WO2009131384A2 - Pharmaceutical composition for preventing and treating malaria, containing compounds that inhibit plasmepsin ii activity, and method of treating malaria using the same - Google Patents

Pharmaceutical composition for preventing and treating malaria, containing compounds that inhibit plasmepsin ii activity, and method of treating malaria using the same Download PDF

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WO2009131384A2
WO2009131384A2 PCT/KR2009/002114 KR2009002114W WO2009131384A2 WO 2009131384 A2 WO2009131384 A2 WO 2009131384A2 KR 2009002114 W KR2009002114 W KR 2009002114W WO 2009131384 A2 WO2009131384 A2 WO 2009131384A2
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amino
pharmaceutical composition
oxo
derivative
plasmepsin
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PCT/KR2009/002114
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WO2009131384A3 (en
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Do-Man Kim
Hee-Kyoung Kang
Do-Won Kim
Giulio Rastelli
Ana-Lucia Da Costa
Vinod Kasam
Vincent Breton
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Industry Foundation Of Chonnam National University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to a pharmaceutical composition containing a compound that binds to active sites of plasmepsin II to inhibit activity. More particularly, the pharmaceutical composition of the invention contains at least one compound selected from the group consisting of an N-alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N-(2- ⁇ [3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino ⁇ phenyl)-4-nitrobenzenesulfonamide.
  • the pharmaceutical composition of the invention contains at least one compound selected from the group consisting of an N-alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N-(2- ⁇ [3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino ⁇ phen
  • the compound contained as an effective ingredient in the pharmaceutical composition of the invention has a use as an inhibitor that by binds to active sites of Plasmodium falciparum protease plasmepsin II to inhibit activity. Furthermore, the present invention relates to a method of preventing and treating malaria by administering an effective dose of the pharmaceutical composition to a mammal.
  • Malaria is a very serious and complex disease threatening human health in the 21 st century. Malaria infects about 3 million people and kills about 1.5 million people around the world. Malaria is an infectious disease caused by four different species of Plasmodia . Of these species, Plasmodium falciparum , also called Plasmodium falciparum malaria, is most dangerous. A successful vaccine for Plasmodium falciparum malaria has not yet been developed, and treatment and prevention for malaria are limited to drugs. However, since malaria having resistance to many anti-malarial drugs is rapidly spreading, new drugs are required.
  • Plasmodium falciparum enters the human body through a wound bitten by a female Anopheles mosquito. Malaria parasites stay in the liver in an early stage to replicate and then multiply further in red blood cells during amplification cycles. In this stage, malaria parasites degrade hemoglobin and use resultant products as nutrients for their growth. Plasmodium falciparum is known to degrade hemoglobin in host cells using its own protease. Parasites use hemoglobin in host red blood cells as important nutrients since they have only a limited ability to biosynthesize amino acid or absorb amino acid in the immediate environment. Parasites consume 25 to 75 % of hemoglobin of host cells during a short period of in vivo life cycle of red blood cells.
  • vacuolar proteases Two species are aspartic proteases, and one species is a cysteine protease
  • Plasmepsin I a first one of the aspartic proteases
  • Plasmepsin II a second aspartic protease
  • Falcipain the cysteine protease
  • Malaria is becoming a more severe threat in developing countries, and particularly, in African countries. Red blood cells infected by malaria parasite are deformed, and when accumulated on the wall of blood vessel, interrupt a flow of blood, thereby causing a complication in the brain, kidney, liver, etc. Therefore, insecticides for eliminating mosquitoes, a source of infection, are under development together with anti-malarial drugs.
  • attacks of malaria are rather increasing due to the increasing resistance of mosquitoes against insecticides and the appearance of variants resistant to anti-malarial drugs.
  • global warming is raising the risk for infection with malaria even in malaria-free areas. Thus, development of novel insecticides and anti-malarial drugs is an emergency request.
  • docking is a new method based on the International Grid (i.e., a new computing infrastructure allowing access to supercomputer power analyses and data around the world) allowing a work, which would last for several tens or hundreds of years when performed using a standard computer, to be finished in only several weeks.
  • International Grid i.e., a new computing infrastructure allowing access to supercomputer power analyses and data around the world
  • a process of acquiring focused compounds libraries using the International Grid and developing drug candidates via in silico and in vitro tests may include the steps of: (1) preparing a database of compounds for checking the levels of binding to subject (disease) proteins (about 40 millions) and a Three-Dimensional (3D) model of the subject proteins and determining binding sites in relation to activity; (2) virtually binding respective chemicals to the binding sites of the subject proteins, computing binding energy, and secondarily analyzing some of the chemicals showing a good binding force (top 15%) in consideration of molecular mechanics; (3) experimenting top 5% of the chemicals showing a most excellent binding force via in vitro tests.
  • the International Grid can preferably use the Enabling Grids for E-science (EGEE) grid.
  • One aspect of the invention is to provide a pharmaceutical composition for preventing and treating malaria, essentially containing, as an effective ingredient, at least one compound that binds to active sites of plasmepsin II to inhibit activity.
  • Another aspect of the invention is to provide a method of preventing and treating malaria by administering an effective dose of the pharmaceutical composition to a mammal.
  • An aspect of the present invention provides a pharmaceutical composition for preventing and treating malaria.
  • the composition may contain, as an effective ingredient, at least one compound selected from the group consisting of an alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N-(2- ⁇ [3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino ⁇ phenyl)-4-nitrobenzenesulfonamide.
  • the compound binds to active sites of plasmepsin II to inhibit activity.
  • Another aspect of the present invention provides a method of preventing and treating malaria.
  • the method may administer an effective dose of the pharmaceutical composition to a mammal.
  • the present invention uses a pharmaceutical composition containing at least one of compounds, which have been found to be able to bind to active sites of plasmepsin II to inhibit the activity, in order to prevent and treat malaria. Accordingly, the present invention is effective to malaria that is resistant to existing anti-malarial drugs.
  • FIG. 1 is a stained picture of SDS electrophoresis on a protein obtained after the cell disruption of a colon bacillus transformed with a plasmepsin II gene (lane 1) and a purified enzyme (lane 2), in which lane M indicates a marker identifying a molecular weight; and
  • FIG. 2 is stained pictures of SDS electrophoresis for testing effects of compounds that inhibit plasmepsin II from degrading hemoglobin, performed to check the effects after a 16 hours of reaction with the final concentration of a chemical reactor 50 ⁇ M, in which lane H indicates hemoglobin, lane C indicates a plasmepsin II + hemoglobin reaction solution, lane P indicates a plasmepsin II + hemoglobin + pepstatin A, lanes 1-30 indicate respective reaction solutions of plasmepsin II + hemoglobin + respective compounds, and lane M is a marker identifying a molecular weight.
  • 1,000 compounds are primarily selected from 500,000 compounds using plasmepsin II as a target through in silico virtual screening in order to discover compounds that bind to active sites of plasmepsin II to inhibit the activity of plasmepsin II.
  • the 1000 compounds are selected through investigation of other targets of plasmepsin II using two docking programs, FlexX and AutoDock.
  • 500 compounds are secondarily selected. At this time, the 500 compounds are selected by investigating interaction of a key residue (major amino acid) of protein. 100 compounds are tertiarily selected.
  • the 100 compounds are selected through virtual screening based on a docking score, an ideal bond mode, and a key residue of protein [Kasam V., Zimmermann, M., Maaa 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].
  • 30 compounds are proved to be excellent in binding to the active sites of plasmepsin II through an in-vitro test.
  • the 30 compounds are available from ChemBridge Corporation of U.S. These compounds show excellent inhibitory activity against plasmepsin II as can be seen from Table 1. Thus, these compounds can be contained in a pharmaceutical composition alone or with a pharmaceutically acceptable carrier, and be used as anti-malarial agents.
  • the inventive compounds are as follows.
  • the compounds 11, 13, 14, 15, 16, 18 and 20 are N-alkylamino derivatives
  • the compounds 7, 8, 10, 12, 17, 19, 23 and 25 are guanidine derivatives
  • the compounds 1, 3, 4, 6, 9, 22, 26 and 30 are amide derivatives
  • the compounds 2, 5, 21, 24, 27 and 29 are urea and thiourea derivatives.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound as an effective component, associated with a role of plasmepsin II, and used for preventing and treating diseases requiring selective inhibition of plasmepsin II.
  • the diseases include malaria.
  • the pharmaceutical composition comprises at least one of the compounds defined herein as an effective component, that is, the inventive anti-malarial pharmaceutical composition can comprise any combination of the inventive compounds.
  • the pharmaceutical composition can be concretely formulated so as to be administered through an arbitrary proper pathway such as oral, rectal, nasal, pulmonary, local, transdermal, intracisternal, intraperitoneal, vaginal, or parenteral (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal) pathway, and preferably an oral pathway.
  • the preferable pathway can be dependent on general conditions and age of a person to be treated, a nature of treated conditions, and selected effective ingredients.
  • the pharmaceutical composition can be administered through an arbitrary proper pathway, for instance an oral pathway in the form of a tablet, capsule, powder, granule, pellet, troche, dragee, globule or lozenge, solution or suspension in aqueous or non-aqueous liquid, oil-in-water or water-in-oil emersion, elixir, syrup, or the like, or a parenteral pathway in the form of an injection solution.
  • Another pharmaceutical composition for the parenteral administration includes a dispersion, suspension or emersion as well as sterile powder dissolved in a sterile injection solution or dispersion prior to use.
  • a depot injection formulation is also regarded to be within the scope of the present invention.
  • Another suitable administration type includes suppository, spray, ointment, cream, gel, inhalant, skin patch, or the like.
  • suppository a suitable administration type
  • spray ointment
  • cream a suitable administration type
  • gel a suitable administration type
  • inhalant a suitable administration type
  • skin patch a suitable administration type
  • methods known in the art can be employed, or arbitrary pharmaceutically acceptable carriers, diluents, excipients or other additives, which are generally used in the art, can be employed.
  • the carrier is typically used when the composition is prepared, and includes, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybezoate, talcum, magnesium stearate, mineral oil, or the like.
  • the composition can additionally comprise an antiseptic, stability improving material, viscosity improving or adjusting material, solubility improving material, sweetener, dye, palatability improving material, osmotic pressure variable salt, buffer solution, antioxidant, and so on.
  • inventive pharmaceutical composition can be used in conjunction with one or more other therapeutically useful materials, for instance other anti-malarial drugs such as quinoline (quinine, chloroquinine, amodiaquine, mefloquine, primaquine, taphenoquine, etc.), peroxide anti-malarial drug (artemisinin derivatives), pyrimethamine-sulfadoxine anti-malarial drugs (e.g. Fansidar), hydroxynaphthoquinone (e.g. atovaquaone), acroline-type anti-malarial drug (e.g. pyronaridine) and so on.
  • other anti-malarial drugs such as quinoline (quinine, chloroquinine, amodiaquine, mefloquine, primaquine, taphenoquine, etc.), peroxide anti-malarial drug (artemisinin derivatives), pyrimethamine-sulfadoxine anti-malarial drugs (e.g. Fansidar),
  • the compounds can be used in any form of free compound, pharmaceutically acceptable salt, solvate including hydrate, ester, or steromer as long as they have the effect inhibiting the activity of plasmepsin II. All of these materials fall within the scope of the present invention.
  • the pharmaceutically acceptable salt can include a pharmaceutically acceptable acid addition salt.
  • the pharmaceutically acceptable acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid, or phosphorous acid, and nontoxic organic acids such as aliphatic mono- and di-carboxylates, phenyl-substituted alkanoate, hydroxyl alkanoate, and alkandioate, aromatic acids, and aliphatic and aromatic sulfuric acids.
  • inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid, or phosphorous acid
  • nontoxic organic acids such as aliphatic mono- and di-carboxylates, phenyl-substituted alkanoate, hydroxyl alkanoate, and alkandioate, aromatic acids, and aliphatic and aromatic sulfuric acids
  • examples of the pharmaceutically acceptable acid addition salt can include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene
  • the present invention provides a method of treating mammalian malaria, which characterized by administering an effective dose of pharmaceutical composition to a mammal.
  • the inventive pharmaceutical composition is administered in the form of a unit dose containing its effective ingredient at an amount between about 1 mg and about 50 mg.
  • the total dose per day of the inventive pharmaceutical composition is within a range from about 1 mg to about 50 mg, and preferably from about 1 mg to about 30 mg.
  • a specific dose beyond such a range can be administered.
  • An optimal dose administered under a specific situation must be decided experimentally.
  • the inventive compounds can be administered once or several times at a dose. Preferably, a dose per day is administered one or twice per day.
  • the inventive compounds can be administered alone or in conjunction with a pharmaceutically acceptable carrier and excipient.
  • the inventive pharmaceutical composition can be formulated into other arbitrary juvantia and excipient known in the art as well as a pharmaceutically acceptable carrier and diluent. This formulation can take the form of a unit dose by a method known in the pharmaceutical field for convenience.
  • the compounds included in the inventive pharmaceutical composition is subjected to a fluorescence resonance energy transfer (FRET) test of plasmepsin II and a hemoglobin degradation test of plasmepsin II, and their efficacies are measured.
  • FRET fluorescence resonance energy transfer
  • Colon bacillus [ E. coli BL21(DE3)pLysS] having PMII-pET3d plasmid was agitated and cultivated in an LB liquid medium of 1 liter containing ampicillin at 37°C until A 600 value reached 0.5. In this state, isopropyl-b-D-thiogalactopyranoside of 400 mM was added, and then shaking culture was performed at 16°C for 18 hours.
  • a lysis buffer solution 50 ml, composition: 50 mM Tris-HCl and 25 mM NaCl, pH 8.0
  • BME b-mercaptoethanol
  • the refolded solution was purified using 50 ml Q-sepharose fast flow (GE Healthcare, USA) equilibrated in 0.1M Tris-HCl (pH 8.5) buffer solution. After resin washing was performed with 100mM Tris-HCl (pH 10) buffer solution, the recombination protein was eluted using a NaCl concentration gradient from 0M to 1M prepared with 100mM Tris-HCl (pH 10) buffer solution.
  • Fractions containing the recombination protein were condensed and were then dialyzed with 10mM Tris-HCl (pH 8.5) buffer solution, to which 5 mM NaCl and 20 mM BME were added. The obtained, purified protein was stored at -20 °C until it was used for analysis.
  • plasmepsin II was translated as a non-active zymogram having an N-terminal pro-sequence of 124 amino acids serving as membrane permeation domains.
  • the pro-sequence in a food vacuole was removed by calpain-like maturase, and then was released as active plasmepsin II [Benerjee, R., Francis, S.E and Goldberg. D.E. Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum . Mol. Biochem. Parasitol. 129: 157-165(2003)].
  • Plasmepsin II as a gene containing glutamic acid 124 next to an initiation codon, Met, was cloned to a pET3d vector, was purified into a single band, and was checked through SDS electrophoresis (FIG. 1).
  • the SDS electrophoresis employed 12% acrylamide gel. Protein (2 mg) was loaded onto the gel for the SDS electrophoresis, and then the SDS electrophoresis was performed, so that plasmepsin II was checked by dying the protein with Coomassie Brilliant Blue.
  • Lane 1 was cell supernatant liquid obtained from the cells expressed after 8M urea treatment, and lane 2 was plasmepsin II purified using the Q-sepharose resin.
  • a size of the protein was determined on the basis of standard protein of Bio-Read Company (USA) [lane M, size marker (myosin, 200 kDa; ⁇ -glactosidase, 116 kDa; phospholinase b, 97 kDa; bovine serum albumin, 66 kDa; ovoalbumin, 45kDa; carbonic anhydrase, 31kDa; soybean trypsin inhibitor, 21 kDa, apoprotein, 7 kDa)]. It was checked that a size of the recombination plasmepsin II was about 37 kDa.
  • the substrate used for plasmepsin analysis was synthetic peptide designed to resemble a cleavage site in hemoglogin (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS; Bachem, USA).
  • DEANS a fluorescence donor
  • DABCYL a fluorescence quencher
  • FRET analysis was performed on 96-well Microplate (Falcon, USA).
  • An analysis buffer solution contains 100 mM Na acetate of pH 4.5, 10 % glycerol, and 0.01 % Tween 20.
  • a culture solution per well contains:
  • Reaction was started by adding the inhibitor, the buffer solution, and the plasmepsin II enzyme. Mixture solutions thereof were cultivated at 37 °C for 30 minutes. Then, FRET substrate was added, followed by cultivation at 37 °C for 30 minutes. The reaction was stopped by adding 10 % (v/v) Tris-base solution to the mixture solutions. Resultant products were monitored by measuring fluorescence intensity (excitation: 405 nm, emission: 510 nm) using a fluorescence microplate Reader Safire 2 (Tescan, Germany). Screening was carried out on inhibitors, which suppress the activity of plasmepsin II at a concentration of 50 % or more, in order to determine a 50% inhibitory concentration (IC 50 ).
  • IC 50 values determined values of respective compounds by non-linear regression.
  • the inhibitory activities of the thirty (30) compounds against plasmepsin II were measured at a concentration on the order of nanometers (Table 1).
  • six (6) species were observed as having a lower value than IC 50 value of pepstatin A of 80 nM.
  • Inhibitor compound No. 14 was observed as having the best inhibitory activity against plasmepsin II. In this case, IC 50 value was 72.17 nM.
  • Hemoglobin degradation analysis was carried out in 0.2 ml tubes.
  • An analysis buffer solution contains 100 mM Na acetate of pH 4.5, 10% glycerol, and 0.01% Tween 20.
  • a culture solution per well contains:
  • Reaction was started by adding the inhibitor, the buffer solution, and the plasmepsin II enzyme. Mixture solutions were cultivated at 37 °C for 30 minutes. Then, hemoglobin was added, followed by cultivation at 37 °C for 16 hours. The reaction was stopped by adding SDS loading dye (60 mM Tris-HCl pH 6.8, 25% glycerol, 14.4 mM 2- mercaptoethanol, 0.1% bromophenol blue) to the mixture solutions. Resultant products were boiled at 100 °C for 5 minutes, and then hemoglobin degradation was checked through SDS-electrophoresis using 15 % acrylamide gel. In FIG.
  • SDS loading dye 60 mM Tris-HCl pH 6.8, 25% glycerol, 14.4 mM 2- mercaptoethanol, 0.1% bromophenol blue
  • Lanes 1 through 30 indicate compounds obtained by reacting mixtures of respective inhibitors, plasmepsin II enzyme and hemoglobin. Markers used in this test were the same as those represented in FIG. 1. After the SDS-electrophoresis, relative abilities to inhibit hemoglobin degradation were observed using a Public Doman NTH image program (USA).
  • Table 2 reports the inhibitory activities of the compounds against hemoglobin degradation measured by performing reaction for 16 hours with a final concentration of 50 ⁇ M.
  • the inhibitory activities (%) against plasmepsin were expressed by ratios (%) of the amount of hemoglobin remaining in a reactor, into which pepstatin A capable of inhibiting hemoglobin degradation was added, with respect to the amount of hemoglobin remaining in an enzyme reactor, into which respective inventive compounds were added.

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Abstract

A pharmaceutical composition containing a compound that binds to active sites of plasmepsin II to inhibit activity, and a method of preventing and treating malaria, including administering an effective dose of the pharmaceutical composition to a mammal. The pharmaceutical composition of the invention contains at least one compound selected from the group consisting of an N- alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N- (2-{[3-(l,3 -benzodioxol- 5 -yl) - 3 -oxo- 1 -propene- 1 -yl] amino } phenyl)-4-nitrobenzene sul¬ fonamide. The compound contained in the pharmaceutical composition is used to prevent and treat malaria since it binds to active sites of plasmepsin II to inhibit activity. The compound is effective to malaria that is resistant to existing anti-malarial drugs.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTING AND TREATING MALARIA, CONTAINING COMPOUNDS THAT INHIBIT PLASMEPSIN II ACTIVITY, AND METHOD OF TREATING MALARIA USING THE SAME
The present invention relates to a pharmaceutical composition containing a compound that binds to active sites of plasmepsin II to inhibit activity. More particularly, the pharmaceutical composition of the invention contains at least one compound selected from the group consisting of an N-alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N-(2-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino}phenyl)-4-nitrobenzenesulfonamide. The compound contained as an effective ingredient in the pharmaceutical composition of the invention has a use as an inhibitor that by binds to active sites of Plasmodium falciparum protease plasmepsin II to inhibit activity. Furthermore, the present invention relates to a method of preventing and treating malaria by administering an effective dose of the pharmaceutical composition to a mammal.
Malaria is a very serious and complex disease threatening human health in the 21st century. Malaria infects about 3 million people and kills about 1.5 million people around the world. Malaria is an infectious disease caused by four different species of Plasmodia. Of these species, Plasmodium falciparum, also called Plasmodium falciparum malaria, is most dangerous. A successful vaccine for Plasmodium falciparum malaria has not yet been developed, and treatment and prevention for malaria are limited to drugs. However, since malaria having resistance to many anti-malarial drugs is rapidly spreading, new drugs are required.
Plasmodium falciparum enters the human body through a wound bitten by a female Anopheles mosquito. Malaria parasites stay in the liver in an early stage to replicate and then multiply further in red blood cells during amplification cycles. In this stage, malaria parasites degrade hemoglobin and use resultant products as nutrients for their growth. Plasmodium falciparum is known to degrade hemoglobin in host cells using its own protease. Parasites use hemoglobin in host red blood cells as important nutrients since they have only a limited ability to biosynthesize amino acid or absorb amino acid in the immediate environment. Parasites consume 25 to 75 % of hemoglobin of host cells during a short period of in vivo life cycle of red blood cells. This is a large amount of catabolism known to occur in a vacuole, namely a unique acidic organelle of pH 5 or less. Three or more species of vacuolar proteases (two species are aspartic proteases, and one species is a cysteine protease) are known to be involved in degrading human hemoglobin into its components. Plasmepsin I, a first one of the aspartic proteases, has been known to initiate degrading hemoglobin by firstly cleaving hemoglobin and then causing molecule breakdown. Thereby, further protein degradation can efficiently occur. Plasmepsin II, a second aspartic protease, is known to cleave hemoglobin with an overlapping specificity. Falcipain, the cysteine protease, is also involved in an early stage of hemoglobin degradation. All the three species of proteases, including plasmepsin I and II and Falcipain, cleave denatured hemoglobin inside a test tube.
Malaria is becoming a more severe threat in developing countries, and particularly, in African countries. Red blood cells infected by malaria parasite are deformed, and when accumulated on the wall of blood vessel, interrupt a flow of blood, thereby causing a complication in the brain, kidney, liver, etc. Therefore, insecticides for eliminating mosquitoes, a source of infection, are under development together with anti-malarial drugs. However, attacks of malaria are rather increasing due to the increasing resistance of mosquitoes against insecticides and the appearance of variants resistant to anti-malarial drugs. Moreover, global warming is raising the risk for infection with malaria even in malaria-free areas. Thus, development of novel insecticides and anti-malarial drugs is an emergency request.
Development of a new drug for solving the above-stated problems is not easy since it requires a long time (10 to 12 years) and a great amount of expenses. As an attempt to make up for the drawbacks, a process of designing new structures of plasmepsin enzymes and drug compounds via simulation using a supercomputer is proceeding. Specifically, in silico virtual screening, using a computer and docking software for this purpose, has great contribution in reducing times and costs for developing new drugs by listing up binding levels of ligands (about 40 millions, drug candidate), which can bind to active sites of target proteins, and by presenting focused compounds libraries so that in vitro tests can be efficiently carried out. In particular, docking is a new method based on the International Grid (i.e., a new computing infrastructure allowing access to supercomputer power analyses and data around the world) allowing a work, which would last for several tens or hundreds of years when performed using a standard computer, to be finished in only several weeks.
A process of acquiring focused compounds libraries using the International Grid and developing drug candidates via in silico and in vitro tests may include the steps of: (1) preparing a database of compounds for checking the levels of binding to subject (disease) proteins (about 40 millions) and a Three-Dimensional (3D) model of the subject proteins and determining binding sites in relation to activity; (2) virtually binding respective chemicals to the binding sites of the subject proteins, computing binding energy, and secondarily analyzing some of the chemicals showing a good binding force (top 15%) in consideration of molecular mechanics; (3) experimenting top 5% of the chemicals showing a most excellent binding force via in vitro tests. In this case, the International Grid (WISDOM) can preferably use the Enabling Grids for E-science (EGEE) grid.
One aspect of the invention is to provide a pharmaceutical composition for preventing and treating malaria, essentially containing, as an effective ingredient, at least one compound that binds to active sites of plasmepsin II to inhibit activity.
Another aspect of the invention is to provide a method of preventing and treating malaria by administering an effective dose of the pharmaceutical composition to a mammal.
An aspect of the present invention provides a pharmaceutical composition for preventing and treating malaria. The composition may contain, as an effective ingredient, at least one compound selected from the group consisting of an alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N-(2-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino}phenyl)-4-nitrobenzenesulfonamide. The compound binds to active sites of plasmepsin II to inhibit activity.
Another aspect of the present invention provides a method of preventing and treating malaria. The method may administer an effective dose of the pharmaceutical composition to a mammal.
The present invention uses a pharmaceutical composition containing at least one of compounds, which have been found to be able to bind to active sites of plasmepsin II to inhibit the activity, in order to prevent and treat malaria. Accordingly, the present invention is effective to malaria that is resistant to existing anti-malarial drugs.
FIG. 1 is a stained picture of SDS electrophoresis on a protein obtained after the cell disruption of a colon bacillus transformed with a plasmepsin II gene (lane 1) and a purified enzyme (lane 2), in which lane M indicates a marker identifying a molecular weight; and
FIG. 2 is stained pictures of SDS electrophoresis for testing effects of compounds that inhibit plasmepsin II from degrading hemoglobin, performed to check the effects after a 16 hours of reaction with the final concentration of a chemical reactor 50μM, in which lane H indicates hemoglobin, lane C indicates a plasmepsin II + hemoglobin reaction solution, lane P indicates a plasmepsin II + hemoglobin + pepstatin A, lanes 1-30 indicate respective reaction solutions of plasmepsin II + hemoglobin + respective compounds, and lane M is a marker identifying a molecular weight.
1,000 compounds are primarily selected from 500,000 compounds using plasmepsin II as a target through in silico virtual screening in order to discover compounds that bind to active sites of plasmepsin II to inhibit the activity of plasmepsin II. At this time, the 1000 compounds are selected through investigation of other targets of plasmepsin II using two docking programs, FlexX and AutoDock. 500 compounds are secondarily selected. At this time, the 500 compounds are selected by investigating interaction of a key residue (major amino acid) of protein. 100 compounds are tertiarily selected. At this time, the 100 compounds are selected through virtual screening based on a docking score, an ideal bond mode, and a key residue of protein [Kasam V., Zimmermann, M., Maaa 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]. Among these compounds, 30 compounds are proved to be excellent in binding to the active sites of plasmepsin II through an in-vitro test.
The 30 compounds are available from ChemBridge Corporation of U.S. These compounds show excellent inhibitory activity against plasmepsin II as can be seen from Table 1. Thus, these compounds can be contained in a pharmaceutical composition alone or with a pharmaceutically acceptable carrier, and be used as anti-malarial agents. The inventive compounds are as follows.
Compound 1:
2-anilino-4-(3-furyl)-6-oxo-N-phenyl-1-cyclohexene-1-carboxamide
Figure PCTKR2009002114-appb-I000001
Compound 2:
3-chloro-N-{[2-(2-methoxybenzoyl)hydrazino]carbonothioyl}-1-benzothiophene-2-carboxamide
Figure PCTKR2009002114-appb-I000002
Compound 3:
2-{[N-(2,3-dihydro-1,4-benzodioxine-6-yl)-N-(methylsulfonyl)glycyl]amino}-N-isobutylbenzamide
Figure PCTKR2009002114-appb-I000003
Compound 4:
N-benzyl-2-{[3-(3-nitrophenyl)acryloyl]amino}benzamide
Figure PCTKR2009002114-appb-I000004
Compound 5:
6-bromo-2,3,4,9-tetrahydro-1H-carbazole-1-one N-phenylthiosemicarbazone
Figure PCTKR2009002114-appb-I000005
Compound 6:
N-(2-methoxyphenyl)-2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)butaneamide
Figure PCTKR2009002114-appb-I000006
Compound 7:
N-{(2,3-dihydro-1,4-benzodioxine-6-ylamino)[(6-methyl-4-oxo-1,4-dihydro-2-pyrimidinyl)amino]methylene}benzamide
Figure PCTKR2009002114-appb-I000007
Compound 8:
N-{(2,3-dihydro-1,4-benzodioxine-6-ylamino)[(6-oxo-4-propyl-1,6-dihydro-2-pyrimidinyl)amino]methylene}-4-methoxybenzamide
Figure PCTKR2009002114-appb-I000008
Compound 9:
4-methoxy-N-(2-[5-(2-nitrophenyl)-2-furyl]-1-{[(4-pyridinylmethyl)amino]carbonyl}vinyl)benzamide
Figure PCTKR2009002114-appb-I000009
Compound 10:
N-{amino[(4,6-dimethyl-2-quinazolinyl)amino]methylene}-2-(4-chlorophenyl)aceteamide
Figure PCTKR2009002114-appb-I000010
Compound 11:
4-amino-N'-(benzyloxy)-N-(4-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide
Figure PCTKR2009002114-appb-I000011
Compound 12:
N,N'-{[(4,6-dimethyl-2-quinazolinyl)amino]methylidene}dipropanamide
Figure PCTKR2009002114-appb-I000012
Compound 13:
N'-[(4-methoxy-3-nitrobenzoyl)oxy]-2-(1-naphtyl)ethaneimidamide
Figure PCTKR2009002114-appb-I000013
Compound 14:
N'-{[(4-chloro-3,5-dimethylphenoxy)acetyl]oxy}-2-(3,4-dimethoxyphenyl)ethaneimidamide
Figure PCTKR2009002114-appb-I000014
Compound 15:
2-(1-naphtyl)-N'-[(4-nitrobenzoyl)oxy]ethaneimidamide
Figure PCTKR2009002114-appb-I000015
Compound 16:
4-amino-N'-[(2,4-dichlorobenzyl)oxy]-N-(2-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide
Figure PCTKR2009002114-appb-I000016
Compound 17:
N-(3,4-dichlorophenyl)-2-[(4,6-dimethyl-2-pyrimidinyl)amino]-3a,4,5,6,7,7a-hexahydro-1H-benzimidazole-1-carboxamide
Figure PCTKR2009002114-appb-I000017
Compound 18:
4-amino-N'-[(2-methyl-1-naphtyl)methoxy]-1,2,5-oxadiazole-3-carboximidamide
Figure PCTKR2009002114-appb-I000018
Compound 19:
N-{[(2,4-dimethylphenyl)amino][(4-oxo-1,4,5,6,7,8-hexahydro-2-quinazolinyl)amino]methylene}benzamide
Figure PCTKR2009002114-appb-I000019
Compound 20:
4-amino-N'-(benzyloxy)-N-(2-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide
Figure PCTKR2009002114-appb-I000020
Compound 21:
N-[(cyclopentylamino)carbonothioyl]-4-ethoxy-3-nitrobenzamide
Figure PCTKR2009002114-appb-I000021
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
Figure PCTKR2009002114-appb-I000022
Compound 23:
N-(3,4-dimethylphenyl)-N'-{imino[(4,6,7-trimethyl-2-quinazolinyl)amino]methyl}thiourea
Figure PCTKR2009002114-appb-I000023
Compound 24:
N-({[6-methyl-2-(4-methylphenyl)-2H-1,2,3-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide
Figure PCTKR2009002114-appb-I000024
Compound 25:
2-(1,3-benzothiazole-2-ylamino)-N-(2-chlorophenyl)-6-oxo-1,4,5,6-tetrahydro-4-pyrimidinecarboxamide
Figure PCTKR2009002114-appb-I000025
Compound 26:
2-anilino-3-chloro-N-phenyl-4-(phenylimino)-2-buteneamide
Figure PCTKR2009002114-appb-I000026
Compound 27:
N',N'''-1,2-phenylenebis[N-(3-chlorophenyl)urea]
Figure PCTKR2009002114-appb-I000027
Compound 28:
N-(2-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino}phenyl)-4-nitrobenzenesulfoneamide
Figure PCTKR2009002114-appb-I000028
Compound 29:
N-({[2-(4-ethylphenyl)-6-methyl-2H-1,2,3-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide
Figure PCTKR2009002114-appb-I000029
Compound 30:
N-(1,3-benzodioxol-5-ylmethyl)-N'-[4-(2-oxo-2H-chromene-3-yl)-1,3-thiazole-2-yl] succinamide
Figure PCTKR2009002114-appb-I000030
Among the compounds, the compounds 11, 13, 14, 15, 16, 18 and 20 are N-alkylamino derivatives, the compounds 7, 8, 10, 12, 17, 19, 23 and 25 are guanidine derivatives, the compounds 1, 3, 4, 6, 9, 22, 26 and 30 are amide derivatives, and the compounds 2, 5, 21, 24, 27 and 29 are urea and thiourea derivatives.
The present invention provides a pharmaceutical composition comprising the compound as an effective component, associated with a role of plasmepsin II, and used for preventing and treating diseases requiring selective inhibition of plasmepsin II. Particularly, the diseases include malaria. The pharmaceutical composition comprises at least one of the compounds defined herein as an effective component, that is, the inventive anti-malarial pharmaceutical composition can comprise any combination of the inventive compounds.
The pharmaceutical composition can be concretely formulated so as to be administered through an arbitrary proper pathway such as oral, rectal, nasal, pulmonary, local, transdermal, intracisternal, intraperitoneal, vaginal, or parenteral (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal) pathway, and preferably an oral pathway. The preferable pathway can be dependent on general conditions and age of a person to be treated, a nature of treated conditions, and selected effective ingredients.
According to the present invention, the pharmaceutical composition can be administered through an arbitrary proper pathway, for instance an oral pathway in the form of a tablet, capsule, powder, granule, pellet, troche, dragee, globule or lozenge, solution or suspension in aqueous or non-aqueous liquid, oil-in-water or water-in-oil emersion, elixir, syrup, or the like, or a parenteral pathway in the form of an injection solution. Another pharmaceutical composition for the parenteral administration includes a dispersion, suspension or emersion as well as sterile powder dissolved in a sterile injection solution or dispersion prior to use. A depot injection formulation is also regarded to be within the scope of the present invention. Another suitable administration type includes suppository, spray, ointment, cream, gel, inhalant, skin patch, or the like. In order to prepare the composition, methods known in the art can be employed, or arbitrary pharmaceutically acceptable carriers, diluents, excipients or other additives, which are generally used in the art, can be employed.
The carrier is typically used when the composition is prepared, and includes, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybezoate, talcum, magnesium stearate, mineral oil, or the like. The composition can additionally comprise an antiseptic, stability improving material, viscosity improving or adjusting material, solubility improving material, sweetener, dye, palatability improving material, osmotic pressure variable salt, buffer solution, antioxidant, and so on.
Further, the inventive pharmaceutical composition can be used in conjunction with one or more other therapeutically useful materials, for instance other anti-malarial drugs such as quinoline (quinine, chloroquinine, amodiaquine, mefloquine, primaquine, taphenoquine, etc.), peroxide anti-malarial drug (artemisinin derivatives), pyrimethamine-sulfadoxine anti-malarial drugs (e.g. Fansidar), hydroxynaphthoquinone (e.g. atovaquaone), acroline-type anti-malarial drug (e.g. pyronaridine) and so on.
The compounds can be used in any form of free compound, pharmaceutically acceptable salt, solvate including hydrate, ester, or steromer as long as they have the effect inhibiting the activity of plasmepsin II. All of these materials fall within the scope of the present invention.
In the present invention, the pharmaceutically acceptable salt can include a pharmaceutically acceptable acid addition salt. The pharmaceutically acceptable acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid, or phosphorous acid, and nontoxic organic acids such as aliphatic mono- and di-carboxylates, phenyl-substituted alkanoate, hydroxyl alkanoate, and alkandioate, aromatic acids, and aliphatic and aromatic sulfuric acids. More specifically, examples of the pharmaceutically acceptable acid addition salt can include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, -hydroxybutyrate, glycolate, maleate, tartrate, methane sulfonate, propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.
Further, the present invention provides a method of treating mammalian malaria, which characterized by administering an effective dose of pharmaceutical composition to a mammal.
Typically, the inventive pharmaceutical composition is administered in the form of a unit dose containing its effective ingredient at an amount between about 1 mg and about 50 mg. The total dose per day of the inventive pharmaceutical composition is within a range from about 1 mg to about 50 mg, and preferably from about 1 mg to about 30 mg. However, in comprehensive consideration of the situation of a patient, and in consideration of the activity of an administered medication, a specific dose beyond such a range can be administered. An optimal dose administered under a specific situation must be decided experimentally.
The inventive compounds can be administered once or several times at a dose. Preferably, a dose per day is administered one or twice per day. The inventive compounds can be administered alone or in conjunction with a pharmaceutically acceptable carrier and excipient. The inventive pharmaceutical composition can be formulated into other arbitrary juvantia and excipient known in the art as well as a pharmaceutically acceptable carrier and diluent. This formulation can take the form of a unit dose by a method known in the pharmaceutical field for convenience.
In order to verify an anti-malarial effect of the inventive pharmaceutical composition, the compounds included in the inventive pharmaceutical composition is subjected to a fluorescence resonance energy transfer (FRET) test of plasmepsin II and a hemoglobin degradation test of plasmepsin II, and their efficacies are measured.
The present invention will now be described more fully with reference to EXAMPLES, which are in no way intended to be in the nature of limitation.
EXAMPLE 1
Expression and Preparation of Recombination Protein
1. Plasmepsin II Gene
Gene for coding plasmepsin II was purchased from MR4/American Type culture Collection, USA [Luker, K.E., Francis, S.E.,Gluzman, I.Y. and D.E. Goldberg. Kinetic analysis of plasmepsin I and II aspartic protease of the Plasmodium falciparum digestive vacuole. Mol. Biochem. Parasitol. 79, 71-78(1996); lstvan, E.S. and Goldberg D.E. Distal substrate interactions enhance plasmepsin activity. J. Biol. Chem. 280, 6890-6896(2005)].
2. Preparation of Recombination Protein
Colon bacillus [E. coli BL21(DE3)pLysS] having PMII-pET3d plasmid was agitated and cultivated in an LB liquid medium of 1 liter containing ampicillin at 37℃ until A600 value reached 0.5. In this state, isopropyl-b-D-thiogalactopyranoside of 400 mM was added, and then shaking culture was performed at 16℃ for 18 hours. After the culture was completed, cells were recovered through centrifugation (8,000 xg, at 4℃ for 10 minutes), and were suspended again into a lysis buffer solution (50 ml, composition: 50 mM Tris-HCl and 25 mM NaCl, pH 8.0) to which b-mercaptoethanol (BME) of 50 ml was added. The suspended cells were disrupted using a sonicator (Ultrasonic Processor 250, Sonics and Materials, Inc., CT, USA; output 4, duty cycle 50%, 30 seconds in ice, repetition of 25 times). After the disrupture, centrifugation was carried out, a pellet containing an inclusion body was washed again in 0.1M Tris (pH 10) buffer solution to which BME of 50 ml was added. The pellet was suspended again in a urea solution of 8M (dissolved into 100 mM Tris- HCl pH 10, 1 mM EDTA, 1mM glycine solution), and the cells were disrupted using the sonicator. After the disrupture, BME of 35 ml was added. This suspension was stored at 4℃ for 18 hours, and then was centrifuged at a speed of 23,000xg at 4℃ for 30 minutes. After the centrifugation, supernatant liquid was obtained, and the supernatant liquid and water were diluted at a ratio of 1:10, were agitated for 18 hours so as to cause refolding of recombination protein. The refolded solution was purified using 50 ml Q-sepharose fast flow (GE Healthcare, USA) equilibrated in 0.1M Tris-HCl (pH 8.5) buffer solution. After resin washing was performed with 100mM Tris-HCl (pH 10) buffer solution, the recombination protein was eluted using a NaCl concentration gradient from 0M to 1M prepared with 100mM Tris-HCl (pH 10) buffer solution. Fractions containing the recombination protein were condensed and were then dialyzed with 10mM Tris-HCl (pH 8.5) buffer solution, to which 5 mM NaCl and 20 mM BME were added. The obtained, purified protein was stored at -20 ℃ until it was used for analysis.
In the parasite state, plasmepsin II was translated as a non-active zymogram having an N-terminal pro-sequence of 124 amino acids serving as membrane permeation domains. The pro-sequence in a food vacuole was removed by calpain-like maturase, and then was released as active plasmepsin II [Benerjee, R., Francis, S.E and Goldberg. D.E. Food vacuole plasmepsins are processed at a conserved site by an acidic convertase activity in Plasmodium falciparum. Mol. Biochem. Parasitol. 129: 157-165(2003)].
Plasmepsin II, as a gene containing glutamic acid 124 next to an initiation codon, Met, was cloned to a pET3d vector, was purified into a single band, and was checked through SDS electrophoresis (FIG. 1). The SDS electrophoresis employed 12% acrylamide gel. Protein (2 mg) was loaded onto the gel for the SDS electrophoresis, and then the SDS electrophoresis was performed, so that plasmepsin II was checked by dying the protein with Coomassie Brilliant Blue. Lane 1 was cell supernatant liquid obtained from the cells expressed after 8M urea treatment, and lane 2 was plasmepsin II purified using the Q-sepharose resin. A size of the protein was determined on the basis of standard protein of Bio-Read Company (USA) [lane M, size marker (myosin, 200 kDa; β-glactosidase, 116 kDa; phospholinase b, 97 kDa; bovine serum albumin, 66 kDa; ovoalbumin, 45kDa; carbonic anhydrase, 31kDa; soybean trypsin inhibitor, 21 kDa, apoprotein, 7 kDa)]. It was checked that a size of the recombination plasmepsin II was about 37 kDa.
EXAMPLE 2
Fluorescence Resonance Energy Transfer (FRET) Analysis of Plasmepsin II
The substrate used for plasmepsin analysis was synthetic peptide designed to resemble a cleavage site in hemoglogin (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS; Bachem, USA). DEANS, a fluorescence donor, and DABCYL, a fluorescence quencher, are bound to this Substrate [Matayoshi, E.D., Wang, G.T., Kraff, G.A. and Erickson, J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science. 247: 954-958(1990)]. Fluorescence is observed only when EDANS group is separated from DABCYL group only when the substrate was cleaved [Luker, K.E., Francis, S.E., Gluzman, I.Y. 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 Microplate (Falcon, USA). An analysis buffer solution contains 100 mM Na acetate of pH 4.5, 10 % glycerol, and 0.01 % Tween 20.
A culture solution per well contains:
- 37.5 ㎕ buffer solution
- 5 ㎕ inhibitor (dissolved into DMSO, observed at a concentration from 10 μM to 1 nM)
- FRET substrate corresponding to 5 ㎕, solved into DMSO with a final concentration 5 nM
- 2.5 nl plasmepsin II enzyme (final content) with 7.5 ng per analysis tube
Reaction was started by adding the inhibitor, the buffer solution, and the plasmepsin II enzyme. Mixture solutions thereof were cultivated at 37 ℃ for 30 minutes. Then, FRET substrate was added, followed by cultivation at 37 ℃ for 30 minutes. The reaction was stopped by adding 10 % (v/v) Tris-base solution to the mixture solutions. Resultant products were monitored by measuring fluorescence intensity (excitation: 405 nm, emission: 510 nm) using a fluorescence microplate Reader Safire 2 (Tescan, Germany). Screening was carried out on inhibitors, which suppress the activity of plasmepsin II at a concentration of 50 % or more, in order to determine a 50% inhibitory concentration (IC50).
IC50 values determined values of respective compounds by non-linear regression. The inhibitory activities of the thirty (30) compounds against plasmepsin II were measured at a concentration on the order of nanometers (Table 1). Among these compounds, six (6) species were observed as having a lower value than IC50 value of pepstatin A of 80 nM. Inhibitor compound No. 14 was observed as having the best inhibitory activity against plasmepsin II. In this case, IC50 value was 72.17 nM.
Table 1 IC50 Value (nM) of Compounds Screened for Plasmepsin II
No. Inhibitor Mw IC50 value (nM)
1 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-benzodioxine-6-yl)-N-(methylsulfonyl)glycyl]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-carbazole-1-one N-phenylthiosemicarbazone 413.34 174.97
6 N-(2-methoxyphenyl)-2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)butaneamide 393.47 91.32
7 N-{(2,3-dihydro-1,4-benzodioxine-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-benzodioxine-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)aceteamide 367.84 241.51
11 4-amino-N'-(benzyloxy)-N-(4-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide 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-naphtyl)ethaneimidamide 379.37 155.54
14 N'-{[(4-chloro-3,5-dimethylphenoxy)acetyl]oxy}-2-(3,4-dimethoxyphenyl)ethaneimidamide 406.86 72.17
15 2-(1-naphtyl)-N'-[(4-nitrobenzoyl)oxy]ethaneimidamide 349.35 123.93
16 4-amino-N'-[(2,4-dichlorobenzyl)oxy]-N-(2-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide 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-naphtyl)methoxy]-1,2,5-oxadiazole-3-carboximidamide 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-carboximidamide 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-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide 446.49 115.62
25 2-(1,3-benzothiazole-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-buteneamide 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-propene-1-yl]amino}phenyl)-4-nitrobenzenesulfonamide 467.46 100.40
29 N-({[2-(4-ethylphenyl)-6-methyl-2H-1,2,3-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide 460.52 114.84
30 N-(1,3-benzodioxol-5-ylmethyl)-N'-[4-(2-oxo-2H-chromene-3-yl)-1,3-thiazole-2-yl] succinamide 477.50 246.37
EXAMPLE 3
Hemoglobin Degradation Test of Plasmepsin II
Hemoglobin degradation analysis was carried out in 0.2 ml tubes. An analysis buffer solution contains 100 mM Na acetate of pH 4.5, 10% glycerol, and 0.01% Tween 20.
A culture solution per well contains:
- 6.5 ㎕ buffer solution
- 1㎕ inhibitor (dissolved into DMSO, final concentration 50 μM)
- Hemoglobin corresponding to 1 ㎕, dissolved into a saline solution with a final solution concentration 10 μg
- 1.5 μl plasmepsin II enzyme (final content) with 75 ng per analysis tube
Reaction was started by adding the inhibitor, the buffer solution, and the plasmepsin II enzyme. Mixture solutions were cultivated at 37 ℃ for 30 minutes. Then, hemoglobin was added, followed by cultivation at 37 ℃ for 16 hours. The reaction was stopped by adding SDS loading dye (60 mM Tris-HCl pH 6.8, 25% glycerol, 14.4 mM 2- mercaptoethanol, 0.1% bromophenol blue) to the mixture solutions. Resultant products were boiled at 100 ℃ for 5 minutes, and then hemoglobin degradation was checked through SDS-electrophoresis using 15 % acrylamide gel. In FIG. 2, lane H indicates hemoglobin, lane C indicates a reaction product of enzyme and hemoglobin, and lane P indicates the addition of enzyme, hemoglobin and pepstatin that inhibits hemoglobin degradation. Lanes 1 through 30 indicate compounds obtained by reacting mixtures of respective inhibitors, plasmepsin II enzyme and hemoglobin. Markers used in this test were the same as those represented in FIG. 1. After the SDS-electrophoresis, relative abilities to inhibit hemoglobin degradation were observed using a Public Doman NTH image program (USA).
When reaction was carried out using 50 μM inhibitor compounds for 16 hours, all compound samples of the invention were observed to inhibit the activity of plasmepsin II compared to control group lane C in which plasmepsin II and hemoglobin were added. The results represent the excellent ability of the inventive compounds to inhibit hemoglobin degradation when compared to lane P in which pepstatin known as a potent inhibitor of hemoglobin degradation was added. Compound No. 5 of the 30 compounds of the invention [6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one N-phenylthiosemicarbazone] represented an inhibitory activity against enzyme of 45.2 %. Compound No. 13 [N'-[(4-methoxy-3-nitrobenzoyl)oxy]-2-(1-naphtyl)ethaneimidamide] represented inhibitory activity against enzyme of 81 %. Compound No. 21 [N-[(cyclopentylamino)carbonothioyl]-4-ethoxy-3-nitrobenzamide] represented an inhibitory activity against enzyme of 53.8 %. The remaining 27 compounds of the invention were reported to have inhibitory activity against plasmepsin up to 100 %. As a result, it was observed that all the 30 compounds of the invention had an excellent inhibitory activity against plasmepsin II (Table 2).
Table 2 Inhibitory Activity of Inventive Compounds against Plasmepsin II (%)
No. Inhibitor IA*
1 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-benzodioxine-6-yl)-N-(methylsulfonyl)glycyl]amino}-N-isobutylbenzamide 100
4 N-benzyl-2-{[3-(3-nitrophenyl)acryloyl]amino}benzamide 100
5 6-bromo-2,3,4,9-tetrahydro-1H-carbazole-1-one N-phenylthiosemicarbazone 45.2
6 N-(2-methoxyphenyl)-2-(5H-[1,2,4]triazino[5,6-b]indole-3-ylthio)butaneamide 100
7 N-{(2,3-dihydro-1,4-benzodioxine-6-ylamino)[(6-methyl-4-oxo-1,4-dihydro-2-pyrimidinyl)amino]methylene}benzamide 100
8 N-{(2,3-dihydro-1,4-benzodioxine-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)aceteamide 100
11 4-amino-N'-(benzyloxy)-N-(4-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide 100
12 N,N'-{[(4,6-dimethyl-2-quinazolinyl)amino]methylidene}dipropanamide 100
13 N'-[(4-methoxy-3-nitrobenzoyl)oxy]-2-(1-naphtyl)ethaneimidamide 81%
14 N'-{[(4-chloro-3,5-dimethylphenoxy)acetyl]oxy}-2-(3,4-dimethoxyphenyl)ethaneimidamide 100
15 2-(1-naphtyl)-N'-[(4-nitrobenzoyl)oxy]ethaneimidamide 100
16 4-amino-N'-[(2,4-dichlorobenzyl)oxy]-N-(2-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide 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-naphtyl)methoxy]-1,2,5-oxadiazole-3-carboximidamide 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-carboximidamide 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-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide 100
25 2-(1,3-benzothiazole-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-buteneamide 100
27 N',N'''-1,2-phenylenebis[N-(3-chlorophenyl)urea] 100
28 N-(2-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino}phenyl)-4-nitrobenzenesulfonamide 100
29 N-({[2-(4ethylphenyl)-6-methyl-2H-1,2,3-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide 100
30 N-(1,3-benzodioxol-5-ylmethyl)-N'-[4-(2-oxo-2H-chromene-3-yl)-1,3-thiazole-2-yl] succinamide 100
Note) IA*: Inhibitory Activity (%)
Table 2 reports the inhibitory activities of the compounds against hemoglobin degradation measured by performing reaction for 16 hours with a final concentration of 50μM. The inhibitory activities (%) against plasmepsin were expressed by ratios (%) of the amount of hemoglobin remaining in a reactor, into which pepstatin A capable of inhibiting hemoglobin degradation was added, with respect to the amount of hemoglobin remaining in an enzyme reactor, into which respective inventive compounds were added.

Claims (8)

  1. A pharmaceutical composition for preventing and treating malaria, comprising, as an effective ingredient, at least one compound selected from the group consisting of an alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N-(2-{[3-(1,3-benzodioxol-5-yl)-3-oxo-1-propene-1-yl]amino}phenyl)-4-nitrobenzenesulfonamide; or one of pharmaceutically acceptable salt, hydride and ester thereof, wherein the compound binds to active sites of plasmepsin II to inhibit activity.
  2. The pharmaceutical composition of claim 1, wherein the N-alkoxyamidine derivative is selected from the group consisting of 4-amino-N'-(benzyloxy)-N-(4-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide, N'-[(4-methoxy-3-nitrobenzoyl)oxy]-2-(1-naphtyl)ethaneimidamide, N'-{[(4-chloro-3,5-dimethylphenoxy)acetyl]oxy}-2-(3,4-dimethoxyphenyl)ethaneimidamide, 2-(1-naphtyl)-N'-[(4-nitrobenzoyl)oxy]ethaneimidamide, 4-amino-N'-[(2,4-dichlorobenzyl)oxy]-N-(2-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide, 4-amino-N'-[(2-methyl-1-naphtyl)methoxy]-1,2,5-oxadiazole-3-carboximidamide, and 4-amino-N'-(benzyloxy)-N-(2-methylphenyl)-1,2,5-oxadiazole-3-carboximidamide.
  3. The pharmaceutical composition of claim 1, wherein the guanidine derivative is selected from the group consisting of N-{(2,3-dihydro-1,4-benzodioxine-6-ylamino)[(6-methyl-4-oxo-1,4-dihydro-2-pyrimidinyl)amino]methylene}benzamide, N-{(2,3-dihydro-1,4-benzodioxine-6-ylamino)[(6-oxo-4-propyl-1,6-dihydro-2-pyrimidinyl)amino]methylene}-4-methoxybenzamide, N-{[(2,4-dimethylphenyl)amino][(4-oxo-1,4,5,6,7,8-hexahydro-2-quinazolinyl)amino]methylene}benzamide, N-{amino[(4,6-dimethyl-2-quinazolinyl)amino]methylene}-2-(4-chlorophenyl)acetamide, N,N'-{[(4,6-dimethyl-2-quinazolinyl)amino]methylidene}dipropanamide, N-(3,4-dichlorophenyl)-2-[(4,6-dimethyl-2-pyrimidinyl)amino]-3a,4,5,6,7,7a-hexahydro-1H-benzimidazole-1-carboxamide, N-{[(2,4-dimethylphenyl)amino][(4-oxo-1,4,5,6,7,8-hexahydro-2-quinazolinyl)amino]methylene}benzamide, N-(3,4-dimethylphenyl)-N'-{imino[(4,6,7-trimethyl-2-quinazolinyl)amino]methyl}thiourea, and 2-(1,3-benzothiazole-2-ylamino)-N-(2-chlorophenyl)-6-oxo-1,4,5,6-tetrahydro-4-pyrimidinecarboxamide.
  4. The pharmaceutical composition of claim 1, wherein the amide derivative is selected from the group consisting of 2-anilino-4-(3-furyl)-6-oxo-N-phenyl-1-cyclohexene-1-carboxamide, 2-{[N-(2,3-dihydro-1,4-benzodioxine-6-yl)-N-(methylsulfonyl)glycyl]amino}-N-isobutylbenzamide, N-benzyl-2-{[3-(3-nitrophenyl)acryloyl]amino}benzamide, N-(2-methoxyphenyl)-2-(5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)butanamide, 4-methoxy-N-(2-[5-(2-nitrophenyl)-2-furyl]-1-{[(4-pyridinylmethyl)amino]carbonyl}vinyl)benzamide, 6-({2-[(5-chloro-2-methoxyphenyl)amino]-2-oxoethyl}thio)-5-cyano-N-(2-methoxyphenyl)-2-methyl-4-phenyl-1,4-dihydro-3-pyridinecarboxamide, 2-anilino-3-chloro-N-phenyl-4-(phenylimino)-2-butenamide, and N-(1,3-benzodioxol-5-ylmethyl)-N'-[4-(2-oxo-2H-chromene-3-yl)-1,3-thiazole-2-yl] succinamide.
  5. The pharmaceutical composition of claim 1, wherein the urea derivative and the thiourea derivative are selected from the group consisting of 3-chloro-N-{[2-(2-methoxybenzoyl)hydrazino]carbonothioyl}-1-benzothiophene-2-carboxamide, 6-bromo-2,3,4,9-tetrahydro-1H-carbazole-1-one N-phenylthiosemicarbazone, N-[(cyclopentylamino)carbonothioyl]-4-ethoxy-3-nitrobenzamide, N-({[6-methyl-2-(4-methylphenyl)-2H-1,2,3-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide, N',N'''-1,2-phenylenebis[N-(3-chlorophenyl)urea], and N-({[2-(4-ethylphenyl)-6-methyl-2H-1,2,3-benzotriazole-5-yl]amino}carbonothioyl)-2-nitrobenzamide.
  6. The pharmaceutical composition of any one of claims 1 through 5, further comprising one of pharmaceutically acceptable carrier, diluent, and forming agent.
  7. A method of preventing and treating malaria, the method comprising administering an effective dose of the pharmaceutical composition as described in claim 6 to a mammal.
  8. The method of claim 7, wherein the effective dose comprises a unit dose containing the effective ingredient at an amount between 1 mg and 50 mg, and a total dose per day at an amount between 1 mg and 50 mg.
PCT/KR2009/002114 2008-04-22 2009-04-22 Pharmaceutical composition for preventing and treating malaria, containing compounds that inhibit plasmepsin ii activity, and method of treating malaria using the same WO2009131384A2 (en)

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