KR20140086355A - Compositions for Preventing or Treating Cancer Comprising Colchicine Derivatives - Google Patents

Abstract

The present invention relates to a novel colchicine derivative or a pharmaceutically acceptable salt thereof and to a composition for preventing or treating cancer comprising the same as an active ingredient. The novel colchicine derivative of the present invention significantly reduces the expression of polo-like kinase (Plk-1) and stimulates the apoptosis of cancer cells while markedly suppressing the growth of the cancer cells by activating p21 and p53, thereby being capable of being usefully applied to an effective treatment composition on various cancers.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating cancer comprising, as an active ingredient,

The present invention relates to a composition for the prophylaxis or treatment of cancer, which comprises a novel callicin derivative as an active ingredient.

Plk (Polo-like kinase) regulates meiosis, mitosis and cytoplasmic breakdown in the cell cycle. Plk plays a role in mitosis and cytoskeleton, Plk2 (Snk) plays a role in DNA-damage response, synaptic plasticity, and G1 / S function Plk3 (Fnk, Pnk) plays a role in regulating DNA-damaging responses, synaptic plasticity and G2 / M function. Among them, Plk-1 is most widely overexpressed in various types of cancer cells and is known to induce cancer.

Plk-1 exhibits a low expression level in the G0, G1, and S phases of the cell cycle, and a gradual increase in expression level in the G2 phase.

In the G2 / M phase, the cell cycle is progressed by the signaling of the cell cycle from the beginning to the end by the cdk1 and the cyclin B complex. When mitosis progresses, the cdk1 / Cyclin B complex, which was not activated, is activated by dephosphorylation of Thr14P / Typ15P of cdk1 in the complex of Cdc25 complex, which was humanized by Myt1 / Wee1, to promote mitosis. At this time, Plk-1 is involved in the activation of Cdc25 and in Myt1 / Wee1. In addition, it is involved in the activity of DNA-damaged checkpoints.

Delayed cdk1 activation activates DNA-damaged checkpoints. Among them, activation of p53, which is the most relevant tumor suppressor gene, activation of ATM / ATR and activation of chk1 / chk2 inhibits cdc25 and at the same time activates Myt1 / Wee1, Cell death is caused by the activity of p53 in conjunction with the / M-phase arrest. Thus, inhibition of Plk-1 can be the target of new cancer therapies.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have found that compounds capable of inhibiting the activity of Plk-1 (Polo-like kinase), which is overexpressed in various kinds of cancer cell lines and cause excessive division and proliferation of cancer cells, In order to develop a composition for preventing or treating the disease. As a result, it has been found that the callithine derivative of the above formula (1) significantly inhibits the proliferation of cancer cells by significantly reducing the expression of Plk-1 and activating p21.

It is therefore an object of the present invention to provide a novel calliXin derivative or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a composition for preventing or treating cancer, which comprises a novel callicin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a colchicine derivative or a pharmaceutically acceptable salt thereof,

Formula 1

In the above formula, R ₁ To R ₄ are each independently hydrogen or C ₁ -C ₅ alkyl, R ₅ is phenyl-C ₁ -C ₃ Alkyl, phenyl C ₁ -C ₃ Alkylcarbonyl, phenyl C ₁ -C ₃ Alkoxycarbonyl, bicyclo [4.4.0] dec-2,4,6-enyl (bicyclo [4.4.0] dec-2,4,6-enyl) C ₁ -C ₃ alkoxycarbonyl or fluorenyl fluorenyl) C ₁ -C ₃ alkoxycarbonyl and X is sulfur or oxygen.

The present inventors have found that compounds capable of inhibiting the activity of Plk-1 (Polo-like kinase), which is overexpressed in various kinds of cancer cell lines and cause excessive division and proliferation of cancer cells, In order to develop a composition for preventing or treating the disease. As a result, it has been found that the callithine derivative of the above formula (1), which has not been known so far, significantly suppresses the proliferation of cancer cells by significantly reducing the expression of Plk-1 and activating p21. Therefore, the composition of the present invention can be an effective preventive and therapeutic agent for various cancers induced by Plk-1.

As used herein, the term " alkyl " means a straight or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isobutyl, pentyl or hexyl. C ₁ -C ₅ Alkyl means an alkyl group having an alkyl unit having 1 to 5 carbon atoms, and when C ₁ -C ₅ alkyl is substituted, the number of carbon atoms of the substituent is not included.

The term " phenyl Alkyl " means an alkyl in which the phenyl group is substituted, for example when R ₅ is " phenyl C ₁ -C ₃ Quot; alkyl ", the phenyl group is substituted on the nitrogen atom and C ₁ -C ₃ Alkyl < / RTI >

As used herein, the term " phenylalkylcarbonyl " means a carbonyl group to which an alkyl group substituted with a phenyl group is bonded, and " phenyl C ₁ -C ₃ Alkyl acyl "is a phenyl group substituted with a group C ₁ -C ₃ Alkyl < / RTI > For example, in the case of " phenylmethylacyl ", a phenylethanoyl group is obtained.

The term " alkoxy " in the specification means a radical formed by removing hydrogen from an alcohol, for example, C ₁ -C ₃ Alkoxy means a radical formed by removing hydrogen from an alcohol having 1 to 3 carbon atoms.

As used herein, the term " phenylalkoxycarbonyl " means a carbonyl group in which a phenyl group is substituted with a substituted alkoxy group, and " phenyl C ₁ -C ₃ Alkoxycarbonyl " refers to C ₁ -C ₃ substituted with a phenyl group in the carbonyl Means that the alkoxy group is bonded.

According to a preferred embodiment of the present invention, R < 1 _> To R ₄ are each independently C ₁ -C ₃ alkyl and R ₅ is phenylmethyl, phenylmethylcarbonyl, phenylmethoxycarbonyl, bicyclo [4.4.0] dec-2,4,6-enyl (bicyclo [4.4.0] dec-2,4,6-enyl) C ₁ -C ₃ Alkoxycarbonyl or fluorenyl C ₁ -C ₃ Alkoxycarbonyl, and X is oxygen.

More preferably, the R < _{1 >} To R < ₄ > are methyl.

According to a preferred embodiment of the present invention, the colchicine derivative represented by the formula (1) of the present invention is selected from the group consisting of the compounds represented by the following formulas (2) to (12)

       (2)

       (4)

      (6)

       (8)

       (10)

       Formula 12

More preferably, the colchicine derivative of the present invention is a compound represented by the above formula (4). According to the present invention, the compound (H-18-C4) represented by the above formula (4) arrests the cell cycle through Plk-1 inhibition, activates p21, induces the death of cancer cells through p53 activation, .

The compound of the present invention can 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. As the free acid, inorganic acid and organic acid can be used.

Preferably, the pharmaceutically acceptable salts of the compounds of the present invention are selected from the group consisting of hydrochloride, bromate, sulfate, phosphate, citrate, acetate, trifluoroacetate, lactate, tartrate, maleate, fumarate, But are not limited to, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, gluturonate, ebonate, glutamate, or aspartate salts, And salts formed using various inorganic acids and organic acids. The compounds of the present invention may also exist in the form of solvates (e.g., hydrates).

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a callicin derivative of the present invention, a pharmaceutically acceptable salt thereof or a solvate thereof.

The composition of the present invention may be provided in the form of a pharmaceutical composition for preventing or treating abnormal proliferative vascular diseases. When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a conventional preparation using pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs May be prepared in unit dosage form or may be manufactured by intrusion into a multi-dose container. Typical formulations are, for example, oral (tablets, capsules, powders), intraoral, sublingual, rectal, vaginal, intranasal, topical or parenteral (intravenous, intracameral, intramuscular, subcutaneous And intravenous) administration formulations. For example, a compound according to the present invention may be in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or as an excipient, or in the form of an elixir or suspension containing a chemical that flavors or colors For example, orally, buccally or sublingually. Liquid preparations may contain suspending agents (e. G., A mixture of a semisynthetic glyceride such as methylcellulose, witepsol or apricot kernel oil and a PEG-6 ester, or a mixture of PEG-8 and caprylic / Such as a glyceride mixture, such as a mixture of glycerides (e.g., a mixture of glycerides). It is also most preferred to use it as a sterile aqueous solution form when injected parenterally, for example, intravenously, intraperitoneally, intramuscularly, subcutaneously, and intrathecally, wherein the solution is administered in order to have isotonicity with the blood It may contain other substances (for example, salts or monosaccharides such as mannitol, glucose).

Since the composition of the present invention exhibits cancer cell growth and proliferation inhibition or cancer cell killing efficacy as described above, the pharmaceutical composition of the present invention can be used for various diseases or diseases related to tumors such as gastric cancer, lung cancer, breast cancer, ovarian cancer , Liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer. Preferably, the cancer treated with the composition of the present invention is colon cancer or lung cancer, most preferably colon cancer.

As used herein, the term " prevention " means inhibition of tumor cell formation, and the term " treatment " refers to: (i) And (ii) relief of a disease or disease associated with tumors resulting from the removal of tumor cells.

According to a preferred embodiment of the present invention, the composition of the present invention inhibits expression of intracellular Plk-1 (Polo-like kinase-1). According to the present invention, the composition of the present invention is over-expressed in various cancer cell lines to reduce the expression amount of Plk-1 causing excessive division and proliferation of cancer cells, thereby effectively preventing or treating various cancers having hyperproliferative characteristics .

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a novel colchicine derivative or a pharmaceutically acceptable salt thereof and a composition for preventing or treating cancer comprising the same as an effective ingredient.

(b) The composition of the present invention significantly reduces the expression of Plk-1 (Polo-like kinase) and activates p21 and p53, thereby remarkably inhibiting the proliferation of cancer cells and accelerating the death of cancer cells. Can be usefully used as an effective therapeutic composition.

FIG. 1 is a graph showing the screening results of a compound inhibiting proliferation of human colon cancer cells. FIG. Cells were seeded at 2 x 10 < ⁴ > / well in 96 well plates and the next day treated with compound (10 [mu] M) and incubated at 37 [deg.] C 5% CO ₂ for 48 hours. Cell proliferation was investigated via alamarBlue assay (Fig. 1a). Secondary screening was performed on compounds that were more than 30% effective at 100 nM by diluting the concentration of the compounds showing inhibitory activity over 40% in the primary screening (FIG. 1B).
Figure 2 shows the effect of selected compounds on Plk-2 and p21 expression. Cells were seeded at 6 x 10 < ⁵ > / well in 6 well plates and treated with the selected compounds (1 [mu] M) the following day and then incubated at 37 [deg.] C 5% CO ₂ for 48 hours. Cells were harvested, total protein extracted and marker gene analyzed by Western blotting.
FIG. 3 is a graph showing the effect of the compounds selected in the present invention on the proliferation of human colon cancer cells. Cells were seeded at 2 x 10 ⁴ / well in 96-well plates and the following day treated with callosine (100 nM-1 μM) and incubated at 37 ° C in 5% CO ₂ for 48 hours. Cell proliferation was examined via alamarBlue assay
FIG. 4 is a graph showing the effect of inhibiting PLK-1 expression in HCT116 cells caused by calli. ELISA analysis showed that the concentration of Plk-1 decreased by calli-chin, and that western blotting analysis showed that calli-chin inhibited the expression of Plk-1.
5 is a graph showing the effect of calli chin on the cell cycle of HCT116. FACs analysis confirmed that the G2 / M group was arrested by callosine (Fig. 5A), and the cell cycle marker genes cdk1 / 2 and cyclin B1 were found to be reduced by calliosis by Western blot analysis 5b).
FIG. 6 is a graph showing Western blotting results showing that callicosine induces apoptosis of human colon cancer cells. FIG.
FIG. 7 is a graph showing screening results for selection of a derivative of callicin inhibiting the proliferation of human colon cancer cells. FIG.
FIG. 8 is a graph showing the effect of selected calliXin derivative compounds on the expression of Plk-1 and p21.
FIG. 9 is a graph showing the effect of the selected callicin derivative, H-18-C4, on the proliferation of human colon cancer cells.
FIG. 10 shows that expression of PLK-1 is inhibited by H-18-C4. ELISA analysis showed that the concentration of Plk-1 decreased by calli-chin, and that western blotting analysis showed that calli-chin inhibited the expression of Plk-1.
Fig. 11 shows the effect of H-18-C4 on the cell cycle of HCT116. The FACs analysis confirmed that the G2 / M group was arrested by H-18-C4 (Fig. 11a). Through Western blotting analysis, the cell cycle marker genes cdk1 / 2 and cyclin B1 were detected by H-18-C4 (Fig. 11 (b)).
FIG. 12 shows Western blotting results showing that H-18-C4 induces apoptosis of human colon cancer cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Screening and inhibitory effect of compounds inhibiting the proliferation of colon cancer cells

1) Culture of colon cancer cells

HCT116 was cultured in a 5% CO ₂ incubator at 37 ° C in McCoy's 5A (containing 10% FBD (fetal bovine serum), 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM L-glutamine)

2) Cell proliferation assay (AlamarBlue assay)

HCT116 is seeded at 2 x 10 < ⁴ > / well in a 96-well culture plate and the compound is treated after 24 hours. Forty-eight hours after the compound treatment, 10 μl of alamarBlue reagent was treated in the medium, incubated for 4 hours, and fluorescence was measured under conditions of 530-560 nm excitation.

Inhibitory effect of PLK-1 on drugs inhibiting the proliferation of colon cancer cells

1) Detection of PLK-1 expression level by ELISA

HCT116 was seeded in a 6-well plate at 2 x 10 < ⁵ > / well and the compound was treated after 24 hours. Cells were lysed in SDS lysis buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS (wt / vol), 10% glycerol and 50 mM dithiothreitol) 48 hours after treatment of the compounds. The expression level of PLK-1 was confirmed using a kit containing specific oligos capable of recognizing PLK-1. The extracted mononuclear (10 μg) was tested according to the manufacturer's manual under each condition and confirmed using a Wallac EnVision microplate reader (PerkinElmer, Finland) at 450 nm.

2) Protein extraction and western blotting analysis

HCT116 was seeded at 6 x 10 < ⁵ > / well in 6-well plate s6-well culture plates and treated after 24 hours. Cells were lysed with SDS lysis buffer (62.5 mM Tris-HCL (pH 6.8), 2% SDS (wt / vol), 10% glycerol and 50 mM dithiothreitol) 48 hours after compound treatment. The lysate was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was transferred to a new tube and the protein was quantified using BCA reagent. The protein (20)) was analyzed with a Mini Gel Protein System (Bio-Rad) using SDS polyacrylamide gel (SDS-PAGE) containing 10% acrylamide. The loaded protein was transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Korea) and transferred at 250 mA in a transition buffer (pH 8.3) (containing 25 mM Tris-HCl, 192 mM glycine and 20% methanol). After transfection, the PVDF membrane was blocked with blocking reagent (Amersham Pharmacia Biotech, Korea, TBS-T) for 1 hour at room temperature, washed with primary antibodies (PLK-1, p53, Cyclin B1, CDK1, p21, PARP) were diluted 1: 1000 in BSA / TBS-T buffer, mixed and incubated with O / N at 4 ° C. After washing, the secondary antibody (horseradish peroxidase-conjugated IgG secondary antibody (New England Biolabs, Boston, Mass.)) Was also diluted 1: 5000 in BSA / TBS-T buffer Followed by incubation at room temperature for 2 hours or more. The membranes were then washed and the membranes were detected via a chemiluminescence reaction (ECL plus kit, Amersham Pharmacia Biotech, KOREA) followed by exposing the membranes with Hyperfilm ECL (mersham Pharmacia Biotech, KOREA).

3) Cell cycle progression analysis

HCT116 was seeded at 2 x 10 < ⁵ > / well in a 6-well culture plate and the compound treated after 24 hours. Forty-eight hours after compound treatment, the cells were fixed with 70% EtOH for 30 minutes and treated with 500 μL of PI / RNase, respectively, after 30 minutes. The treated samples were incubated at room temperature for 30 minutes and then measured by FACs.

Experiment result

One. Callchin  Derivatives H-18-C

One) Of calloushen (H11-G04)  Selection

Experiments were conducted to find compounds that inhibited colon cancer cell proliferation in compound libraries. Each compound was fixed to colon cancer cells at a concentration of 10 [mu] M, treated with alamarBlue for 48 hours, and compounds that inhibited by 40% or more were selected (Fig. 1a). Compounds showing inhibitory activity of 40% or more in the primary screening were selected, and the compounds were screened by diluting the concentration at 100 nM for 30% or more (Fig. 1B). In order to select compounds that inhibit the proliferation of colon cancer significantly depending on the concentration, compounds that are overexpressed in various kinds of cancer cells and inhibit Plk-1 that promotes cancer cell formation, they express the expression of Plk-1 (Fig. 2).

We found a compound that inhibits Plk-1 and activates p21 in selected compounds, and the compound was identified as callosine. Therefore, more specifically, the mechanism of inhibition of Plk-1 of callithine was examined.

2) Callous  Colorectal cancer cell proliferation inhibition

<콜치신의 화학구조>

<Chemical structure of callusin>

In HCT116 treated with callosine, cell proliferation was significantly inhibited by concentration (FIG. 3).

3) PLK -1 of Intracellular  Suppression of expression

In addition to inhibiting the proliferation of colon cancer cells by calliosis, overexpression in cancer cells resulted in continuous mitosis, which in turn inhibited the expression of Plk-1, which promotes cancer cell formation (Fig. 4). In addition, when Plk-1 is inhibited, an arrest of the G2 / M group will occur and signal transduction activation of DNA damage checkpoint proceeds together. In order to confirm this, the amount of p21 expression was examined and it was confirmed that signal transduction was significantly activated depending on the concentration (FIG. 4). Therefore, we decided to confirm the apoptosis signaling whether arrest of G2 / M phase due to inhibition of Plk-1 and activation of p53 inhibited by Plk-1.

4) Callous  Cell cycle regulation

Since we observed that Plk-1 was inhibited by callosine, cell cycle progression assay was performed to confirm whether calli-sine regulates cell cycle. It was observed that G2 / M group corresponding to mitosis in the cell cycle was arrested by calli-chin (Fig. 5A). These results indicated that calliosis inhibited the proliferation of HCT116, and that the inhibition of proliferation was due to the G2 / M phase of the cell cycle. FACs analysis confirmed that the G2 / M group was arrested, and that the typical marker genes cdk1 / 2 and cyclin B1 at this time in the signal transduction pathway were reduced by calliosis (Fig. 5B). Thus, it was confirmed that the suppression of cell proliferation by callicosine was due to the G2 / M group of the cell cycle.

5) p53 Induction of apoptosis through activation of

As a result of confirming that apoptosis is induced, it was confirmed that the expression amount of p53 as a marker gene was increased according to the concentration, and it was confirmed that cleavage occurred when cell death was caused in PARP gene in proportion thereto 6). Therefore, Colechin acts as a Plk-1 inhibitor in colorectal cancer cells, inhibiting cell proliferation and inducing G2 / M-phase arrest in the cell cycle through inhibition of Plk-1, Induced apoptosis by activating p53, which was inhibited by.

2. Callchin  The derivative H-18- C4

One) Callchin  The derivative H-18- C4 Selection of

It has been confirmed that Colechicine induces arrest of G2 / M group by inhibiting Plk-1 and induces apoptosis through p53. Therefore, compounds having more excellent Plk-1 inhibitory effect than callicin . Each compound was fixed to colon cancer cells at a concentration of 1 μM, and after 48 hours, the compound was found to inhibit by 40% or more through alamarBlue analysis (FIG. 7A).

Compounds inhibited by more than 40% in the first screening were selected, and the compounds were screened for compounds having an effect of 30% or more at 100 nM by diluting the concentration.

Screening results of colchicine derivatives inhibiting the proliferation of human colon cancer cells No code IC ₅₀ (μM) One H11-G04 4 2 H18-E03 6.3 3 H18-B04 3.7 4 H18-C04 3.7 5 H17-F11 2.8 6 H17-C11 1.5 7 H17-E11 3.2 8 H17-G11 3.7 9 H17-G12 2.6 10 H17-E12 2.9 11 H18-D04 3.7 12 H18-D05 2.6

Inhibition of Plk-1 expression in order to finally select a compound that inhibits the proliferation of colon cancer significantly depending on the concentration, and to select a compound that is overexpressed in various kinds of cancer cells to promote the formation of cancer cells and inhibits Plk-1 Respectively.

We found a compound that inhibits Plk-1 and activates p21 in the selected derivatives (Fig. 8). Among the compounds found, a compound that simultaneously inhibits Plk-1 and p21 is selected as H-18-C4 , An experiment was carried out to determine whether this compound had a better inhibitory effect of Plk-1 compared with colchicine.

 2) Callchin  The derivative H-18- C4 Of colon cancer cell growth inhibition

In HCT116 treated with H-18-C4, a derivative of colchicine, cell proliferation was significantly inhibited in a concentration-dependent manner as in calli-chine (FIG. 9).

3) PLK -1 of Intracellular  Suppression of expression

H-18-C4 inhibited the proliferation of colon cancer cells as well as induced continuous mitosis of cancer cells, thereby inhibiting the expression of Plk-1, which promotes cancer cell formation. In addition, when Plk-1 is suppressed, an arrest of G2 / M group will occur, and signal transduction of the DNA damage checkpoint is progressed simultaneously. To confirm this, the expression level of p21 was examined, (Fig. 10). Therefore, we examined cell death signaling to determine whether G2 / M group was arrested due to the suppression of Plk-1 and whether p53 activated by Plk-1 was activated to induce apoptosis.

4) Cell cycle regulation of H-18- C4

Since we observed that Plk-1 was inhibited by H-18-C4, cell cycle progression analysis was performed to see if the calli cells regulate cell cycle. We observed that the G2 / M group, which corresponds to mitosis in cell cycle, was arrested by H-18-C4. These results indicate that H-18-C4 inhibits the proliferation of HCT116, and the inhibition of proliferation is due to the G2 / M group of the cell cycle. FACs analysis confirmed that the G2 / M group was arrested and CDK1 / 2 and cyclin B1, which are typical marker genes of this period in the signal transduction pathway, were reduced by H-18-C4. Thus, it was confirmed that inhibition of cell proliferation by H-18-C4 was due to the G2 / M group of the cell cycle.

5) p53 Induction of apoptosis through activation of

As a result of confirming the induction of apoptosis, it was confirmed that the expression level of p53, which is a marker gene, increased with the concentration of H-18-C4, and cleavage occurred when PARP gene was apoptotic. (Fig. 12). Therefore, H-18-C4, a derivative of colchicine, acts as a Plk-1 inhibitor in colorectal cancer cells, inhibiting cell proliferation and inducing G2 / M-phase arrest in the cell cycle through inhibition of Plk-1 In addition, it was confirmed that cell death was induced by activating p53, which was inhibited by Plk-1 in cancer cells.

Structure and activity of colchicine derivative compounds number Code rescue data (bp / mp or NMR) IC ₅₀ (μM) One H11-G04

Colchicine 4 2 H18-E03

_{1H NMR (300 MHz, CDCl 3} )
δ 1.68 (m, 1H), 2.13 (m, 1H), 2.38 (m, 1H), 2.47 (m, 1H), 3.68 (s, 2H), 3.73 (s, 3H), 3.89 (s, 3H), 3.93 (s, 3H), 3.99 (s, 3H), 4.62 (m, 1H), 5.98 (d, J = 6.9 Hz, 1H), 6.51 (s, 1H), 6.79 (d, J = 10.5 Hz, 1H ), 7.32 (m, 7H) MS (m / e, M &lt; + &gt;): 475 6.3 3 H18-B04

_{1H NMR (300 MHz, CDCl 3} )
δ 1.70 (m, 1H), 2.20 (m, 1H), 2.41 (m, 2H), 3.41 (d, J = 12.9Hz, 1H), 3.46 (m, 1H), 3.54 (s, 3H), 3.72 ( d, J = 12.9Hz, 1H) , 3.90 (s, 6H), 4.00 (s, 3H), 6.52 (s, 1H), 6.80 (d, J = 10.8Hz, 1H), 7.20 (m, 6H), 7.92 (s, 1H) MS (m / e, M &lt; + &gt;): 447 3.7 4 H18-C04

^{1 H-NMR (300 MHz,} CDCl3) δ 1.71 (m, 1H), 2.19 (m, 1H), 2.28 (s, 3H), 2.41 (m, 2H), 3.40 (d, J = 12.9Hz, 1H) , 3.45 (m, 1H), 3.49 (s, 3H), 3.63 (d, J = 12.9Hz, 1H), 3.91 (s, 3H), 3.92 (s, 3H), 4.00 (s, 3H), 6.53 ( s, 1H), 6.80 (d , J = 10.8Hz, 1H), 7.06 (m, 3H), 7.1, J = 10.8Hz 7.23 (d, J = 10.8Hz, 1H), 7.92 (s, 1H) MS ( m / e, M &lt; + &gt;): 461 3.7 5 H17-F11

1 H-NMR (300 MHz, CDCl 3)
1H), 6.94 (s, 3H), 3.91 (s, 3H), 3.91 (s, 6.88 (t, 1H), 6.95 (d, J = 10.5Hz, 1H), 7.15 (m, 3H), 7.27 (m, 2H), 7.44 (d, J = 10.5 Hz, 1H), 8.07 (s, 1H ), 8.26 (s, IH) Mass (m / e) M: 476 2.8 6 H17-C11

^{1 H-NMR (300MHz, CDCl} 3)
δ 1.74 (m, 1H), 2.26 (m, 1H), 2.49 (m, 1H), 2.52 (m, 1H), 3.65 (s, 3H), 3.90 (s, 3H), 3.94 (s, 3H), 3.98 (s, 3H), 4.45 (m, 1H), 4.91 (d, J = 12.3 Hz, 1H), 5.07 (d, J = 12.3 Hz, 1H), 5.38 (d, J = 7.2 Hz, 1H), 1H), 7.52 (s, 1H), 6.89 (d, J = 10.8 Hz,
MS (m / e, M &lt; + &gt;): 491 1.5 7 H17-E11

^{1 H-NMR (300MHz, CDCl} 3)
δ 1.91 (m, 1H), 2.39 (m, 2H), 2.52 (m, 1H), 3.68 (s, 3H), 3.88 (s, 3H), 3.91 (s, 3H), 3.95 (s, 3H), 4.47 (m, 1H), 4.77 (d, J = 12.0Hz, 1H), 4.97 (d, J = 12.0 Hz, 1H), 6.33 (d, J = 6.9 Hz, 1H), 6.55 (s, 1H), 6.80 (d, J = 10.8 Hz , 1H), 7.08 (d, J = 8.4Hz, 2H), 7.29 (d, J = 10.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.64 ( s, 1 H) MS (m / e, M &lt; + &gt;): 571 3.2 8 H17-G11

^{1 H-NMR (300MHz, CDCl} 3)
δ 1.73 (m, 1H), 2.27 (m, 1H), 2.41 (m, 1H), 2.50 (m, 1H), 3.64 (s, 3H), 3.90 (s, 3H), 3.94 (s, 3H), J = 12.0 Hz, 1H), 5.03 (d, J = 12.0 H, 1 Hz), 5.26 (d, J = 7.2 Hz, 1H) (M, 2H), 7.28 (s, 1H), 6.81 (d, J = 10.8 Hz, 1H, 7.02 (dd, J = 8.7 Hz, 10.8 Hz, 2H) e, M &lt; + &gt;): 509 3.7 9 H17-G12

&Lt; 1 &gt; H NMR (300 MHz, CDCl3)
δ 1.71 (m, 1H), 2.28 (m, 1H), 2.39 (m, 1H), 2.49 (m, 1H), 3.65 (s, 3H), 3.77 (s, 3H), 3.90 (s, 3H), 3.94 (s, 3H), 3.98 (s, 3H), 4.45 (m, 1H), 4.83 (d, J = 11.7 Hz, 1H), 5.01 (d, J = 11.7 Hz, 1H), 5.35 (d, J = 7.2Hz, 1H), 6.53 ( s, 1H), 6.81 (d, J = 10.8 Hz, 1H), 6.86 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 10.8 Hz, 1H), 7.51 (s, 1H). MS (m / e, M +): 521 2.6 10 H17-E12

_{1H NMR (300 MHz, CDCl 3} )
δ 1.88 (m, 1H), 2.39 (m, 2H), 2.54 (m, 1H), 3.66 (s, 3H), 3.90 (s, 3H), 3.91 (s, 3H), 3.97 (m, 4H), (D, J = 10.8 Hz, 1H), 7.25 (m, 2H), 7.51 (m, , 7H). MS (m / e, M &lt; + &gt;): 658 2.9 11 H18-D04

_{1H NMR (300 MHz, CDCl 3} ) δ 1.78 (m, 5H), 2.28 (m, 1H), 2.36 (m, 1H), 2.47 (m, 1H), 2.73 (m, 2H), 3.02 (m, 1H 1H), 3.63 (s, 3H), 3.94 (s, 3H), 3.63 5.33 (d, J = 7.2Hz, 1H), 6.54 (s, 1H), 6.81 (d, J = 10.8 Hz, 1H), 7.16 (m, 4H), 7.26 (d, J = 10.8 Hz, 1H), 7.52 (s, 1H) MS (m / e, M &lt; + &gt;): 545 3.7 12 H18-D05 &Lt; ¹ &gt; H-NMR (300 MHz, CDCl3)
δ 1.72 (m, 1H), 2.27 (m, 1H), 2.40 (m, 1H), 2.48 (m, 1H), 3.66 (s, 3H), 3.80 (s, 6H), 3.90 (s, 3H), (D, J = 7.1 Hz, 1H), 6.45 (s, 1H), 6.53 (s, 3H) , 1H), 6.80 (d, J = 11.0 Hz, 1H), 7.18 (d, J = 8.9Hz, 1H), 7.27 (d, J = 11.0Hz, 8.9 Hz, 2H), 7.50 (s, 1H) MS (m / e, M &lt; + &gt;): 551 2.6

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A colchicine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
Formula 1

In the above formula, R ₁ To R ₄ are each independently hydrogen or C ₁ -C ₅ alkyl, R ₅ is phenyl-C ₁ -C ₃ Alkyl, phenyl C ₁ -C ₃ Alkylcarbonyl, phenyl C ₁ -C ₃ Alkoxycarbonyl, bicyclo [4.4.0] dec-2,4,6-enyl (bicyclo [4.4.0] dec-2,4,6-enyl) C ₁ -C ₃ alkoxycarbonyl or fluorenyl fluorenyl) C ₁ -C ₃ alkoxycarbonyl and X is sulfur or oxygen.
2. The compound according to claim 1, wherein R &lt; 1 _&gt; To R ₄ are each independently C ₁ -C ₃ alkyl and R ₅ is phenylmethyl, phenylmethylcarbonyl, phenylmethoxycarbonyl, bicyclo [4.4.0] dec-2,4,6-enyl (bicyclo [4.4.0] dec-2,4,6-enyl) C ₁ -C ₃ alkoxycarbonyl or fluorenyl C ₁ -C ₃ alkoxycarbonyl, and X is oxygen. Derivative or a pharmaceutically acceptable salt thereof.
3. The compound according to claim 2, wherein R &lt; 1 _&gt; Or R &lt; ₄ &gt; is methyl.
The callusin derivative according to claim 1, wherein the callicin derivative represented by the formula (1) is selected from the group consisting of compounds represented by the following formulas (2) to (12): or a pharmaceutically acceptable salt thereof:
(2)

(4)

(6)

(8)

(10)

Formula 12

The callusin derivative or a pharmaceutically acceptable salt thereof according to claim 4, wherein the callusin derivative is the compound represented by the formula (4).
A pharmaceutical composition for preventing or treating cancer comprising the calli chin derivative according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient.
7. The composition of claim 6, wherein the cancer is colon cancer or lung cancer.
7. The composition of claim 6, wherein said composition inhibits the expression of Polk-like kinase-1 (Plk-1) in the cell.

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