KR20020061846A - Sesquicillin derivatives that regulate cell cycle and cell cycle inhibiting pharmaceutical compositions containing the same as effective ingredients - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for inhibition of a cell cycle containing sesquicillin derivatives and pharmaceutically acceptable salts is provided which can be effectively used in inhibition of the growth of abnormal cells such as cancer cells and treatment and prevention of various diseases containing cancer. CONSTITUTION: The pharmaceutical composition for inhibition of a cell cycle contains sesquicillin derivatives for selectively inhibiting only a G1 group of cell cycles represented by the formula 1 and a pharmaceutically acceptable salt. In formula, R1, R2, R3, R4, R5, R6 and R7 are preferably each H, C1-6 straight or branched alkyl or aryl.

Description

Sesquicillin derivatives that regulate cell cycle and cell cycle inhibiting pharmaceutical compositions containing the same as effective ingredients

The present invention relates to a sesquecillin derivative (Sesqucillin) derivative that regulates the cell cycle and a pharmaceutical composition for cell cycle inhibitors using the same as an active ingredient, specifically, select only G1 phase of the cell cycle that plays an important role in abnormal cell growth process to inhibiting the invention relates to the cell cycle inhibitor pharmaceutical composition for cancer treatment and prevention that the compound acceptable salt thereof with the compound in three skew ache derivatives and pharmaceutical of formula (I) as an active ingredient.

<Formula 1>

In Chemical Formula 1

R1 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R2 is hydrogen, a C _{1 to} C ₆ straight or crushed alkyl group or an aryl group,

R3 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R4 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R5 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R6 is hydrogen, a C _{1 to} C ₆ straight or crushed alkyl group or an aryl group,

R7 is preferably hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group.

The division and differentiation of the cells that make up the body is the basis for maintaining life. In order to function properly, the proliferation and growth of these cells must be regulated by sophisticated intracellular signaling systems. This series of processes is responsible for the various signals (PLC, PKC, Shc, Grb2, Raf, MAPK, MEK, etc.) and molecular mediators (GTP, cAMP, etc.) to deliver to the cell clock in the nucleus. When an abnormality occurs in any part of this process, it is balanced by its own regulatory mechanisms, but in many cases it develops into a disease. In particular, the cell cycle present in the nucleus of the cell is one of the important processes to control the life of the cells.

The cell cycle is divided into four distinct phases, as the clock we use everyday is divided into second hand, minute hand and hour hand. That is, Gap1 (G1), DNA Synthesis (S), Gap2 (G2), and Cleavage (M) are the cells that are present at high density or long term in low concentration growth factors. It enters a growth stop (G ₀ ) state called dormant phase. This series of intranuclear reactions is called the cell cycle.

The clock is vibrated by electric or physical force in the clock, and accordingly, the second hand, minute hand, and hour hand move sequentially to inform us the correct time. To this end, a constant force must be applied to the watch weight, and various accessories that control it constitute the watch. Similarly, the cell cycle has a complex network called checkpoints that allows the cell clock to proceed in exactly the order of G1-S-G2-M. Lack of these checkpoint regulatory mechanisms increases genetic instability, leading to unregulated cell growth and sometimes to inadequate cell growth such as cancer.

Good signaling and nutrition from outside the nucleus increases the cell size of the G1 phase and enters the cell cycle. Cell cycle operation occurs at the G1 restriction point, which is called START in yeast and restriction point in mammalian cells. After this point, if there is no special braking, the cell automatically replicates and divides its genome through four stages of the cell cycle.

Looking at these steps in mammalian cells in detail: As a preparation step to create a new group G1 cell checkpoints exist, at this time it is picked to stop the cell cycle fails to supply sufficient growth factors and nutrients, and the turn of the growth of resting G ₀ state. However, when sufficient nutrition is provided and various growth factors are prepared, the cell cycle proceeds to S phase. At this time, the cell replicates its genome to make two copies of the chromosome, as well as several factors in the cytoplasm to divide into two cells. At the end of S phase, the cell enters the G2 stage known as the second checkpoint. The main mechanism of action during this G2 period regulates DNA replication and completion and prepares for entry into the cleavage phase. Therefore, various factors necessary for the construction of the cell are produced at this time. After the necessary factors for dividing into two cells are made, it proceeds to the M stage, which is the dividing stage where the actual division of the cell occurs. In the M phase, the genome that is replicated in the shortest and most dramatic stage in time is separated into the cell's anode and two daughter cells are made. Losing is a step. This series of processes is a very important process for maintaining the life of cells because all cells go through in order for one cell to grow and divide into two cells. Therefore, the study of the cell cycle and the development of substances regulating them are essential for the study of cell growth mechanisms and the development of a preventive and therapeutic agent for cancer occurring in the cell cycle abnormality ( Science , 274: 1643-1677, 1996).

As mentioned above, the normal cell cycle progression of mammalian cells is determined at the time limit of G1 phase, and abnormal progression at this time point is associated with the development of diseases such as cell aging and cancer. At this restriction point, cyclin D (cyclin D1, D2 and D3) proteins play an important role and bind to cyclin dependent kinase (CDK) 4 or CDK6 to regulate the activity of the enzyme. Cyclin D and CDK4 have the function of phosphorylating Rb (retinoblastoma) protein, a cell cycle brake device, to inactivate it. Therefore, regulating the activity of cyclin D and CDK4 is a very important step in regulating the entire cell cycle. In particular, studies have shown that refractory diseases such as tumors are caused by abnormalities of these genes, and their importance is increasing ( Biotechnol. Prog ., 14: 807-833, 1998).

Accordingly, the present inventors have found that the sesquicillin derivative produced and isolated from the fungus Sesquicillin sp. F60063 (KCTC 0810BP) isolated and identified in soil selectively inhibits G1 phase in the cell cycle. The present invention has been completed by confirming that a pharmaceutical composition comprising a sescuricin derivative as an active ingredient can be developed as a treatment and prevention agent for various diseases including cancer showing abnormal cell growth.

An object of the present invention is to inhibit abnormal cell growth, such as cancer cells, and ultimately seskyucillin (sesquicillin) derivatives that can be used as a treatment and prevention of various diseases including cancer and pharmaceuticals for cell cycle inhibitors using the same as an active ingredient To provide a composition.

Figure 1a is a result of investigating the effect of seskyusulin on the protein expression level of the kinase CDK (cyclin-dependent ptotein kinase) in the present invention, Western blot analysis (western blot analysis),

Figure 1b is the result of investigating the effect of seskyusulin on the expression level of the cyclin (cyclin) protein, a regulatory protein of CDK in the present invention,

2 is a result of Western blot analysis of the effect of seskyusulin of the present invention on the degree of phosphorylation of Rb (retinoblastoma), a regulatory protein of CDK / cycline,

Figure 3 shows the results of investigating the effect of ^seskyusulin of the present invention on the protein expression level of p21 ^{waf / cip1} and p27 ^kip2 which are inhibitors of CDK / cyclin and the protein expression level of p53 that induces apoptosis by Western blot analysis. will be.

In order to achieve the above object, the present invention provides a seskyusulin derivative and a pharmaceutically acceptable salt thereof that selectively inhibit only Gl group in the cell cycle.

The present invention also provides a pharmaceutical composition for the treatment and prevention of cancer using the compound as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention provides seskyusulin derivatives and pharmaceutically acceptable salts thereof that selectively inhibit only Gl phase in the cell cycle.

First, the present invention is a seskyucillin derivative represented by the following formula (1 ) and a pharmaceutically acceptable salt thereof secreted and produced from the fungus Sesquicillin sp. To provide.

<Formula 1>

In Chemical Formula 1

R1 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R2 is hydrogen, a C _{1 to} C ₆ straight or crushed alkyl group or an aryl group,

R3 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R4 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R5 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group,

R6 is hydrogen, a C _{1 to} C ₆ straight or crushed alkyl group or an aryl group,

R7 is preferably hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group.

Representative compounds in the seskyucillin derivative of Formula 1 are R1 = CH ₃ , R2 = CH ₃ , R3 = CH ₃ , R4 = CH ₃ , R5 = CH ₃ COO, R6 = CH ₃ , and R7 = CH ₃ It is a compound having a molecular formula of C ₂₉ H ₄₂ O _{5 as} a sescuryrin represented by the formula (2 ). Sesquicillin is a known compound isolated and identified from Sesquicillin sp. Fungi strains ( J. Microbiol. Biotech. 9: 119-121, 1999) . Selective inhibition of G1 phase only was first identified.

<Formula 2>

The production and separation method of seskyucillin of the present invention,

1) culturing the microorganisms producing sescuricin in a suitable medium;

2) removing the microorganisms from the medium; And

3) isolating sescuricin from the medium from which the microorganisms have been removed.

Step 1) is Seskyusiumum, a fungal strain isolated and identified in soil.Sesquicillin sp.) It is a step of producing sescuryrin from a culture obtained by culturing F60063 (KCTC 0810BP). In a preferred embodiment of the present invention, the fungal strain is cultured on a potato dextrose agar plate (PDA plate) medium to form a surface mycellium, and the surface mycelium is formed of glucose, Inoculated into a medium consisting of tryptone, yeast extract (yeast extract), malt extract (malt extract). After inoculation, the cells were incubated at 25 ° C. for 3 days, and then inoculated in a medium having the same composition as above for 6 days at 25 ° C.

Step 2) is a step of removing the microorganisms from the medium, by using a filtration method (filtration) to remove the micellium from the culture.

Step 3) is a step of separating the seskyucillin from the medium from which the microorganisms have been removed.In a preferred embodiment of the present invention, the medium from which the mycelium is removed in step 2) is adsorbed onto a Diaion HP-20 column. The active material was eluted with methanol. In this case, organic solvents such as ethanol and acetone may be used instead of methanol. After removing methanol under reduced pressure, the organic layer extracted with ethylacetate was concentrated and adsorbed onto silica gel chromatogrphy and activated under the solvent condition of hexane-ethylacetate (3: 2). The material was eluted. At this time, an organic solvent such as methanol, ethanol and acetone may be used instead of ethyl acetate, and hexane-ethylacetate or chloroform-methanol may be used as the solvent condition of the chromatography. The eluted material was adsorbed onto Sephadex LH-20 chromatography, eluted the active fractions with 100% methanol, and finally HPLC (high perforamnce liquid chromatography) was performed using a reverse phase column. . The active material was eluted under acetonitrile solvent conditions containing trifluoroacetic acid (TFA) to obtain 16 mg of the active material in the form of a white powder.

Physical and chemical properties and structure determination of the active material obtained as described above showed a molecular ion peak at m / z 470 (M ⁺ ) by EI-MS analysis and HREI-MS analysis The molecular formula was determined to be C ₂₉ H ₄₂ O ₅ , and the maximum absorption wavelengths in the UV spectrum were observed at 210 nm and 290 nm. 2 carbonyl, 8 quarternary carbon, 4 methine, 8 methylene, and 7 methyl in ¹⁴ C NMR (nuclear magnetic resonance) and DEPT spectra It was confirmed. In addition, two single peaks of 4.45 ppm and 4.18 ppm, shown as terminal methylenes, were observed by the ¹ H NMR spectrum. Finally, as a result of determining the structure of the isolated compound using the HMBC experiment and the database based on the analysis result, the active material isolated from the fungus strain of Securusium is a known substance having the structure of Chemical Formula 2 . It was confirmed that it was sescuricin (see Table 1 ).

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as salts, addition salts formed by pharmaceutically acceptable free acid are 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, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartariac acid, maleic acid, and 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.

In another aspect, the present invention provides a pharmaceutical composition for cell cycle inhibitors for the treatment and prevention of cancer, the seskyusulin derivative of Formula 1 and its pharmaceutically acceptable salts as an active ingredient.

Sescuricin derivatives and pharmaceutically acceptable salts thereof of the present invention can be administered orally or parenterally during clinical administration and can be provided in the form of general pharmaceutical formulations.

Sescuricin derivatives of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used, or Formulated using excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc. are included, and these solid preparations are more excipients at least one of the one or more compounds of formula (I), for example, starch, calcium carbonate (calcium carbonate), can Prepared with a mixture of sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrene talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the seskyucillin derivative of Formula 1 and its pharmaceutically acceptable salt is 10 to 50 mg / kg, preferably 20 to 40 mg / kg. The pharmaceutical composition for the treatment and prevention of cancer comprising the sesquicirium derivative and its pharmaceutically acceptable salts as an active ingredient is 20 to 50 mg / kg, preferably 20 to 30 mg / kg of adult weight per day. It may be administered one to several times.

In order to investigate whether the seskyucillin derivative of the present invention can be usefully used for the treatment and prevention of cancer, the cell cycle is analyzed by treating the cancer cell line and the normal cell line with seskyucillin, which is a representative compound of the sesquicillin derivative, and analyzing CDKs (cyclin The expression levels of -dependent protein kinases and cyclins, CDKs regulatory proteins, were investigated. In addition, the degree of phosphorylation of Rb (retinoblastoma) protein and the expression level of CDK inhibitory protein were analyzed.

In general, CDKs in eukaryotic cells are components of a system that regulates the cell division cycle, whereby each CDK phosphorylates substrate proteins when cells divide according to the phase of the cell division cycle. . In addition, CDKs can function as protein kinases only when proteins called cyclins are bound. The cyclin is gradually increased in concentration at certain stages of the cell cycle and then suddenly broken down by targeted proteolysis at specific times, and this sudden cyclin stops the kinase action of CDK, a cyclin partner. . To date, cyclins in higher animals have been identified for cyclins A, B, C, D, E and F, and cyclin A is known to regulate the S group and cyclins E and D control the G1 group.

Rb protein is a molecule present in large quantities in the nucleus of a mammalian cell and binds to cell cycle regulatory proteins, and its binding capacity depends on the degree of phosphorylation of the Rb protein. That is, when Rb protein is dephosphorylated and becomes hypophosphorylation, it binds to a series of cell cycle regulatory proteins to inhibit the activity of regulatory proteins. When Rb protein is phosphorylated and becomes hyperphosphorylation, It is isolated so that the regulatory protein can function.

In addition, p16 ^INK4 protein and p21 ^{waf / cip1} protein are representative as inhibitory proteins that regulate the activity of CDK kinase. The two inhibitory proteins bind to CDK / cyclin protein in cells and inhibit the phosphorylation activity. Indicates.

Therefore, investigating the effects of the seskyusulin derivatives of the present invention on the expression of CDKs protein, cyclin regulatory protein, and Rb protein may confirm the role of seskyusulin acting on the cell cycle.

Therefore, the present inventors treated the seskyusulin of the present invention to human breast cancer cell line and mouse fibroblast cell line and treated with DMSO as a control at the same time, and then collected the cells by time and compared the cell cycle analysis. The G1 / S phase was specifically arrested only for breast cancer cell lines (see Table 2 and Table 3 ).

In order to identify the cell cycle inhibitory mechanism of sescuricin as described above, the expression level of the protein related to the cell cycle was investigated. Specifically, sesquecillin treatment of human breast cancer cell line and DMSO treatment at the same time control, the cells were collected by time, and protein was separated from the cell line by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blot analysis Western blot analysis was performed.

As a result, the protein expression levels of CDK 1, 2, 4, and 6, which are protein kinase enzymes, did not show a difference in the control cell line treated with seskyusulin (see FIG. 1A ). In contrast, the protein expression levels of cyclins D1, A and E, the regulatory proteins of CDK acting on the G1 and S phases of the cell cycle, were significantly different from those of the control group in the sescuricin-treated cell line. In the case of cyclin D1 acting as a regulatory protein of CDK4 during the progression to the S phase, the amount of expression rapidly decreased from 12 hours (see FIG. 1B ). In addition, in the case of cyclin A, which acts as a regulatory protein of CDK2 in the S phase, the protein expression decreases rapidly from 24 hours after sescuricin treatment. Cycline E, on the other hand, showed a slight decrease from 24 hours.

In addition, to investigate the activity of CDK4 kinase from the cell line, the degree of phosphorylation of Rb protein, an intracellular matrix protein of CDK / cyclin D enzyme, was investigated. Was observed and only the low phosphorylated form was observed at 36 and 48 hours (see FIG. 2 ). This increase in low phosphorylated Rb protein means an increase in CDK4 without enzyme activity.

Finally, we analyzed the amount of p21 ^{waf / cip1} and p27 ^kip2 protein, which are inhibitors of phosphatase, and found that the p27 ^kip2 protein amount was not different between the ^sesequillin treatment group and the control group, whereas the p21 ^{waf / cip1} protein was increased at 24 hours. Since the amount of protein has increased significantly (see FIG. 3 ). In addition, p53 protein, which is known to induce cell death by being expressed by several damaging factors on cells, rapidly increased expression from 24 hours.

From the above results, the pharmaceutical composition comprising the seskyusulin derivatives of the present invention and pharmaceutically acceptable salts thereof as an active ingredient has various diseases including cancers showing abnormal cell growth by selectively inhibiting only G1 phase in the cell cycle. It has been confirmed that it can be developed as a therapeutic and prophylactic for.

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

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

Example 1 Production and Separation of Seskyusulin

<1-1> Production of Seskyusulin

Sesquicillium sp. F60063 (KCTC0810BP) isolated and identified in soil was inoculated onto PDA plate medium and grown at 25 ° C. for 5 days to obtain surface mycelium. For seeding, surface micelles were inoculated into 50 ml of sterile medium consisting of 0.1% glucose, 0.5% tryptone, 0.3% yeast extract, and 0.3% malt extract (pH 6.5) to 25 ml in a 250 ml Erlenmeyer flask. Shake culture was carried out for 3 days at ℃. The seed culture solution was inoculated in 3 L of the same medium and incubated for 6 days at 25 ° C., 150 rpm, and 1.0 vvm in a 5 L fermenter.

<1-2> Seskyucillin Isolation

In order to separate the sescuryrin of the present invention, first, the culture solution was filtered to remove mycelium, adsorbed onto a Diaion HP-20 column, and the active material was eluted with 90% methanol. The solvent was removed under reduced pressure, the active material was extracted with ethylacetate, and the organic layer was separated and concentrated. The concentrated organic layer was adsorbed on silica gel chromatography, and the active fraction was eluted under the solvent condition of hexane-ethylacetate (hexan-ethylacetate, 3: 2). The eluted active fractions were adsorbed onto Sephadex LH-20 chromatography, eluted the active fractions with 100% methanol and finally by HPLC using a reversed phase column to acetonitrile containing 0.05% TFA. The active substance was eluted. The eluted active material was lyophilized to give 16 mg of the active material in the form of a white powder.

Example 2 Physicochemical Properties and Structure Determination of Seskyusulin

In Example 1, the physical and chemical properties of the active material produced and separated from Sescurium F60063 were examined and shown in Table 1 below.

Physicochemical Properties Exterior White powder Molecular formula C ₂₉ H ₄₂ O ₅ EI-MS (M) ⁺ 470 UVλ _max in MeOH (nm) 210, 292 IRν _max in cm (cm ^-1 ) 968, 2935, 2877, 17331670, 1567, 1382, 12411114, 1074, 1027, 985 Solubility Soluble in MeOH, EtOH, Me ₂ CO, EtOAc Hexane, insoluble in water R _f value in silica TLC 0.35 (hexane-EtOAc, 3: 2)

Table 1As shown in EI-MI Analysis result m / z 470 (M⁺), Molecular ion peaks were observed, and HREI-MS analysis showed that the molecular formula was C.₂₉H₄₂O₅The maximum absorption wavelengths in the UV spectrum were observed at 210 nm and 290 nm.¹⁴In the C NMR (nuclear magnetic resonance) and DEPT spectra, two carbonyls, eight quarternary carbons, four methines, eight methylenes and seven methyls Confirmed. Also,^OneTwo single peaks of 4.45 ppm and 4.18 ppm, seen as terminal methylene, were observed by the H NMR spectrum. Finally, the structure of the active substance was identified by using HMBC experiment and database.Formula 2It was confirmed that sesquicillin (sesquicillin) is a known substance having a structure of.

<Formula 2>

Example 3 Cell Cycle Analysis Treated with Seskyusulin

<3-1> Cell Culture

In order to analyze the cell cycle change caused by seskyusulin isolated in Example 1, the following experiment was performed.

Human breast cancer cell line MCF-7 and mouse fibroblast cell line NIH3T3 were cultured in RPMI1640 and DMEM medium containing 10% FBS (fetal bovine serum), respectively. 7.5 ml of the medium was dispensed in a T25 flask and incubated for 12 hours at 37 ° C., and 7.5 μl of DMSO was added to the cell culture medium to be used as a control at a final concentration of 0.1% and dissolved in DMSO at 20 mg / ml. 7.5 μl of sescuricrin was added. After treatment with DMSO and seskyusulin in the control and treatment groups, cells were separated at 12, 24, 36 and 48 hours, respectively, and used for cell cycle analysis.

<3-2> FACS analysis

In Example <3-1>, after removing the medium from the cell lines of the control group and the treatment group cultured by time zone, cells were separated from the flask with trypsin and centrifuged at 300 g for 5 minutes. The separated cells were washed twice with cooled phosphate buffer (PBS) to remove the media components, and treated with 3 ml of 70% ethanol and left at -20 ° C. for 12 hours to fix the cells. Cells fixed with ethanol were centrifuged at 300 g for 3 minutes and washed twice with cooled PBS again to remove residual ethanol. The cells were suspended in 500 μl of PBS, treated with 50 μl of 100 μg / ml RNase A, and reacted at 37 ° C. for 30 minutes. Then, 10 μl of 1 mg / ml propidium iodide was added to stain cell DNA. It was.

Stained cells were measured for the cell cycle of 20,000 cells using a Becton-Dickinson FACS calibur (Becton-Dickinson, San Jose, Calif., USA), and a Becton-Dickinson Modifit cell. Using the cycle analysis program, the amounts of cells present in the G1, S, G2 + M phases of the cell cycle are calculated as percentages, and are shown in Tables 2 and 3 below.

Cell Cycle Analysis of Normal Cell Line NIH3T3 time Cell distribution (%) G1 S G2 / M 12 hours DMSO 42.32 48.54 9.14 Seskyusulin 48.13 40.2 11.67 24 hours DMSO 45.03 46.29 8.67 Seskyusulin 56.48 34.87 6.65 36 hours DMSO 43.46 43.15 13.39 Seskyusulin 60.84 29.04 10.12 48 hours DMSO 41.46 46.5 11.1 Seskyusulin 61.80 28.0 10.2

Cell cycle analysis of breast cancer cell line MCF-7 time Cell distribution (%) G1 S G2 / M 12 hours DMSO 56.58 34.03 8.89 Seskyusulin 68.47 20.43 11.09 24 hours DMSO 58.08 31.06 10.86 Seskyusulin 87.41 6.46 6.13 36 hours DMSO 52.09 36.56 11.35 Seskyusulin 93.83 3.49 2.68 48 hours DMSO 55.62 34.63 9.74 Seskyusulin 92.94 4.34 2.72

As shown in Table 2 , seskyusulin did not affect the cell cycle of the normal cell lines as control, whereas as shown in Table 3 , cell cycle G1 only in breast cancer cell lines by increasing the cells of G1 phase over time. It can be seen that the / S group is selectively arrested. From these results, it was confirmed that seskyusulin can be usefully used for developing various drugs by cell cycle regulation by selectively inhibiting G1 phase in the cell cycle in cancer cells induced by abnormal cell growth.

Example 4 Analysis of Expression of Proteins That Control Cell Cycle

<4-1> Western blot analysis

In order to investigate the cell cycle inhibitory activity mechanism of seskyusulin observed in Example 3, the morphology of the protein that regulates the cell cycle was examined by SDS-PAGE and Western blot analysis.

After culturing the human breast cancer cell line MCF-7 in the same manner as in Example <3-1>, 7.5 μl of DMSO was added to the control medium at a final concentration of 0.1% in the control group and 20 mg / ml in DMSO to the treated group. 7.5 μl of the dissolved sescuricin was added. Cells were isolated at 12, 24, 36 and 48 hours after treatment with DMSO and seskyusulin in the control and treatment groups, respectively.

The isolated cells were treated with Tris-HCl (5 mM), NP-40 (1%), Na-deoxychloate (Na-deoxychloate, 0.25%), NaCl (150 mM), EDTA (1 mM), PMSF (1 mM). ), Na ₃ VO ₄ (1 mM), NaF (1 mM), Aprotinin (Aprotinin, 1 μg / ml), Leupeptin (1 μg / ml), Pepstatin (pepstatin, 1 μg / ml) Treatment with hemolysis solution (lysis buffer) to remove the cell membrane and the total protein was isolated. Proteins were analyzed by electrophoresis on 10% of the total protein in a 12% SDS-polyacrylamide gel. The protein bands of electrophoretic SDS-gels were transferred to PVDF (Roche, Germany) membranes and 1 at 25 ° C. A blocking solution consisting of maleic acid in which 10% casein protein was dissolved was treated for a period of time. After reacting for 2 hours at 25 ℃ by adding a primary antibody of the protein to be irradiated to the PVDF membrane and washed 5 times with TBST (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20) solution It was. After the secondary antibody binding to the primary antibody of the PVDF membrane was added and reacted for 1 hour at 25 ℃, the membrane was washed with TBST solution. The expression level of the protein to be investigated on the PVDF membrane was observed using the chemiluminescence detection kit (Romi, Germany).

<4-2> Analysis of CDK Protein Expression

To investigate the effect of seskyusulin on protein expression of protein kinase, CDK 1, 2, 4 and 6 primary antibodies (anti-rabbit, rabbit produced antibodies, Santa Cruz, USA) were 1: 1000. By diluting at a ratio of, Western blot analysis was performed according to the method described in Example <4-1> to investigate the expression level of CDK, and the results are shown in FIG. 1A . As shown in Figure 1a , the protein expression of CDK 1, 2, 4 and 6 did not show a difference between the cell line treated with seskyusulin and the control cell line.

<4-3 > Analysis of CDK Regulatory Protein Expression

To investigate the effect of seskyusulin on the expression of cyclin protein, a CDK regulatory protein, the primary antibodies of cyclin D1, A and E (anti-rabbit, rabbit-produced antibody, Santa Cruz, USA) were 1: 500. By diluting at a ratio of and performing Western blot analysis according to the method described in Example <4-1>, the expression level of cyclin was investigated.1bShown in1bAs shown in FIG. 2, cyclin D1, which acts as a regulatory protein of CDK4, rapidly decreased in expression of sescuricin-treated groups at 12 hours during the progression from G1 to S phase. In addition, in the case of cyclin A, which acts as a regulatory protein of CDK2 in the S phase, the protein expression decreases rapidly from 24 hours after sescuricin treatment. In the case of cyclin E, on the other hand, only slight decrease was observed from 24 hours.

From the results of Examples <4-2> and <4-3>, the mechanism by which seskyusulin selectively inhibits G1 phase in the cell cycle does not affect the expression of CDK, a phosphatase, but cyclin, a regulatory protein of CDK4. It was found that this was due to the decrease in the expression levels of D1 and A.

<4-4> Analysis of phosphorylation degree of Rb protein

To investigate the effect of seskyusulin on the degree of phosphorylation of Rb protein that binds to cell cycle regulatory proteins and modulates protein kinase activity, the primary antibody of Rb (anti-mouse, Pharminge, USA) is 1: 500. was diluted in a ratio of by the method described in example <4-1> to perform Western blot analysis were investigated the expression level of the cyclin the results are shown in Figure 2. As shown in FIG. 2, in the treatment group treated with seskyusulin, the Rb protein of the highly phosphorylated form was gradually decreased over time, which means that the activity of CDK gradually decreased.

<4-5> Analysis of expression level of CDK inhibitory protein

To investigate the effects of ^seskyusulin on the expression of CDK inhibitory protein and p53 protein that induces cell death, the primary antibodies of p21 ^{waf / cip1} , p27 ^kip2 and p53 (anti-mouse, p21 and p27 antibodies were selected from Transduction laboratory , USA, p53 antibody purchased from Santa Cruz, USA) 1: diluted in a ratio of 1: 1000 Western blot analysis according to the method described in Example <4-1> to express the inhibitory protein and apoptosis protein Was investigated and the result is shown in FIG . As shown in FIG. 3 , the protein expression ^level of p27 ^kip2 was not significantly different between the control group and the control group, but the protein expression ^level of p21 ^{waf / cip1} increased rapidly in the treated group after 24 hours and the protein expression level of p53 was also increased. There was a sharp increase in the treatment group after 24 hours.

These results ^suggest that ^seskyusulin increases p21 ^{waf / cip1} , a CDK inhibitory protein.

Increased p21 ^{waf / cip1 binds} to the CDK / cyclin protein and shows inhibitory activity, suggesting that it selectively inhibits the progression of G1 phase in the cell cycle. In addition, the expression of p53, a tumor suppressor protein, and p21, a CDK inhibitory protein, is increased by seskyusulin, thereby inhibiting the activity of the CDK / cyclin protein acting on G1 phase, thereby selectively inhibiting the progression of G1 phase in the cell cycle. do.

As described above, the sesquicillin derivative of the present invention inhibits the expression of cyclin D1 and phosphorylation of Rb (retinoblastoma) protein, a protein that regulates the progression of G1 to S phase in the cell cycle. In addition, by increasing the expression of p21 ^{waf1 / cip1} , a CDK (cyclin-dependnet protein kinase) inhibitory protein, it was proved to be a substance that selectively inhibits G1 phase only in the cell cycle. Therefore, the pharmaceutical composition for cell cycle inhibitors comprising the sescuricin derivative of the present invention and a pharmaceutically acceptable salt thereof inhibits abnormal cell growth such as cancer cells and ultimately treats and prevents various diseases including cancer. It can be usefully used.

Claims (7)

A sesquicillin derivative and a pharmaceutically acceptable salt thereof that selectively inhibit only G 1 group in the cell cycle represented by the following formula (1 ). <Formula 1> In Chemical Formula 1 R1 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group, R2 is hydrogen, a C _{1 to} C ₆ straight or crushed alkyl group or an aryl group, R3 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group, R4 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group, R5 is hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group, R6 is hydrogen, a C _{1 to} C ₆ straight or crushed alkyl group or an aryl group, R7 is preferably hydrogen, C ₁ ~C ₆ is a straight or branched chain alkyl group or an aryl group. According to claim 1, with _{R1 = CH 3, R2 = CH} 3, R3 = CH 3, R4 = CH 3, R5 = CH 3 COO, R6 = CH 3, and R7 = CH ₃ in the formula 2 of the formula Sescuricin and pharmaceutically acceptable salts thereof represented. <Formula 2> The method of claim 1, wherein by reducing the expression level of cyclin (D1), a cyclin-dependent protein kinase (CDK) regulatory protein, seskyucillin derivatives and pharmacologically characterized by selectively inhibiting G1 phase in the cell cycle Acceptable salts thereof. 2. The seskyusulin derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein the sesquicillin derivative and pharmaceutically acceptable salt thereof are selectively inhibited only during G 1 phase in the cell cycle by hypophosphorylation of Rb (retinoblastoma), a CDK / cycline regulatory protein. The ^ceskyusulin derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein the ^ceskyusulin derivative and pharmaceutically acceptable salt thereof are selectively inhibited only in G1 phase of the cell cycle by increasing the expression ^level of p21 ^{waf / cip1} , which is a CDK / ^cycline inhibitory protein. A pharmaceutical composition for cell cycle decontamination for the treatment and prevention of cancer comprising the seskyucillin derivative of claim 1 and a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for cell cycle decontamination for the treatment and prevention of cancer, comprising the seskyucillin of claim 2 and a pharmaceutically acceptable salt thereof as an active ingredient.