KR20020045821A - Apicidin derivatives which inhibit histone deacetylase activity and cancer metastasis, pharmaceutical compositions containing the same and use thereof - Google Patents

Abstract

PURPOSE: Provided are Apicidin derivatives which inhibit cell growth selectively, histone deacetylase(HDAC) activity concentration dependently and MMP-2 activity effectively. And, provided are pharmaceutical compositions containing them and their use for the inhibition of HDAC and cancer metastasis. CONSTITUTION: Apicidin derivative is represented by the formula(1), wherein R is methoxy group; hydroxy group; C2-C6 dialkylamino group; C2-C6 linear or branched hydroxyalkyl; C3-C6 linear or branched dihydroxyalkyl group; C3-C6 alkoxyalkyl group; and substituted or unsubstituted 5 or 6 membered hetero cyclic compound including 1-3 of hetero atoms selected among unsubstituted or C1-C3 alkyl group substituted N, O, and S.

Description

Apicidin derivatives which inhibit histone deacetylase activity and cancer metastasis, pharmaceutical compositions containing the same and use about

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to apicidin derivatives that exhibit histone deacetylase (hereinafter abbreviated as "HDAC") inhibition and cancer metastasis inhibitory effects, pharmaceutical compositions comprising the same, and their use, specifically As a fungal metabolite isolated from a microorganism of the genus Fusarium as an antiprotozoal agent, an apisidine derivative represented by the following Chemical Formula 1 effectively inhibiting HDAC activity and cancer metastasis activity, and a pharmaceutical composition comprising the same And uses thereof.

<Formula 1>

In Chemical Formula 1

R is a methoxy group; Hydrogen; Hydroxyl group; C2-C6 dialkylamino group; C2-C6 linear or crushed hydroxyalkyl group; C3-C6 linear or crushed dihydroxyalkyl group; C3-C6 alkoxyalkyl group; It is preferably a saturated or unsaturated 5 or 6 membered heterocyclic compound containing 1 to 3 heteroatoms selected from N, O, S substituted with an alkyl group which is unsubstituted or substituted with C1 to C3.

Tumors are a group of cells that exhibit autonomous overgrowth, and are classified into malignant tumors (so-called cancers) and benign tumors that may lead to death. In particular, cancer is a genetic disease caused by mutations in genes such as oncogenes and tumor suppressor genes, which are caused at the cellular level. Cancer gene products (oncogene products) play a role in regulating the signaling system of cell division or differentiation by forming a network in the cells. In other words, if their regulation is abnormal, the cell differentiation process is interrupted and indefinite division continues. If the signal transduction pathway can be reduced normally, an excellent therapeutic effect with fewer side effects can be obtained. It is expected to be.

Currently, the three major treatments for cancer are surgical, pharmacotherapy, and radiotherapy. In reality, therapies such as laser therapy and the like are widely used, but pain, metastasis, etc. Considering this, it is natural that expectations for drug therapy are high. Therefore, although many anticancer drugs have been developed and used to cope with this, none of them can be said to be effective for all kinds of cancers, and they cannot be used alone for a long time due to their strong side effects. Generally used a lot. Therefore, there is a need for the development of new anticancer agents, especially anticancer agents having a high anticancer effect and long-term use in a simple manner.

One of the most important biological properties of cancer is that it can cause metastasis, which is considered to be the biggest obstacle to treating cancer. In fact, about 60% of all solid tumor patients already have cancers that have been clinically metastasized at the time of primary tumor diagnosis, and it is well known that the major cause of death in most cancer patients is cancer metastasis. Neovascularization, which is accompanied by infiltration of local tissues of cancer during metastasis, involves tumor vasculature, and blood vessels newly formed by the tumor are easily invaded by cancer cells. do. In addition, cancer invasion and metastasis processes involve numerous cancer cell surface receptors such as laminin receptors required for adhesion to tissue matrix and basement membrane, type IV collagenase, plasminogen activator and cathepsin Expression of numerous enzymes, growth factors, autocrine motility factors and cancer genes required to dissolve normal tissue epilepsy such as B is required.

Studies on the mechanism of cancer development and cancer metastasis have been carried out mainly in the pure scientific aspects, and in recent years, researches using molecular biological methods have been actively conducted. As a method for treating, preventing and inhibiting cancer from a molecular biological point of view, there are methods using antibodies or peptides, but the effects thereof have been questioned, and the problem of effective drug delivery and in the body during administration There is a lot of research that needs to be solved, such as the problem of producing antibodies. Moreover, the conventional anticancer agent aims at the anticancer effect which suppresses the proliferation of cancer in a primary or metastasis place, and the cancer metastasis suppression effect is not reported as a currently available anticancer agent. Accordingly, it can be seen that the anti-cancer effect and cancer metastasis inhibiting effect are based on different actions, and it is urgent to develop cancer metastasis inhibitors separately from anti-cancer agents.

To date, the expectation for the development of a drug having such a cancer metastasis inhibitory effect is very high, but in practice, there are very few examples of the development of a drug for the purpose of inhibiting cancer metastasis. Sulfated polysaccharides, N-diazoacetylglycine derivatives, noiraminitase and fibronectin degrading enzymes (FNS) have been reported to have such inhibitory effects, but there are no reports of their practical use, and they have anticancer effects themselves. There is no. In addition, Arg-Gly-Asp (RGD) analog, a common binding peptide of the integrin molecule, which is a mediator of cell adhesion, has been focused on the development of cancer metastasis inhibitors. As a result, RGD peptidomimetics, RGD Disintegrin isolated from polymers and snake venom has been developed, but since these peptides and proteins are easily hydrolyzed, they are difficult to orally administer, have very short action half-lives, and shock due to immune activity when intraperitoneally administered. There are side effects that have not yet been developed as a new drug.

On the other hand, uncontrolled degradation of the extracellular matrix and the basement membrane, which is an essential part of the metastatic mechanism, is associated with cancer cell invasion, among which matrix metalloproteinases, especially the main collagen structure of the basement membrane MMP-2, a 72 kDa type IV collagenase (gelatin A) that degrades IV collagen, and MMP-9, a type IV collagenase (gelatin B) of 92 kDa, play an important role in cancer cell metastasis. In addition, the promoter has a binding site for AP-1 such as c-fos and c-jun, and it is known that c-fos and c-jun increase the expression of matrix metalloproteinase (MMP). Therefore, it would be effective to prevent cancer metastasis if it could inhibit the activity of MMP proteins, a kind of collagenase, or inhibit the expression of c-fos and c-jun .

It is also known that histone acetylation and deacetylation play a very important role in the regulation of transcription in eukaryotic cells (Grunstein M., Nature , 389, 349-352, 1997), and trapoxin ( Natural HDAC inhibitors, such as trapoxin, trichostatin A, and depudecin, have been known to have detransforming activity against cancer cells. Among them, the anti-angiogenic activity of dipudecin has been shown both in vivo and in vitro, and cell cycle arrest, changes in gene expression and apoptosis. The cellular response of HDAC inhibitors, including induction of apoptosis, is known.

Accordingly, the present inventors have tried to develop a cancer metastasis inhibitor that can effectively inhibit cancer metastasis by inhibiting the activity of MMP protein and HDAC, and as a result, the fungal metabolite, apisidine derivative, specifically downregulates MMP-2. In addition to significantly inhibit the invasive activity of the cell, and also effectively inhibit the HDAC activity to induce the morphological degeneration and growth inhibition of the cell, and the apisidine derivatives and pharmaceutical compositions comprising the same as HDAC inhibitors and cancer metastasis inhibitors The present invention has been completed by revealing that it can be used as.

It is an object of the present invention to provide HDAC inhibitors and apisidine derivatives as cancer metastasis inhibitors which can inhibit HDAC activity and effectively inhibit cancer metastasis, pharmaceutical compositions comprising them and their use.

1 is a graph showing the effect of apisidine of the present invention inhibits cell growth in H- ras MCF10A cells,

2 is a graph showing the effect of apisidine of the present invention on inhibiting HDAC activity in H- ras MCF10A cells,

3 is a graph showing the number of H- ras MCF10A cells invading polycarbonate filter paper according to the apicidine treatment concentration of the present invention,

4 is a photograph showing the effect of apisidine of the present invention on the morphological changes of H- ras MCF10A cells,

A: control group (H- ras MCF10A not treated with apisidine)

B: Experimental group (H- ras MCF10A treated with apisidine)

C: comparative group (MCF10A)

5 is a graph showing the effect of apisidine of the present invention on the activity of MMP-2 and MMP-9 in H- ras MCF10A cells.

■: MMP-9

●: MMP-2

In order to achieve the above object, the present invention provides apisidine derivatives and pharmaceutically acceptable salts thereof that effectively inhibit HDAC activity and cancer metastasis activity.

The present invention also provides a pharmaceutical composition for HDAC inhibitors, cancer metastasis inhibitors and anti-inflammatory containing the compound as an active ingredient.

In addition, the present invention provides the use of the compounds as HDAC inhibitors, cancer metastasis inhibitors and anti-inflammatory agents.

In addition, the present invention provides the use of the compounds as HDAC inhibitors and cancer metastasis inhibitors.

Hereinafter, the present invention will be described in detail.

The present invention provides an apisidine derivative of formula (1) and a pharmaceutically acceptable salt thereof that effectively inhibit HDAC activity and cancer metastasis activity.

<Formula 1>

In Chemical Formula 1

R is a methoxy group; Hydrogen; Hydroxyl group; C2-C6 dialkylamino group; C2-C6 linear or crushed hydroxyalkyl group; C3-C6 linear or crushed dihydroxyalkyl group; C3-C6 alkoxyalkyl group; It is preferably a saturated or unsaturated 5 or 6 membered heterocyclic compound containing 1 to 3 heteroatoms selected from N, O, S substituted with an alkyl group which is unsubstituted or substituted with C1 to C3.

In Formula 1 , apicidine, in which R is a methoxy group (apicidin, cyclo [NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl]) is Fusarium (Fusarium) It is a fungal metabolite isolated from an antiprotozoal agent in a microorganism of the genus and is structurally very similar to trapoxin, an HDAC inhibitor represented by the following formula (2 ).

<Formula 2>

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Compounds of formula (1 ) may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Organic acids and inorganic acids may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid or phosphoric acid may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, or fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

The present invention also provides a pharmaceutical composition for HDAC inhibitors, cancer metastasis inhibitors and anti-inflammatory containing the compound as an active ingredient.

The apisidine derivatives and pharmaceutically acceptable salts thereof can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

In other words, the apisidine derivatives and pharmaceutically acceptable salts thereof of the present invention can be administered in a variety of parenteral formulations, which, when formulated, are commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants. It is prepared using diluents or excipients. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the apisidine derivatives and pharmaceutically acceptable salts thereof may be used in admixture with various carriers accepted as pharmaceuticals, such as physiological saline or organic solvents, and glucose and sucrose in order to increase stability or absorption. Or carbohydrates such as dextran, ascorbic acid or antioxidants such as glutathione, chelating agents, small molecule proteins or other stabilizers can be used as medicaments.

The effective dose of the apisidine derivative and the pharmaceutically acceptable salt thereof is 0.01-5 mg / kg for injectables and 2-200 mg / kg for oral administration and may be administered 1-6 times per day.

The total effective amount of the apisidine derivative and pharmaceutically acceptable salts thereof in the pharmaceutical composition of the present invention is administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. The concentration of the apisidine derivative and pharmaceutically acceptable salts thereof is determined in consideration of various factors such as the age and health of the patient as well as the route of administration and the number of treatments of the drug. One of ordinary skill in the art will be able to determine appropriate effective dosages for the particular use of the apisidine derivatives and pharmaceutically acceptable salts thereof as pharmaceutical compositions.

In addition, the present invention provides the use of the compounds as HDAC inhibitors, cancer metastasis inhibitors and anti-inflammatory agents.

The inventors conducted the following experiments to determine whether the apisidine derivatives and their pharmaceutically acceptable salts can effectively inhibit cancer metastasis by inhibiting the activity of MMP protein and HDAC activity.

First, the present inventors have found that a spontaneously immortalized (spontaneously immortalized) normal (normal) human breast epithelial cell line, and cell line MCF10A and H- ras are installed using the transformed H- ra s MCF10A cell line cell growth inhibition Bahia when Dean As a result of measuring the effects, it was confirmed that the apisidine treatment inhibited the proliferation of H- ras MCF10A cells in a dose-dependent manner (see FIG. 1 ). Apisidine had a selective effect on cell growth. Only about 50% of H- ras MCF10A cells survived by apisidine treatment, whereas non-transformed MCF10A cells remained 95% after the same treatment. % Survived. From this, the IC ₅₀ value of apicidin for inhibiting 50% growth of H- ras MCF10A cells was set to 190 nM. It can be seen from the above results that the apisidine of the present invention inhibits the growth of H- ras MCF10A cells very effectively.

In order to confirm the possibility of using the apisidine derivative of the present invention as an HDAC inhibitor for the treatment of cancer, the effect of apisidine on the in vitro HDAC activity was investigated. As a result, in the H- ra s MCF10A cells IC ₅₀ values for HDAC activity of Bahia when Dean was 102 nM, a concentration-inhibited HDAC activity of H- ras by MCF10A dependent aspects effectively (see Fig. 2). The inhibitory activity of this apicidine was much higher than sodium butyrate used as a positive control.

As reported previously, genes of the activating ras gene family can induce complex signaling mechanisms that induce a variety of cellular responses including proliferation, differentiation, apoptosis, metastasis and invasion. The results of the present invention show that HDAC is involved in ras signal transduction, considering that unregulated ras activity is the most common genetic defect in human cancer cells. It can be used for use as an HDAC inhibitor.

In order to measure the cancer metastasis inhibiting activity of apicidin on the cells, the present invention investigated the effect of apicidin on the H- ras -induced invasive phenotype of MCF10A cells by extracellular invasive assay. It was confirmed that the number of infiltrated cells was significantly reduced by apicidin in a concentration-dependent manner (see FIG. 3 ). The IC ₅₀ value of the anti-invasive activity of apicidin in H- ras MCF10A cells was 450 nM, and no invasive change was observed in the MCF10A cells not transformed with H- ras by apicid treatment.

Malignant transformation is mainly accompanied by rapid morphological changes in the cells, many HDAC inhibitors are known to revert the morphology of the transformed cells. In order to determine whether apicidin induces transformative deactivation activity in mammalian cells transformed by oncogenes , we have identified a transformed phenotype as a result of the structural activity of the H- ras signal. The effect of apisidine on the morphology of H- ras MCF10A cells was investigated.

As a result, it could be seen that the morphology of MCF10A cells transfected with H- ras by apisidine treatment was apparently similar to MCF10A used as the normal cell comparison group (see FIG. 4 ). Apicidine treatment transformed the spindle-like, foci-formation form of H-ras MCF10A cells into a flat shape similar to that of parental MCF10A, but altered the morphology of parental MCF10A. I didn't let you. Several other HDAC inhibitors, including Trapoxin, Trichostatin A, and Depudecin, are known to restore spindle-like, transformed cells to normal cell form (Yoshida H., Jpn. J. Cancer Res., 83, 324-328, 1992; Sugita K., Cancer Res., 52, 168-172, 1992; Kwon HJ., Proc. Natl. Acad. Scil USA, 95, 3356-3361, 1998). It suggests that the transformation inhibitory activity of the seedin is also due to the inhibition of HDAC.

On the other hand, since it is known that proteolytic enzymes belonging to MMPs play an essential role in cancer invasion, the present inventors have various concentrations to investigate the relationship between MMP-2 and / or MMP-9 in the anti-invasive effect of apisidine. when the Bahia Dean-treated H- ras MCF10A cells, the enzyme activity of MMP-2 and MMP-9 secreted by the enzyme was measured by gelatin gel electrophoresis assay (I aunt g) (A. Moon et al., Int. J. Cancer, 85, 176-181, 2000).

As a result, a significant decrease in MMP-2 activity by apicidine was observed by gelatin zyogram, whereas no change in activity of MMP-9 was observed (see FIG. 5 ). These results indicate that MMP-2 is more closely related to the H- ras induced invasive phenotype than MMP-9 in human breast epithelial cells. In addition, cell invasiveness was almost completely inhibited by apisidine treatment, while MMP-2 activity still remained about 50% of the control group, indicating that MMP-2 was responsible for the antiinvasive effect of apisidine in the cells. It is not the only factor and indicates that other mechanisms of action are involved. The results show that in H- ras MCF10A human pectoral epithelial cells in which MMP-2 is much more involved than MMP-9, the invasive phenotype of the cells can be inhibited by apisidine treatment.

As discussed above, the apisidine derivatives and pharmaceutically acceptable salts thereof of the present invention not only inhibit cell growth and HDAC activity, induce anti-invasive and transformation inhibitory activity, but also effectively inhibit the activity of MMP-2. Therefore, since the cancer cell metastasis inhibiting effect is excellent, a pharmaceutical composition containing the same as an active ingredient may be usefully used as an HDAC inhibitor and a cancer metastasis inhibitor.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Measurement of Cell Growth Inhibition Effect of Apisidine

In order to confirm the effect of the apisidine derivatives of the present invention on the growth of cells, the present inventors have investigated the human breast epithelial cell line MCF10A cell line (Michigan Cancer Foundation, Michigan, USA) and H- ras transfected with the cell line H-. ras MCF10A cell line (Moon et al., Int. J. Cancer , 85, 176-181, 2000) was used. First, MCF10A and H- ras MCF10A cells with 5% horse serum (horse serum), 0.5 ㎍ / ㎖ hydrocortisone (hydrocortisone), 10 ㎍ / ㎖ insulin, 2 ng / ㎖ EGF, 0.1 ㎍ / ㎖ cholera enterotoxin (enterotoxin ), 2 mM L-glutamine, 100 units / ml penicillin-streptomycin and 0.5 μg / ml fungizone were incubated in DMEM / F12 medium. The cells were cultured in a humid atmosphere of 37 ° C., 5% CO ₂ , and apicidine was supplied from Professor Lee In-won of Seoul National University and dissolved in DMSO (dimethyl sulfoxide).

Cells at 1.5 x 10 ⁵ cells / ml were added to 48-well plates, and then cultured for 48 hours in the presence of 0.16 to 1.6 μM of apisidine dissolved in DMSO, and control cells were treated with DMSO only. Cells were trypsinized after apicidine treatment and cell viability was measured by Trypan Blue exclusion assay, where the cell number was counted using a hemacytometer. From this, the survival rate of the apisidine treated cells relative to the control cells was obtained and the results are expressed as a percentage.

As a result, proliferation of H- ras MCF10A cells was inhibited in a concentration-dependent manner by 48 hours of apisidine treatment ( FIG. 1 ). The inhibitory effect of apisidine on cell growth was selective. Only about 50% of H- ras MCF10A cells survived by 160 nM of apisidine treatment, whereas non-transformed MCF10A cells survived. 95% survived after the same treatment. From this, the IC ₅₀ value of apicidin for inhibiting 50% growth of H- ras MCF10A cells in the present invention was set to 190 nM.

From the above results, it was found that the apisidine of the present invention inhibits the growth of H- ras MCF10A cells very effectively.

Example 2 Measurement of the Effect of Apicidine on Extracellular HDAC Activity

In order to confirm the possibility of using the apisidine derivative of the present invention as an HDAC inhibitor for the treatment of cancer, the effect of apisidine on the in vitro HDAC activity was investigated.

Extracellular activity of human HDACs was secreted from [ ³ H] acetylated histones according to Inoue and Fujimoto's methods (Inoue A., Jufimoto D., Biochim. Biophys. Acta, 220, 307-316, 1970). It was measured as the amount of acetic acid to be [ ³ H] acetic acid. Briefly, cells of 7 × 10 ⁸ cells / ml concentration are dissolved in HDAC buffer (pH 7.5) containing 15 mM potassium phosphate, 5% glycerol and 0.2 mM EDTA, followed by nuclei. The nuclei were recovered and redissolved in HDAC buffer containing 1 M (NH ₄ ) 2 SO ₄ . Protein was precipitated by (enzyme fraction) was dialyzed dissolved in HDAC buffer solution, ^[3 H] acetylated histone to 1 × 10 was prepared from ⁸ cells / ㎖ concentration of cells the Bahia when Din is not added or is added Added to enzyme fraction. The mixture was incubated at 37 ° C. for 10 minutes, and the reaction was terminated by addition of 10 μl of concentrated HCl, followed by extraction of secreted [ ³ H] acetic acid with 1 ml ethyl acetate to measure radioactivity.

As a result, IC ₅₀ values for HDAC activity of Bahia when Dean from H- ra s MCF10A cells with 102 nM concentration-dependent manner with aspects were effectively inhibit HDAC activity of H- ras cells MCF10A sodium butyrate used as a positive control (sodium butyrate) showed much higher HDAC inhibitory activity ( FIG. 2 ).

Example 3 Determination of Apisidine Inhibition of Cancer Cell Metastasis

In order to investigate the cancer cell metastasis inhibiting ability of the apisidine derivative of the present invention, an in vitro invasion assay, a transformation inhibitory assay (detransformation assay) and gelatin zymography were performed.

<3-1> Extracellular Invasive Assay

Extracellular invasion assays were performed according to previously reported methods using a 24-well transwell unit with a polycarbonate filter (CorningCoster, Cambridge, Mass.) (Moon A. et. al., Int. J. Cancer, 85, 176-181, 2000). Cells of 1.5 × 10 ⁵ cells / ml treated with various concentrations of apisidine were suspended in 100 μl of DMEM / F12 containing the corresponding concentrations of apisidine and then placed on top of the transwell plate. After incubating the cells for 17 hours, transwells were stained and observed at 400-fold magnification under a microscope to determine the invasive phenotype by counting the number of cells moved to the bottom of the filter. Each field was counted in 13 fields and each sample was repeated three times.

As a result, the apicidine of the present invention significantly reduced the number of cells invading through the reconstituted basement membrane in a concentration-dependent manner ( FIG. 3 ). In particular, 480 nM and 1,600 nM in concentration during the Bahia Dean H- ras MCF10A cells, the treatment was a decrease in the number of one compared to the control group in statistically significant invasive cells (P <0.05). The IC ₅₀ value of the anti-invasive activity of apicidin in H- ras MCF10A cells was 450 nM, and no invasive change was observed by apicidine treatment in MCF10A cells not transfected with H- ras . As shown in Example 1, it can be seen that the cells act selectively.

<3-2> Transformation Inhibition Activity Measurement

Malignant transformation is known to be accompanied mainly by rapid morphological changes of the cells. Thus, the present inventors have transformed the apicidine derivatives as a result of the structural activity of the H- ras signal in order to determine whether the induction of transformation deactivation activity in mammalian cells transformed by the oncogene . The effect of apisidine on the morphology of H- ras MCF10A cells showing phenotyping was investigated.

As a result, when 480 nM apisidine was treated for 48 hours, the morphology of MCF10A cells transfected with H- ras was recovered similarly to that of MCF10A used as a normal cell comparison group ( FIG. 4 ). . Apicidine transformed the spindle-like, foci-formation form of H-ras MCF10A cells into a flat shape similar to that of parental MCF10A, but did not alter the morphology of parental MCF10A. .

As a result, the form of MCF10A cells transformed by H- ras by the apisidine treatment of the present invention is apparently recovered similarly to normal cells, and the concentration of the number of cells invading through the reconstituted basement membrane is determined. By significantly reducing the dependent aspect, it was confirmed that the apicidine of the present invention can be effectively used for inhibiting metastasis of cancer cells.

<3-3> gelatin zymogram (enzyme electrophoresis)

In order to investigate how the anti-invasive effect of the apisidine derivatives of the present invention identified in Example <3-1> is related to proteolytic enzymes belonging to MMPs, secreted by apisidine-treated cells The enzymatic activity of the prepared MMP-2 and MMP-9 was measured by gelatin zymogram analysis. 0.16 to 1.6 μM was under Dean Bahia when the presence of the H- ras MCF10A cell density culture for 48 hours, 0.1% Coomassie blue (Coomassie blue) to blue lysis area (lysis area) was stained and discolored with 10% acetic acid Observation as a white band against a colored background. Relative activity of MMP-2 and MMP-9 was determined by measuring the area of white band shown in gelatin zymogram analysis.

As a result, MMP-2 was significantly reduced in MMP-2 by apisidine, whereas no change in activity of MMP-9 was observed ( FIG. 5 ). These results indicate that MM-2 is more closely related to H- ras induced invasive phenotype than MMP-9 in human breast epithelial cells. In addition, in Example <3-1>, the invasion of the cells was almost completely inhibited by 1.6 μM apisidine treatment, but MMP-2 activity remained about 50% of the control group, which was MMP. -2 is not the only factor responsible for the anti-invasive effect of apicidin in these cells, indicating that other mechanisms of action are involved.

That in the above results, MMP-9 than can at least in part, MMP-2 is effectively inhibited the expression of the invasive Bahia when Dean cells transfected in a much more involved in the expression and H- ras transfected invasive human MCF10A breast epithelial cells Shows.

Example 4 Parenteral Administration Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended parenterally with apisidine of the present invention in 0.5% methylcellulose solution at a dose of 1 g / kg / 15 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, the apicidine of the present invention did not show a toxic change in rats up to 100 mg / kg, and the parenteral administration minimum dose (LD ₅₀ ) was determined to be a safe substance of 1,000 mg / kg or more.

Preparation Example 1 Manufacturing Method of Syrup

A syrup containing an apisidine derivative of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts, saccharin and sugars of the apisidine derivatives were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Apisidine derivatives or Apisidine derivatives, hydrochloride ... 2 g

Saccharin 0.8 g

25.4 g of sugar · ・ ・ ・ ··················

Glycerin ... 8.8 g

Spices ················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ······················

Preparation Example 2 Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

Apicidine derivatives or 250 g of apisidine derivatives and hydrochlorides were mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Apisidine derivatives or Apisidine derivatives, hydrochloride 250 g

Lactose ............... 175.9 g

Potato starch · 180 g ················

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch ... 160 g

Talc · 50 g

Magnesium stearate ... 5 g

Preparation Example 3 Manufacturing Method of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

Apicidine derivative or 1 g of apisidine derivative, hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Apisidine derivatives or Apisidine derivatives, hydrochloride ... 1 g

Sodium Chloride ·············· 0.6 g

Ascorbic acid 0.1 g

Distilled water ······················

As described above, the apisidine derivatives of the present invention selectively inhibit cell growth, concentration-dependently inhibit the enzyme activity of HDAC, induce anti-invasive and transformation inhibitory activity, as well as inhibit the activity of MMP-2. Since it effectively inhibits, it can be usefully used as an HDAC inhibitor and a cancer metastasis inhibitor.

Claims (5)

Apicidine derivatives represented by the following general formula (1) having HDAC inhibitory activity and cancer metastasis inhibitory activity and pharmaceutically acceptable salts thereof. <Formula 1> In Chemical Formula 1 R is a methoxy group; Hydrogen; Hydroxyl group; C2-C6 dialkylamino group; C2-C6 linear or crushed hydroxyalkyl group; C3-C6 linear or crushed dihydroxyalkyl group; C3-C6 alkoxyalkyl group; Saturated or unsaturated 5- or 6-membered heterocyclic compounds containing 1 to 3 heteroatoms selected from N, O and S unsubstituted or substituted with C1 to C3 alkyl groups. 2. Api according to claim 1, wherein R is an apicidine (cyclo [NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl]) which is a methoxy group. Cydine derivatives and pharmaceutically acceptable salts thereof. A pharmaceutical composition for inhibiting HDAC, comprising the apisidine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for inhibiting cancer metastasis comprising the apisidine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. An anti-inflammatory pharmaceutical composition comprising the apisidine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.