KR20090073511A - Malaria therapeutic pharmaceutical composition containing flavonoid compound as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating malaria comprising a flavonoid compound and a derivative thereof as an active ingredient. Synthetic flavonoid compounds according to the present invention has an excellent malaria therapeutic effect and can be effectively used as a therapeutic agent for malaria.

Description

Pharmaceutical composition containing flavonoid compounds for treating malarial disease}

The present invention relates to a pharmaceutical composition for treating malaria comprising a flavonoid compound and a derivative thereof as an active ingredient, and more particularly, confirms the excellent antimalarial effect of three flavone compounds and two flavanone compounds, It relates to a pharmaceutical composition for treating malaria containing as a component.

Malaria is an infectious disease commonly found in tropical and subtropical regions of the Americas, Asia, and Africa. It is a very dangerous disease that kills between 400 and 900 million people annually, killing 1-3 million (Breman, 2001). Human malaria is called Plasmodium falciparum), the P. malaria (P. malariae), P. Ovalle (P. ovale), P. Non-boxes (P. vivax) there is onset by, in particular, it is among the eight P. Shifa room is the most important invention It is reported that infection by this protozoa accounts for about 80% of the total and about 90% of deaths are caused by this protozoa (Mendis et al., 2001).

The first treatment for malaria caused by P. falciparum originated from the use of the 17th-century cinchona tree, whose quinine is very resistant to malaria. It is known to have a therapeutic effect. Since the chemical synthesis of kinine was reported in 1944 (Woodward and Doering, 1944), several different types of antimalarial drugs have been synthesized (Wataya et al., 2005). It is used in the clinic.

However, as P. falciparum becomes more resistant to existing antimalarial drugs, efforts are underway to develop new therapeutics from natural or synthetic compounds (Gessler et al., 1994; Tran et al., 2003). . When introducing the results of the antimalarial effect of the compound similar to the flavonoid compound synthesized in the present invention,

1) 5,7,3-trihydroxy-4 ', 5'-(2 ''''. 2 ''''-dimethylpyran)-8,2'-di (3-methyl-2-butenyl)-(2S )-flavanone, 5,7,3-trihydroxy-4'-methoxy-8,2'-di (3-methyl-2-butenyl)-(2S) -flavanone and 7,3 ', 4-trihydroxy-6- The IC ₅₀ value of methoxy-8,2'-di (3-methyl-2-butenyl) -2S) -flavan is 2.6-3.3 μg / ml (Kanokmedhakul et al., 2004) and 2) (R) -4 ''- The methoxydalbergione, obtusafuran, 7,4'-dihydroxy-3'-methoxyisoflavone and isoliquiritigenin are all reported with IC ₅₀ values of 5.8-8.7 μM (Beldjoudi et al., 2003), and 3) 27 synthetic piperazinyl flavones Has an IC ₅₀ value of 0.18-41.0 μM (Auffret et al., 2007), and 4) reports on the antimalarial effects of 28 natural and synthetic flavonoids (Tasdemir et al., 2006).

In addition to the flavonoid compounds described above, many compounds were isolated from natural products or chemically synthesized to detect antimalarial effects, but P. palmiparum of the flavonoid compounds synthesized in the present invention There are no previous studies on the effects of growth inhibition.

Accordingly, the present inventors conducted a study to find a compound having better side effects and less side effects than conventionally known antimalarial substances. As a result, the present inventors confirmed the antimalarial inhibitory effect of specific flavonoid compounds and derivatives thereof, and continued the study. It was completed.

That is, an object of the present invention is to provide a pharmaceutical composition containing a flavonoid compound and its derivatives that can treat malaria by inhibiting the growth of P. falciparum, which causes malaria.

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating cancer.

It is another object of the present invention to provide a method for preparing flavone and flavanone compounds and derivatives thereof which can treat malaria by inhibiting the growth of P. falciparum which causes malaria.

In order to achieve the above object, according to an aspect of the present invention,

It provides a flavonoid compound consisting of flavones and flavanone derivatives and a pharmaceutical composition for the treatment and prevention of malaria comprising the derivative as an active ingredient. In other words, it is characterized in that it contains a specific flavonoid compound and its derivatives in an amount useful for treating malaria, these compounds can treat malaria by inhibiting the growth of P. falciparum, the main protozoa of malaria.

The chemical formula of the applicable flavonoid compound and its derivatives is as follows:

In the formula, compounds 1 to 3 are flavone derivatives, compounds 4 to 5 are flavanone derivatives, specifically compound 1 is 3-bromo-3,4-dimethoxyflavone, and compound 2 is 7,3 , 4-trimethoxyflavone, compound 3 is 6,7-dimethyl-3,4-dimethoxyflavone, compound 4 is 6-methyl-3,4-dimethoxyflavanone, compound 5 is It is named 7,3,4-trimethoxyflavanone.

According to the second aspect of the present invention,

EC ₅₀ of low FM3A cytotoxicity using 6,7-dimethyl-3,4-dimethoxyflavone as the active ingredient It provides a pharmaceutical composition for preventing and treating cancer, characterized in that it has a value.

According to the third aspect of the present invention, the flavone derivatives of one aspect and the second aspect

(a) synthesizing 2-hydroxykelcon from benzaldehyde and 2-hydroxyacetophenone;

(b) ring-closing the 2-hydroxykelcon;

(c) drying the obtained organic layer with anhydrous forget-me-not, filtration and concentrating under reduced pressure to obtain a residue; And

(d) purifying the obtained residue by silica gel column chromatography to synthesize a flavone derivative; It provides a method for producing a pharmaceutical composition for the treatment and prevention of malaria, characterized in that obtained by performing.

According to the fourth aspect of the present invention, the flavanone derivative of one aspect

Putting 2-hydroxyacetophenone and benzaldehyde in dimethylformamide and refluxing boric acid, piperidine and silicon dioxide at about 120 ° C .;

Checking that the substrate disappeared on thin layer chromatography during reflux, cooling to room temperature, diluting with acetone, and filtering; And

Drying the obtained filtrate with anhydrous forget-me-not and filtering the residue obtained by concentrating the filtrate under reduced pressure by silica gel column chromatography to obtain a flavanone derivative; It provides a manufacturing method characterized in that obtained by performing.

Hereinafter, the present invention will be described in detail.

Flavonoid compounds are one of a large group of polyphenolic secondary metabolites produced in plants. This compound has two aromatic rings bonded to each other with a heterocyclic ring having three carbons, and is largely composed of six compound groups (flavones, flavanones, isoflavones, flavonols, flavanols, and anthocyanins). Divided into Flavonoid compounds are generally known to have antioxidant activity and are effective in the prevention and treatment of various adult diseases. In addition, antibacterial, diuretic, lowering blood pressure, anti-allergic, 瀉 下, and 鎭 痙 effects have been reported (三橋 博 et al., 1985). ).

The present invention finds that some compounds have excellent antimalarial effects in the course of synthesizing flavonoid compounds having the above-mentioned efficacy and studying various pharmacological effects which are not yet known, and are characterized by providing a pharmaceutical composition containing them.

The compounds used in the present invention are obtained by modifying known methods by the following steps:

That is, synthesizing 2-hydroxykelcon; Ring-closing 2-hydroxykelcon; Drying the obtained organic layer with anhydrous forget-me-not, filtration and concentrating under reduced pressure to obtain a residue; And purifying by silica gel column chromatography.

Specifically, the synthesis of 2-hydroxykelcon except for the hydroxyl group substituted in the benzaldehyde and the 2-hydroxyacetophenone position 2 except for the hydroxyl group in the acetone solvent in the reflux of potassium carbonate and chloromethyl methyl ether and protected The hydroxyl group at position 2 of the acetophenone is protected by reacting chloromethylmethyl ether at 0 ° C. using sodium hydride as a catalyst in a tetrahydrofuran solvent; And a second step in which the aldehyde derivative and the acetophenone derivative having the hydroxyl group protected in the first step are added to potassium hydroxide in ethanol to carry out the Klaisen-Schmid condensation reaction. It is preferable to synthesize | combine the flavone and flavanone derivative which have.

2'-hydroxychalcone thus synthesized is dissolved in dimethyl sulfoxide and then refluxed by adding iodine; After confirming that the substrate is lost by the thin layer chromatography, sequentially adding sodium thiosulfate, potassium hydroxide, and ice water; Diluting the obtained aqueous solution with ethyl acetate, washing the organic layer with an aqueous sodium chloride solution, drying with anhydrous forget-me-not, and filtration, and concentrating the filtrate under reduced pressure to obtain a residue; And purifying by silica gel column chromatography (solvent: hexane solution of 10% ethyl acetate) from the obtained residue to obtain a flavone derivative. By performing the flavone derivatives having an antimalarial effect can be synthesized.

In addition, 2-hydroxyacetophenone and benzaldehyde in the dimethylformamide boric acid, piperidine and silicon dioxide and the step of refluxing at about 120 ℃; Checking that the substrate disappeared on thin layer chromatography during reflux, cooling to room temperature, diluting with acetone, and filtering; And drying the obtained filtrate with anhydrous forget-me-not and filtering the residue obtained by concentrating the filtrate under reduced pressure by silica gel column chromatography (solvent: hexane solution of 10% ethyl acetate) to obtain a flavanone derivative. By carrying out, it is possible to synthesize a flavanone derivative having an antimalarial effect.

In the pharmaceutical composition of the present invention, the solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the liquid preparations for oral use include suspensions, contents, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as wetting agents, sweeteners, fragrances, preservatives, and the like.

The effective amount of the compound in the pharmaceutical composition of the present invention can be determined within a wide range depending on the route of administration, the dosage form, the purpose of use, the degree of disease of the patient, and the like. In general, however, the content may be formulated to be administered at a concentration of 1.0-2.0 mg / kg / week, preferably 1.5 mg / kg / week.

Formulations for administering the above compound within the above range may have a conventional form, for example, may be used in the form of a pill or capsule. They can be administered for the treatment of malaria via oral or various parenteral routes of administration, and are suitable for the dosage form and are already well known to those skilled in the art and can be readily selected by those skilled in the art and can be readily selected by those skilled in the art. And the like.

Certain flavonoid compounds of the present invention can treat malaria diseases by inhibiting the growth of P. falciparum protozoa, which is effectively used as an antimalarial agent, and 6,7-dimethyl-3,4- which is a kind of flavone derivative in this process. EC ₅₀ with low FM3A cytotoxicity with dimethoxyflavones It is confirmed that the value and can be provided with a pharmaceutical composition for preventing and treating cancer.

Certain flavonoid compounds of the present invention can treat malaria diseases by inhibiting the growth of P. falciparum protozoa, and thus can be effectively used as antimalarial agents, and certain flavone derivatives can be applied as cancer prevention and treatment agents.

[ Example  1] 2'- Of hydroxycalcon  synthesis

The hydroxyl groups except for the 2-position of 2-hydroxyacetophenone and the hydroxyl group substituted in benzaldehyde are protected by adding reflux with potassium carbonate and chloromethylmethyl ether under acetone solvent, and the hydroxyl group at the 2-position of acetophenone is tetrahydro Sodium hydride was used as a catalyst in a furan solvent to protect chloromethylmethylether by reaction at 0 ° C. The aldehyde derivative and the acetophenone derivative having a protected hydroxyl group were added to potassium hydroxide in 96% ethanol to synthesize 2'-hydroxychalcone through Klaisen-Schmid condensation reaction.

[ Example  2] Synthesis of Flavone Derivatives

Dissolve 2'-hydroxychalcone in dimethylsulfoxide, add iodine and reflux at 150 ° C for 1 hour. After confirming that the substrate disappeared by thin layer chromatography, sodium thiosulfate, potassium hydroxide and ice water were added. The aqueous solution was diluted with ethyl acetate, the organic layer was washed with aqueous sodium chloride solution, dried over anhydrous forget-me-not, filtered and the residue obtained by concentrated under reduced pressure was subjected to silica gel column chromatography (solvent: hexane solution of 10% ethyl acetate) to flavone. A derivative was obtained.

[ Example  3] Flavanon  Synthesis of Derivatives

2-hydroxyacetophenone and benzaldehyde are added to dimethylformamide with boric acid, piperidine and silicon dioxide, and refluxed at 120 ° C. After confirming the disappearance of the substrate on thin layer chromatography, the reaction was cooled at room temperature, then diluted with acetone and filtered. The obtained filtrate was dried over anhydrous forget-me-not, and the filtrate obtained by filtration was concentrated under reduced pressure. The residue obtained was subjected to silica gel column chromatography (solvent: hexane solution of 10% ethyl acetate) to obtain a flavanone derivative.

< Experimental Example  1> Cultivation of Malaria Protozoa

Plasmodium, the malaria protozoa used in this experiment The falciparum (ATCC 30932, FCR-3 strain) was used by modifying the method of Trager and Jensen (Trager et al., 1976).

That is, 5% hematocrit of type A human red blood cells was suspended in RPMI 1640 medium and supplemented with 10% type A human serum activated by heat. Plates were incubated at 37 ° C. in an incubator containing 5% carbon dioxide-5% oxygen-90% nitrogen and the medium was changed daily until 5 every 100 erythrocytes infected with protozoa (5% parasitemia).

< Experimental Example  2> antimalarial effect measurement

Plasmodium cultured in Experimental Example 1 Palmiparum was used to detect antimalarial activity by slightly modifying the following known methods (Kim et al., 1998; 1999).

To this end, the synthesized flavonoid compounds were prepared in various concentrations of dimethylsulfoxide solution, and 5 μl of this solution was added to each well of 24-well multi-dish. Erythrocytes, on the order of 0.3% parasitemia, were added to each well containing 995 μl of medium, resulting in a final hematocrit level of 3%. These plates were incubated at 37 ° C. for 72 hours in an incubator containing 5% carbon dioxide-5% oxygen-90% nitrogen. To measure the antimalarial activity of the samples, thin blood films were made on each culture plate and stained with Giemsa. A total of 10,000 red blood cells per one blood film were observed under a microscope.

All measured sample compounds were measured twice at a final concentration of 5.0-5.8 μg / ml. At the same time, the drug-free control group was also measured and all data were calculated as average values. Parasitemia of the control group was adjusted to 4-5% at 72 hours.

EC ₅₀ The value means the effect of inhibiting the increase of protozoa by 50% compared to the control for 72 hours.

< Experimental Example  3> Culture of Mammalian Tumor Cells

Tumor cells of mice [FM3A cells; wild-type, subclone F28-7 (Yoshioka et al., 1987)] was supplied by the Japanese Cancer Research Resources Bank, and these FM3A cells were cultured in suspension medium at 37 ° C under 5% carbon dioxide conditions. At this time, the cells were cultured in a plastic bottle containing ES medium containing serum of fetal bovine inactivated by 2% heat.

< Experimental Example  4> Mammals On tumor cell lines  Cytotoxicity Measurements for

FM3A cells were grown over about 12 hours twice. Prior to exposure to the sample compound, the cell concentration was adjusted to 5 × 10 ⁴ cells / ml. 995 μl of cell suspension was divided into test plates and various concentrations of compound dissolved in dimethylsulfoxide (5 μl) were added to each well of a 24-well plate.

This plate was loaded at 37 ° C with 5% CO ₂ Under Incubated for 48 hours. All sample compounds were measured twice at each concentration. Cell number was measured by Micro cell counter CC-130 (Toa Medical Electric Co., Japan) and all data were calculated as the average value. EC ₅₀ Values refer to the concentration of compound required to inhibit 50% cell growth in the control group for 48 hours. Selectivity is average EC ₅₀ for FM3A cells The value is divided by the mean EC ₅₀ value for P. falciparum.

The above results are shown in Table 1 below.

Compound number Final concentration (μg / ml) P. falciparum inhibitory effect (% inhibition / EC ₅₀ value) FM3A Cytotoxic EC ₅₀ (μg / ml) Choice index One 5.3 40 Non-toxic - 2 5.8 37 3.2 - 3 5.2 13 2.0 - 4 5.0 100 / 1.6 > 4.8 > 3 5 5.7 36 Non-toxic -

From Table 1, compound 4 (6-methyl-3,4-dimethoxyflavanone) of the compound of the present invention inhibited the growth of P. falciparum by 100% at a final concentration of 5.0 μg / ml and had an EC ₅₀ value. Also shown as very low 1.6 μg / ml showed a very good antimalarial effect.

In particular, this compoundP.EC for Falsiparum₅₀ Value of FM3A cytotoxic EC₅₀ The index of choice divided by the value is> 3, suggesting the possibility of developing a very powerful antimalarial drug.

On the other hand, compound 1 (3-bromo-3,4-dimethoxyflavone). Compound 2 (7,3,4-trimethoxyflavone) and Compound 5 (7,3,4-trimethoxyflavanone) showed similar growth effects of P. falciparum with 36-40%. Only compound 3 (6,7-dimethyl-3,4-dimethoxyflavone) was relatively weak. However, compound 3 is an EC _{50 with} FM3A cytotoxicity. It is evaluated that the mouse tumor cell inhibitory effect is excellent because the value is lower than other compounds.

<References>

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As described above, the specific flavonoid compounds of the present invention can be used effectively as an antimalarial agent by treating the malaria disease by inhibiting the growth of P. falciparum protozoa, and the specific flavone derivatives can be applied as a cancer prevention and treatment agent. Can be.

Claims (9)

Flavonoid compound consisting of flavones and flavanone derivatives and pharmaceutical compositions for malaria treatment and prevention comprising the derivative as an active ingredient. The group of claim 1, wherein the flavone derivative is a group consisting of 3-bromo-3,4-dimethoxyflavone, 7,3,4-trimethoxyflavone and 6,7-dimethyl-3,4-dimethoxyflavone A composition characterized in that it is selected from. The composition of claim 1, wherein the flavanone derivative is selected from the group consisting of 6-methyl-3,4-dimethoxyflavanone, and 7,3,4-trimethoxyflavanone. The pharmaceutical composition of any one of claims 1 to 3, wherein the pharmaceutical composition is formulated at a concentration of 1.0-2.0 mg / kg / week to provide anti-malarial treatment and prophylactic effects by P. palmiparum inhibition. Having a composition. A 6,7-dimethyl-3,4-dimethoxyflavone as an active ingredient, characterized by having a low FM3A cytotoxic EC ₅₀ value, cancer prevention and treatment pharmaceutical composition. The flavone derivatives of any one of claims 1, 2, 4, 5 (a) synthesizing 2-hydroxykelcon from benzaldehyde and 2-hydroxyacetophenone; (b) ring-closing the 2-hydroxykelcon; (c) drying the obtained organic layer with anhydrous forget-me-not, filtration and concentrating under reduced pressure to obtain a residue; And (d) purifying the obtained residue by silica gel column chromatography to synthesize a flavone derivative; Method of producing a pharmaceutical composition for treating and preventing malaria, characterized in that obtained by performing. The method of claim 6, wherein (a) the synthesis of 2-hydroxykelcon is The hydroxyl group except for the 2-position of the hydroxyl group and 2-hydroxyacetophenone substituted in the benzaldehyde is protected by putting reflux with potassium carbonate and chloromethylmethyl ether under acetone solvent, and the hydroxyl group at the 2-position of acetophenone A first step of protecting and reacting chloromethylmethyl ether at 0 ° C. using sodium hydride as a catalyst in a tetrahydrofuran solvent; And And a second step of performing the Klyzen-Schmid condensation reaction by adding potassium hydroxide in ethanol to the aldehyde derivative and the acetophenone derivative having the hydroxyl group protected in the first step. The method of claim 6, wherein steps (b) to (d) Dissolving the 2'-hydroxychalcone of claim 6 in dimethyl sulfoxide and then adding iodine to reflux; After confirming that the substrate is lost by the thin layer chromatography, sequentially adding sodium thiosulfate, potassium hydroxide, and ice water; Diluting the obtained aqueous solution with ethyl acetate, washing the organic layer with an aqueous sodium chloride solution, drying with anhydrous forget-me-not, and filtration, and concentrating the filtrate under reduced pressure to obtain a residue; And Purifying by silica gel column chromatography from the obtained residue to obtain a flavone derivative; Method for carrying out the. The flavanone derivative of any one of claims 1, 3 and 4 Putting 2-hydroxyacetophenone and benzaldehyde in dimethylformamide and refluxing boric acid, piperidine and silicon dioxide at about 120 ° C .; Checking that the substrate disappeared on thin layer chromatography during reflux, cooling to room temperature, diluting with acetone, and filtering; And Drying the obtained filtrate with anhydrous forget-me-not and filtering the residue obtained by concentrating the filtrate under reduced pressure by silica gel column chromatography to obtain a flavanone derivative; Production process characterized in that obtained by performing.