle phase. During this stage, the malaria protozoa breaks down hemoglobin and uses its breakdown products as nutrients for growth. Plasmodium falciparum is known to degrade their hemoglobin in host cells using their proteolytic enzymes. Parasites have limited ability to biosynthesize amino acids or take amino acids from immediate environment. Thus, these parasites use hemoglobin in host erythrocytes as a major nutrient source. Parasites consume 25-75% of host cell hemoglobin for a short period of internal red blood cell sleep cycles. This mass breakdown metabolism is known to occur in unique acidic organelles below pH 5 called digestive vacuoles. Three or more vacuoles (two are aspartic proteases, one is cysteine proteases) are known to be involved in the degradation of human hemoglobin to its constituent amino acids. Plasmepsin I, one of the aspartic proteases, has been known to initiate hemoglobin degradation by first cleaving hemoglobin and releasing molecules to allow further proteolysis to proceed efficiently. Plasxinsin II, the second aspartic protease, is known to cleave hemoglobin with overlapping specificity. The cysteine protease Falcipain is also involved in the early stages of hemoglobin degradation. All three of these protease Plasxin I, II and falsipine cleave denatured hemoglobin in vitro.

Malaria is a serious problem, especially in developing countries such as Africa. Malaria strains cause red blood cells to deform and accumulate on the walls of blood vessels, blocking the flow of blood and causing complications in the brain, kidneys, and liver. Therefore, insecticides and antimalarial drugs for controlling mosquitoes as infectious agents have been developed. However, the incidence of malaria is increasing due to increased resistance of mosquitoes to existing insecticides and the emergence of resistant strains to antimalarial drugs. In addition, the development of new mosquito insecticides and antimalarial drugs is urgently required as malaria is more likely to occur outside of malaria due to global warming.

The development of new therapeutics to solve such problems is not easy because of the long time required (10-12 years) and high cost. As a method to compensate for these drawbacks, a process of designing the structures of these plasticine enzymes and new drug compounds by using a supercomputer is being conducted. That is, in silico virtual screening (virtual screening) uses a computer and docking software to determine the degree of binding of ligands (about 40 million, candidate candidates) to the active site of the target protein. By providing concentrated drug compounds for listing and making in vitro tests more efficient, it contributes significantly to the time and cost savings of new drug development. In particular, docking using the international grid (supercomputing power analytics on the planet, a new computing infrastructure that provides access to data) is a new way to complete decades-hundreds of years using standard computers. This can be called.

An example of the process of developing a library of concentrated chemicals using the international grid and developing new drug candidates for in silico and in vitro experiments is as follows: (1) a database of 40 million chemicals to determine the degree of binding to the target (disease) protein; Determining binding sites associated with preparation and activity of tertiary structural models of the target protein, (2) virtually binding each chemical to the binding site of the target protein, calculating binding energies, and then chemistry showing good binding Secondary analysis of the materials (Top 15%) in consideration of molecular dynamics, and (3) the in vitro experiment of the top 5% or less of the most binding materials. In this case, the international grid (WISDOM) may preferably use a grid of EGEE.

Accordingly, an aspect of the present invention is to provide a pharmaceutical composition for preventing and treating malaria, comprising as an active ingredient at least one compound that binds to the active site of Plascexin II and inhibits activity.

Another aspect of the invention is to provide a method for preventing and treating malaria comprising administering the pharmaceutical composition to a mammal in an effective amount.

According to one aspect of the invention alkoxyamidine derivatives, guanidine derivatives, amide derivatives, urea or thiourea derivatives, and N- (2-{[3- (1,3-benzodioxol-5-yl) -3- At least one compound selected from the group consisting of oxo-1-propen-1-yl] amino} phenyl) -4-nitrobenzene sulfonamide, characterized in that it binds to the active site of flamycin II and inhibits its activity Provided is a pharmaceutical composition for preventing and treating malaria comprising as an active ingredient.

According to another aspect of the invention there is provided a method for preventing and treating malaria comprising administering the pharmaceutical composition to a mammal in an effective amount.

The present invention finds a compound that binds to the active site of Plascexin II and inhibits its activity. Thus, a pharmaceutical composition comprising the same is used for the prevention and treatment of malaria. It can have a valid effect.

, A., Schwichtenberg, H., Wolf, A., Jacq, N., Breton, V. and Hofmann-Apitius, M. Design of new plasmepsin inhibitors: a virtual high throughput screening approach on the EGEE grid. Journal of Chemical Information and Modeling (2007) 47:1818-1828]. 이들 중 플라스멥신 II의 활성 부위에 결합하는 능력이 우수한 30 개의 화합물들은 시험관(in vitro) 테스트를 통해 그 효과를 확인하였다. For the discovery of compounds that bind to and inhibit the activity of Plascecin II, insilico ( in silico ) 1,000 compounds were screened first out of 500,000 compounds by targeting Plascinsin II through virtual screening (Virtual Screening). Two other docking programs, FlexX and AutoDock, were used to select different targets of Plasticine II. Secondly 500 compounds were selected. At this time, the interaction between the key amino acids (key residue) of the protein was examined and selected. 100 compounds were selected in the third order. This was selected by talking score, ideal binding mode, virtual screening through the main amino acids of the protein [Kasam V., Zimmermann, M., Maa

, A., Schwichtenberg, H., Wolf, A., Jacq, N., Breton, V. and Hofmann-Apitius, M. Design of new plasmepsin inhibitors: a virtual high throughput screening approach on the EGEE grid. Journal of Chemical Information and Modeling (2007) 47: 1818-1828. Of these, 30 compounds with excellent ability to bind to the active site of Plascinsin II were tested in vitro. In vitro ) tests confirmed the effect.

The thirty compounds can be obtained from Chembridge Corporation, USA, which have excellent activity to inhibit Plascecin II, as can be seen in Table 1. Accordingly, the compounds may be used alone or in combination with a pharmaceutically acceptable carrier to treat malaria. Compounds of the present invention are as follows.

Compound 1: 2-anilino-4- (3-furyl) -6-oxo-N-phenyl-1-cyclohexene-1-carboxamide

Compound 2: 3-chloro-N-{[2- (2-methoxybenzoyl) hydrazino] carbonothioyl} -1-benzothiophene-2-carboxamide

Compound 3: 2-{[N- (2,3-dihydro-1,4-benzodioxin-6-yl) -N- (methylsulfonyl) gylsil] amino} -N-isobutylbenzamide

Compound 4: N-benzyl-2-{[3- (3-nitrophenyl) acryloyl] amino} benzamide

Compound 5: 6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one N-phenylthiosemicarbazone

Compound 6: N- (2-methoxyphenyl) -2- (5H- [1,2,4] triazino [5,6-b] indol-3-ylthio) butanamide

Compound 7: N-{(2,3-dihydro-1,4-benzodioxin-6-ylamino) [(6-methyl-4-oxo-1,4-dihydro-2-pyrimidinyl) Amino] methylene} benzamide

Compound 8: N-{(2,3-dihydro-1,4-benzodioxin-6-ylamino) [(6-oxo-4-propyl-1,6-dihydro-2-pyrimidinyl) Amino] methylene} -4-methoxybenzamide

Compound 9: 4-methoxy-N- (2- [5- (2-nitrophenyl) -2-furyl] -1-{[(4-pyridinylmethyl) amino] carbonyl} vinyl) benzamide

Compound 10: N- {amino [(4,6-dimethyl-2-quinazolinyl) amino] methylene} -2- (4-chlorophenyl) acetamide

Compound 11: 4-amino-N '-(benzyloxy) -N- (4-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide

Compound 12: N, N '-{[(4,6-dimethyl-2-quinazolinyl) amino] methylidene} dipropanamide

Compound 13: N '-[(4-methoxy-3-nitrobenzoyl) oxy] -2- (1-naphthyl) ethaneimideamide

Compound 14: N '-{[(4-chloro-3,5-dimethylphenoxy) acetyl] oxy} -2- (3,4-dimethoxyphenyl) ethaneimideamide

Compound 15: 2- (1-naphthyl) -N '-[(4-nitrobenzoyl) oxy] ethaneimideamide

Compound 16: 4-amino-N '-[(2,4-dichlorobenzyl) oxy] -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide

Compound 17: N- (3,4-dichlorophenyl) -2-[(4,6-dimethyl-2-pyrimidinyl) amino] -3a, 4,5,6,7,7a-hexahydro-1H- Benzimidazole-1-carboxamide

Compound 18: 4-amino-N '-[(2-methyl-1-naphthyl) methoxy] -1,2,5-oxadiazole-3-carboxamideamide

Compound 19: N-{[(2,4-dimethylphenyl) amino] [(4-oxo-1,4,5,6,7,8-hexahydro-2-quinazolinyl) amino] methylene} benzamide

Compound 20: 4-amino-N '-(benzyloxy) -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide

Compound 21: N-[(cyclopentylamino) carbonothioyl] -4-ethoxy-3-nitrobenzamide

Compound 22: 6-({2-[(5-chloro-2-methoxyphenyl) amino] -2-oxoethyl} thio) -5-cyano-N- (2-methoxyphenyl) -2-methyl 4-phenyl-1,4-dihydro-3-pyridinecarboxamide

Compound 23: N- (3,4-dimethylphenyl) -N '-{imino [(4,6,7-trimethyl-2-quinazolinyl) amino] methyl} thiourea

Compound 24: N-({[6-methyl-2- (4-methylphenyl) -2H-1,2,3-benzotriazol-5-yl] amino} carbonothioyl) -2-nitrobenzamide

Compound 25: 2- (1,3-benzothiazol-2-ylamino) -N- (2-chlorophenyl) -6-oxo-1,4,5,6-tetrahydro-4-pyrimidinecarbox mid

Compound 26: 2-anilino-3-chloro-N-phenyl-4- (phenylimino) -2-butenamide

Compound 27: N ', N' ''-1,2-phenylenebis [N- (3-chlorophenyl) urea]

Compound 28: N- (2-{[3- (1,3-benzodioxol-5-yl) -3-oxo-1-propen-1-yl] amino} phenyl) -4-nitrobenzenesulfonamide

Compound 29: N-({[2- (4ethylphenyl) -6-methyl-2H-1,2,3-benzotriazol-5-yl] amino} carbonothioyl) -2-nitrobenzamide

Compound 30: N- (1,3-benzodioxol-5-ylmethyl) -N '-[4- (2-oxo-2H-chromen-3-yl) -1,3-thiazole-2- General] Succinamide

Among the compounds, compounds 11, 13, 14, 15, 16, 18 and 20 are N-alkylamino derivatives, compounds 7, 8, 10, 12, 17, 119, 23 and 25 are guanidine derivatives, compounds 1, 3, 4 , 6, 9, 22, 26 and 30 are amide derivatives, and 2, 5, 21, 24, 27 and 29 are compounds belonging to urea and thiourea derivatives.

According to the present invention there is provided a pharmaceutical composition for the prevention and treatment of a disease containing the compound as an active ingredient and associated with the role of Plascinsin II and in need of selective inhibition of Plascinsin II, in particular the disease is associated with malaria. Include. The pharmaceutical composition comprises at least one compound of the compounds described herein as an active ingredient, that is, the pharmaceutical composition for treating malaria according to the present invention may include the compound of the present invention in any combination.

The pharmaceutical composition may be administered by oral, rectal, nasal, lung, topical, transdermal, intracisternal, intraperitoneal, vaginal and parenteral (subcutaneous, intramuscular, intrathecal, intravenous and intradermal) routes. It may be specifically formulated to be administered by any suitable route such as, preferably by oral route. Preferred routes will depend on the general conditions and age of the subject being treated, the nature of the condition being treated and the active ingredient selected.

The pharmaceutical compositions prepared according to the invention may be in any suitable route, for example in tablets, capsules, powders, granules, pellets, oral tablets, dragees, pills or tablets, aqueous or non-aqueous liquids. Parenteral administration in the form of oral or injectable solutions in the form of aqueous solutions or suspensions, or oil-in-water or water-in-oil liquid emulsions, elixirs, syrups and the like. Other pharmaceutical compositions for parenteral administration include sterile powders composed of sterile injectable solutions or dispersions prior to use as well as dispersions, suspensions or emulsions. Depot injection formulations are also considered to be within the scope of the present invention. Other suitable dosage forms include suppositories, sprays, ointments, creams, gels, inhalants, skin patches, and the like. Methods known in the art may be used to prepare such compositions, and any pharmaceutically acceptable carrier diluent, excipient or other additive commonly used in the art may be used.

Such carriers are conventionally used in the preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroli Don, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, and the like. The composition may further contain a preservative, a stability improving substance, a viscosity improving or controlling substance, a solubility improving substance, a sweetener, a dye, an aesthetic improvement compound, a salt which changes the osmotic pressure, a buffer, an anti-oxidant, and the like.

In addition, the pharmaceutical compositions of the present invention can be used in one or more other therapeutically useful substances, such as quinoline (quinine, chloroquinine, amodiaquine, mefloquinine, primaquine, tafenoquine, etc.), peroxide anti-malaria Drugs (artemisinin derivatives), pyrimethamine-sulpadoxin anti-malaria drugs (e.g., pancidar, etc.), hydroxynaphthoquinones (e.g., atorbacuon, etc.), acroline-type anti-malarial drugs In combination with other antimalarial drugs such as, for example, pyrinaridine.

The compounds may be used in any form of free compounds, pharmaceutically acceptable salts, solvates including hydrates, esters, stereoisomers, as long as they exhibit the desired inhibitory effect of Plascinsin II, all of which are present inventions. It is within the scope of.

Pharmaceutically acceptable salts in the present invention may include pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy al It may be one obtained from non-toxic organic acids such as canoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. Specific examples include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate , Propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate, sebacate, fumarate , Maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate, Terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfo Yate, Phenyl Acetate, Phenylpropionate, Phenylbutyrate, Citrate, Lactate, β-hydroxybutyrate, Glycolate, Maleate, Tartrate, Methanesulfonate, Propanesulfonate, Naphthalene-1-sulfonate, Naphthalene -2-sulfonate or mandelate.

The present invention also provides a method for treating malaria in a mammal, characterized in that the pharmaceutical composition is administered to the mammal in an effective amount.

Typically the pharmaceutical compositions of the present invention are administered in unit dosage form containing the active ingredient in an amount of about 1 μg to 50 mg. The total daily dose is usually from about 1 mg to 50 mg, preferably 1 mg to 30 mg of the active compound of the present invention, provided that the above range is considered in consideration of the patient's situation and also considering the activity of the drug to be administered. Certain amounts may not be administered. Determining the optimal dose for a particular situation should be determined experimentally.

The compound of the present invention may be administered in one or several doses, and preferably, the daily dose is divided once or twice daily. It may be administered alone or in combination with a pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions according to the invention may be formulated according to the prior art with any other known auxiliaries and excipients as well as pharmaceutically acceptable carriers or diluents. The formulations may conveniently be presented in unit dosage form by methods known in the art of pharmacy.

In order to verify the malaria therapeutic effect of the pharmaceutical composition of the present invention, the fluorescence resonance energy transfer (FRET) of hemoglobin resolution of Plascinsin II and the hemoglobin resolution of Plascinsin II, etc. of the compounds included in the pharmaceutical composition were carried out. Measured.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1. Expression and Preparation of Recombinant Proteins

1. Plascinsin II Gene

Genes encoding Plascinsin II (PMII) were distributed by the MR4 / American Type Culture Collection (USA) [Luker, KE, Francis, SE, Gluzman, IY and DE Goldberg. Kinetic analysis of plasmepsin I and II aspartic protease of the Plasmodium falciparum digestive vacuole. Mol. Biochem. Parasitol. 79, 71-78 (1996); lstvan, ES and Goldberg DE Distal substrate interactions enhance plasmepsin activity. J. Biol. Chem. 280, 6890-6896 (2005).

2. Preparation of Recombinant Protein

Escherichia coli with the PMII-pET3d plasmid [E. coli BL21 (DE3) pLysS] is incubated with stirring at 37 ° C. until A ₆₀₀ reaches 0.5 in LB liquid medium containing ampicillin. 400 μM isopropyl-β-D-thiogalactopyranoside was added thereto, followed by shaking culture at 16 ° C. for 18 hours. After incubation, the cells were recovered by centrifugation (8,000xg, 10 minutes at 4 ° C), and 50 ml of lysis buffer solution (50 ml, composition: 50) was added to β-mercaptoethanol (BME). resuspended in mM Tris-HCl, 25 mM NaCl, pH 8.0). Suspended cells are disrupted using an ultrasonic crusher (Ultrasonic processor 250, Sonics and Materials, Inc., CT, USA; output 4, duty cycle 50%, 30 seconds in ice, 25 repetitions). After crushing and centrifugation, pellets containing inclusion bodies were washed again in 0.1 M Tris pH 10 buffer solution with 50 μl of BME. The pellet is resuspended in 8M urea solution (dissolved in 100 mM Tris-HCl pH 10, 1 mM EDTA, 1 mM glycine solution), and the cells are disrupted with an ultrasonic crusher. After crushing, 35 μl of BME is added. The suspension is stored at 4 ° C. for 18 hours and then centrifuged at 4 ° C. at 23,000 × g for 30 minutes. After centrifugation, the supernatant is obtained, the supernatant and water are diluted at a ratio of 1:10, and refolded of the recombinant protein while stirring for 18 hours. The refolded solution was purified using 50 ml Fast Flow Q-Sepharose Fast Flow (GE Healthcare, USA) equilibrated in 0.1 M Tris-HCl pH 8.5 buffer solution. After resin washing with 100 mM Tris-HCl (pH10) buffer solution, the recombinant protein was eluted using a 0 to 1M NaCl concentration gradient made with 100 mM Tris-HCl (pH 10) buffer solution. Fractions containing the recombinant protein were concentrated, dialyzed with 10 mM Tris-HCl (pH 8.5) buffer solution with 5 mM NaCl and 20 mM BME, and the obtained purified protein was then subtracted to 20 ° C until used for analysis. Store at ℃.

In the parasitic state, Plascinsin II is translated as an inactive zymogram with an N-terminal prosequence of 124 amino acids that acts as a membrane-permeable domain. In the food vacuole, the pro-sequence is removed by calpain-like maturase and released into active plasmin II [Benerjee, R., Francis, SE and Goldberg. DE Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum . Mol. Biochem. Parasitol. 129: 157-165 (2003).

Plascinsin II was cloned into the pET3d vector as a gene containing the initiation codon Met followed by glutamic acid 124 and purified by single band to confirm by SDS-electrophoresis (FIG. 1). SDS-electrophoresis was performed using 12% acrylamide gel. Proteins (2 μg) were loaded onto SDS-electrophoresis gel, followed by electrophoresis and confirmed by Coomassie Brilliant Blue staining. Lane 1 is a cell supernatant obtained from cells expressed after 8M urea treatment, and lane 2 is Plascinsin II purified using Q-Sepharose resin. The size of the protein was determined by Bio-Red (USA) standard protein [Lane M, size marker (myosin, 200 kDa; β-galactosidase, 116 kDa; phosphinase b, 97 kDa; bovine serum). Albumin, 66 kDa; egg white albumin, 45 kDa; carbonic unhydrated, 31 kDa; kontrypsin inhibitor, 21 kDa, apoprotein 7 kDa)], and the size of the recombinant plasmincin II was about 37 kDa. It was.

Example 2. Fluorescence Resonance Energy Transfer (FRET) Analysis for Plascinsin II

Substrates used for plasmin analysis are synthetic peptides designed to mimic cleavage sites present in hemoglobin (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS; Bachem, USA). The substrate combines the fluorescence donor EDANS with the fluorescence destructor DABCYL [Matayoshi, ED, Wang, GT, Kraff, GA and Erickson, J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science. 247: 954-958 (1990). Fluorescence is identified only when the EDANS group is separated from the DABCYL group due to substrate cleavage [Luker, KE, Francis, SE, Gluzman, IY and Goldberg. Kinetic analysis of plasmepsin I and II aspartic protease of the Plasmodium falciparum digestive vacuole. Mol. Biochem. Parasitol. 79: 71-78 (1996).

FRET analysis was performed on 96 well microplates (Falcon, USA). Assay buffer solution consists of 100 mM Na acetate pH 4.5, 10% glycerol, 0.01% Tween 20.

Cultures per well are as follows.

-37.5 μ ㅣ buffer solution

-5 μ ㅣ inhibitor (soluble in DMSO, found at concentrations from 10 μM to 1 nM)

FRET substrate corresponding to 5 μl dissolved in DMSO at a final concentration of −5 μM

-2.5 μl Plascecin II enzyme with 7.5 ng final content per assay tube

The reaction begins with the addition of an inhibitor, a buffer solution, and Plascinsin II enzyme. These mixed solutions are incubated at 37 ° C for 30 minutes. After incubation, the FRET substrate was added and then incubated at 37 ° C. for 30 minutes. The reaction is stopped by addition of 10% (v / v) Tris-base solution. The reaction product is monitored by measuring the fluorescence intensity (exitation 405 nm, emission 510 nm) using a fluorescence microplate reader Safire 2 (Tecan, Germany) instrument. Inhibitor materials that gave reduced activity of Plascinsin II at 50% or higher concentrations were screened to determine 50% inhibitory concentrations (IC ₅₀ ).

IC ₅₀ The value determined the value of each compound in a nonlinear regression manner. Thirty compounds were measured for inhibitory activity of Plasxinsin II at nanomolar concentrations (Table 1). IC ₅₀ of pepstatin a among these It was confirmed that the compound which has a value lower than 80 nM which is a value is 6 types. Inhibiting compound No. 14 was found to have the best inhibitory activity against Plascecin II. IC ₅₀ The value was 72.17 nM.

Table 1.IC ₅₀ for Compounds Selected in Plascinsin II Value (nM)

number Inhibitor Molecular Weight IC ₅₀ value (nM) One 2-anilino-4- (3-furyl) -6-oxo-N-phenyl-1-cyclohexene-1-carboxamide 372.43 154.91 2 3-chloro-N-{[2- (2-methoxybenzoyl) hydrazino] carbonothioyl} -1-benzothiophene-2-carboxamide 419.91 146.03 3 2-{[N- (2,3-dihydro-1,4-benzodioxin-6-yl) -N- (methylsulfonyl) gylsil] amino} -N-isobutylbenzamide 461.54 209.90 4 N-benzyl-2-{[3- (3-nitrophenyl) acryloyl] amino} benzamide 401.43 101.32 5 6-Bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one N-phenylthiosemicarbazone 413.34 174.97 6 N- (2-methoxyphenyl) -2- (5H- [1,2,4] triazino [5,6-b] indol-3-ylthio) butanamide 393.47 91.32 7 N-{(2,3-dihydro-1,4-benzodioxin-6-ylamino) [(6-methyl-4-oxo-1,4-dihydro-2-pyrimidinyl) amino] methylene } Benzamide 405.42 86.61 8 N-{(2,3-dihydro-1,4-benzodioxin-6-ylamino) [(6-oxo-4-propyl-1,6-dihydro-2-pyrimidinyl) amino] methylene } -4-methoxybenzamide 463.50 98.72 9 4-methoxy-N- (2- [5- (2-nitrophenyl) -2-furyl] -1-{[(4-pyridinylmethyl) amino] carbonyl} vinyl) benzamide 498.50 248.84 10 N- {amino [(4,6-dimethyl-2-quinazolinyl) amino] methylene} -2- (4-chlorophenyl) acetamide 367.84 241.51 11 4-amino-N '-(benzyloxy) -N- (4-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide 323.36 107.80 12 N, N '-{[(4,6-dimethyl-2-quinazolinyl) amino] methylidene} dipropanamide 327.39 127.31 13 N '-[(4-methoxy-3-nitrobenzoyl) oxy] -2- (1-naphthyl) ethaneimideamide 379.37 155.54 14 N '-{[(4-chloro-3,5-dimethylphenoxy) acetyl] oxy} -2- (3,4-dimethoxyphenyl) ethaneimideamide 406.86 72.17 15 2- (1-naphthyl) -N '-[(4-nitrobenzoyl) oxy] ethaneimideamide 349.35 123.93 16 4-amino-N '-[(2,4-dichlorobenzyl) oxy] -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide 392.25 85.48 17 N- (3,4-dichlorophenyl) -2-[(4,6-dimethyl-2-pyrimidinyl) amino] -3a, 4,5,6,7,7a-hexahydro-1H-benzimidazole -1-carboxamide 433.34 73.65 18 4-amino-N '-[(2-methyl-1-naphthyl) methoxy] -1,2,5-oxadiazole-3-carboxamideamide 297.32 82.59 19 N-{[(2,4-dimethylphenyl) amino] [(4-oxo-1,4,5,6,7,8-hexahydro-2-quinazolinyl) amino] methylene} benzamide 415.50 74.56 20 4-amino-N '-(benzyloxy) -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide 323.36 72.24 21 N-[(cyclopentylamino) carbonothioyl] -4-ethoxy-3-nitrobenzamide 337.40 71.24 22 6-({2-[(5-chloro-2-methoxyphenyl) amino] -2-oxoethyl} thio) -5-cyano-N- (2-methoxyphenyl) -2-methyl-4- Phenyl-1,4-dihydro-3-pyridinecarboxamide 575.09 163.50 23 N- (3,4-dimethylphenyl) -N '-{imino [(4,6,7-trimethyl-2-quinazolinyl) amino] methyl} thiourea 392.53 99.56 24 N-({[6-methyl-2- (4-methylphenyl) -2H-1,2,3-benzotriazol-5-yl] amino} carbonothioyl) -2-nitrobenzamide 446.49 115.62 25 2- (1,3-benzothiazol-2-ylamino) -N- (2-chlorophenyl) -6-oxo-1,4,5,6-tetrahydro-4-pyrimidinecarboxamide 399.86 88.23 26 2-anilino-3-chloro-N-phenyl-4- (phenylimino) -2-butenamide 375.86 94.42 27 N ', N' ''-1,2-phenylenebis [N- (3-chlorophenyl) urea] 415.28 75.62 28 N- (2-{[3- (1,3-benzodioxol-5-yl) -3-oxo-1-propen-1-yl] amino} phenyl) -4-nitrobenzenesulfonamide 467.46 100.40 29 N-({[2- (4ethylphenyl) -6-methyl-2H-1,2,3-benzotriazol-5-yl] amino} carbonothioyl) -2-nitrobenzamide 460.52 114.84 30 N- (1,3-benzodioxol-5-ylmethyl) -N '-[4- (2-oxo-2H-chromen-3-yl) -1,3-thiazol-2-yl] succinate amides 477.50 246.37

Example 3. Confirmation of Hemoglobin Resolution for Plascinsin II

Hemoglobin resolution analysis was performed in 0.2 ml tubes. Assay buffer consists of 100 mM Na acetate pH 4.5, 10% glycerol, 0.01% Tween 20.

Cultures per tube are as follows.

-6.5 μl buffer

-1 μl inhibitor (soluble in DMSO, final concentration 50 μM)

Hemoglobin corresponding to 1 μl dissolved in saline at a final concentration of -10 μg

-1.5 μl Plascecin II enzyme with 75 ng final content per assay tube

The reaction begins with the addition of an inhibitor, a buffer solution, and Plascinsin II enzyme. These mixed solutions are incubated at 37 ° C for 30 minutes. After incubation, hemoglobin was added and then incubated at 37 ° C. for 16 hours. The reaction is stopped by adding SDS loading stain (60 mM Tris-HCl pH 6.8, 25% glycerol, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue). The reaction product was boiled at 100 ° C. for 5 minutes, and the hemoglobin resolution was confirmed by SDS-electrophoresis using 15% acrylamide gel. In Figure 2, lane H is hemoglobin and lane C is the reaction product of the enzyme and hemoglobin. Lane P is the addition of pepstatin which inhibits the degradation of enzymes, hemoglobin and hemoglobin. Lanes 1 to 30 are the products reacted after mixing each inhibitory compound, Plascinsin II enzyme and hemoglobin. The marker used at this time is the same as the marker shown in FIG. After SDS-electrophoresis of hemoglobin resolution, the relative inhibitory activity was confirmed using the Public Doman NIH image program (USA).

After 16 hours of reaction with 50 μM inhibitory compounds, it can be seen that the inhibition of Plascecin II activity in all compound samples of the present invention was compared with Control Lane C with Plascecin II and hemoglobin, which was found to be hemoglobin. Compared to lane P with addition of pepstatin, which is known to inhibit the degradation of the compound, the compound of the present invention has excellent hemoglobin degradation inhibitory ability. Compound 5 of the 30 compounds of the present invention [6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one N-phenylthiosemicarbazone] inhibited 45.2% of enzyme activity Compound 13, [N '-[(4-methoxy-3-nitrobenzoyl) oxy] -2- (1-naphthyl) ethaneimideamide], exhibited an activity inhibitory property of 81%, and 21 Burn compound [N-[(cyclopentylamino) carbonothioyl] -4-ethoxy-3-nitrobenzamide] exhibits 53.8% of enzyme activity inhibiting properties, and all other 27 compounds of the present invention. Was shown to inhibit 100% plascincin activity, and as a result, all 30 compounds of the present invention can be confirmed that the inhibitory ability against Plascinsin II activity (Table 2).

Table 2. Inhibition of Plascecin II activity of the compounds of the present invention (%)

number Inhibitor Plasticine
% Inhibition ^* One 2-anilino-4- (3-furyl) -6-oxo-N-phenyl-1-cyclohexene-1-carboxamide 100 2 3-chloro-N-{[2- (2-methoxybenzoyl) hydrazino] carbonothioyl} -1-benzothiophene-2-carboxamide 100 3 2-{[N- (2,3-dihydro-1,4-benzodioxin-6-yl) -N- (methylsulfonyl) gylsil] amino} -N-isobutylbenzamide 100 4 N-benzyl-2-{[3- (3-nitrophenyl) acryloyl] amino} benzamide 100 5 6-Bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one-N-phenylthiosemicarbazone 45.2 6 N- (2-methoxyphenyl) -2- (5H- [1,2,4] triazino [5,6-b] indol-3-ylthio) butanamide 100 7 N-{(2,3-dihydro-1,4-benzodioxin-6-ylamino) [(6-methyl-4-oxo-1,4-dihydro-2-pyrimidinyl) amino] methylene } Benzamide 100 8 N-{(2,3-dihydro-1,4-benzodioxin-6-ylamino) [(6-oxo-4-propyl-1,6-dihydro-2-pyrimidinyl) amino] methylene } -4-methoxybenzamide 100 9 4-methoxy-N- (2- [5- (2-nitrophenyl) -2-furyl] -1-{[(4-pyridinylmethyl) amino] carbonyl} vinyl) benzamide 100 10 N- {amino [(4,6-dimethyl-2-quinazolinyl) amino] methylene} -2- (4-chlorophenyl) acetamide 100 11 4-amino-N '-(benzyloxy) -N- (4-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide 100 12 N, N '-{[(4,6-dimethyl-2-quinazolinyl) amino] methylidene} dipropanamide 100 13 N '-[(4-methoxy-3-nitrobenzoyl) oxy] -2- (1-naphthyl) ethaneimideamide 81% 14 N '-{[(4-chloro-3,5-dimethylphenoxy) acetyl] oxy} -2- (3,4-dimethoxyphenyl) ethaneimideamide 100 15 2- (1-naphthyl) -N '-[(4-nitrobenzoyl) oxy] ethaneimideamide 100 16 4-amino-N '-[(2,4-dichlorobenzyl) oxy] -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide 100 17 N- (3,4-dichlorophenyl) -2-[(4,6-dimethyl-2-pyrimidinyl) amino] -3a, 4,5,6,7,7a-hexahydro-1H-benzimidazole -1-carboxamide 100 18 4-amino-N '-[(2-methyl-1-naphthyl) methoxy] -1,2,5-oxadiazole-3-carboxamideamide 100 19 N-{[(2,4-dimethylphenyl) amino] [(4-oxo-1,4,5,6,7,8-hexahydro-2-quinazolinyl) amino] methylene} benzamide 100 20 4-amino-N '-(benzyloxy) -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide 100 21 N-[(cyclopentylamino) carbonothioyl] -4-ethoxy-3-nitrobenzamide 53.8 22 6-({2-[(5-chloro-2-methoxyphenyl) amino] -2-oxoethyl} thio) -5-cyano-N- (2-methoxyphenyl) -2-methyl-4- Phenyl-1,4-dihydro-3-pyridinecarboxamide 100 23 N- (3,4-dimethylphenyl) -N '-{imino [(4,6,7-trimethyl-2-quinazolinyl) amino] methyl} thiourea 100 24 N-({[6-methyl-2- (4-methylphenyl) -2H-1,2,3-benzotriazol-5-yl] amino} carbonothioyl) -2-nitrobenzamide 100 25 2- (1,3-benzothiazol-2-ylamino) -N- (2-chlorophenyl) -6-oxo-1,4,5,6-tetrahydro-4-pyrimidinecarboxamide 100 26 2-anilino-3-chloro-N-phenyl-4- (phenylimino) -2-butenamide 100 27 N ', N' ''-1,2-phenylenebis [N- (3-chlorophenyl) urea] 100 28 N- (2-{[3- (1,3-benzodioxol-5-yl) -3-oxo-1-propen-1-yl] amino} phenyl) -4-nitrobenzenesulfonamide 100 29 N-({[2- (4ethylphenyl) -6-methyl-2H-1,2,3-benzotriazol-5-yl] amino} carbonothioyl) -2-nitrobenzamide 100  30 N- (1,3-benzodioxol-5-ylmethyl) -N '-[4- (2-oxo-2H-chromen-3-yl) -1,3-thiazol-2-yl] succinate amides 100

The table above The final concentration of the reactor was 50 μM, and after 16 hours of reaction, each compound confirmed the effect of inhibiting hemoglobin degradation, and the degree of inhibition of plascine was added to a reactor containing pepstatin A, which is known to have hemoglobin degradation inhibitory ability. The ratio of the amount of hemoglobin remaining and the amount of hemoglobin remaining undecomposed in the enzymatic reactor in which each compound of the present invention is added is shown.

1 is a staining picture of SDS-electrophoresis of the protein (lane 1) and the purified enzyme (lane 2) obtained after cell disruption of E. coli transformed with Plaschexin II gene, lane M is a marker for confirming the molecular weight Indicates.

Figure 2 is a SDS-electrophoresis staining picture for confirming the effect of the compound inhibiting hemoglobin degradation by Plascecin II, after confirming the effect of the compound that inhibits hemoglobin degradation after 16 hours of reaction at a reactor final concentration of 50μM Lane H is hemoglobin, lane C is Plascinsin II + hemoglobin reaction, Lane P is Plascinsin II + hemoglobin + pepstatin A, lanes 1-30 is Plascinsin II + hemoglobin + each compound reaction, lane M is molecular weight Marker for confirming.

Claims (8)

delete 4-amino-N '-(benzyloxy) -N- (4-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide, N'-[(4-methoxy-3-nitrobenzoyl ) Oxy] -2- (1-naphthyl) ethaneimideamide, N '-{[(4-chloro-3,5-dimethylphenoxy) acetyl] oxy} -2- (3,4-dimethoxyphenyl ) Ethaneimideamide, 2- (1-naphthyl) -N '-[(4-nitrobenzoyl) oxy] ethaneimideamide, 4-amino-N'-[(2,4-dichlorobenzyl) oxy] -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxymidamide, 4-amino-N '-[(2-methyl-1-naphthyl) methoxy] -1,2 , 5-oxadiazole-3-carboxamideamide, and 4-amino-N '-(benzyloxy) -N- (2-methylphenyl) -1,2,5-oxadiazole-3-carboxamideamide At least one compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: Pharmaceutical composition for preventing and treating malaria, including as an active ingredient. delete delete delete The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier, diluent or excipient. delete delete

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