KR20050055415A - Anticancer agent containing benzimidazole derivatives as an effective component - Google Patents

Abstract

The present invention relates to an anticancer agent containing a benzimidazole derivative as an active ingredient, and provides an anticancer agent containing a compound represented by the following Chemical Formula 1 having proton pump inhibitory activity as an active ingredient:

<Formula 1>

Wherein R ¹ and R ² are each independently hydrogen, methoxy, difluoromethoxy, ethoxy, ethoxycarbonyl; R ³ to R ⁵ are each independently hydrogen, methyl, methoxy, ethoxy, trifluoroethoxy.

An anticancer agent containing a compound of the present invention as an active ingredient selectively kills only tumor cells and thus can be used as a useful anticancer agent.

Description

Anticancer agent containing benzimidazole derivatives as active ingredient {ANTICANCER AGENT CONTAINING BENZIMIDAZOLE DERIVATIVES AS AN EFFECTIVE COMPONENT}

The present invention relates to an anticancer agent containing a benzimidazole derivative as an active ingredient, and more particularly, to an anticancer agent containing a benzimidazole derivative having a proton pump inhibitory activity as an active ingredient.

Cancer, along with acquired immunodeficiency syndrome (AIDS), is one of the incurable diseases that modern medicine remains to address. Although the incidence and frequency of cancers are increasing year by year in Korea, cancer is a major cause of death due to its low healing rate. The world is making huge investments to cure cancer, but no satisfactory treatment is being developed. Therefore, it is essential to develop cancer drugs with excellent medicinal effects and fewer side effects for more efficient and effective healing.

Most anticancer agents are drugs that interfere with various metabolic pathways of cancer cells and mainly inhibit the synthesis of nucleic acids or exhibit anticancer activity. However, these anticancer agents not only act selectively on cancer cells but also damage normal cells, particularly tissue cells with active cell division, resulting in various side effects such as bone marrow dysfunction, gastrointestinal disorders, and alopecia.

Currently, local therapies such as surgery, radiation, and systemic therapies such as chemotherapy and immunotherapy are used to treat cancer. Among them, chemotherapy is widely used as an adjuvant of topical therapy or monotherapy at the time of metastasis of various tumors and solid tumors that occur primarily in various organs including gastrointestinal, liver and lungs. As chemotherapy by anticancer drugs will continue to play a key role in cancer treatment in the future, it is required to develop an ideal anticancer agent that can selectively select and destroy only cancer cells without toxicity to normal cells.

Proton pump inhibitors (PPIs), on the other hand, are potent gastric acid secretion inhibitors and are used for the treatment of gastric acid related diseases in humans, such as, for example, gastric ulcer and duodenal ulcer. Benzimidazole derivatives that exhibit proton pump inhibitory activity include pantoprazole (European Patent No. 166 287), Omeprazole (European Patent No. 5129), Lansoprazole (European Patent No. 174 726), and Reminoprazole (England Patent No. 2 163 747), all of which are only disclosed as potent gastric acid secretion inhibitors, and there are no reports showing anti-cancer activity related to growth inhibition and death of cancer cells.

The present invention has been made in view of the above requirements, and an object of the present invention is to provide an anticancer agent containing a benzimidazole derivative having a proton pump inhibitory activity excellent in cancer cell selectivity as an active ingredient.

In order to achieve the above object, the present invention provides an anticancer agent containing a compound represented by the following formula (1) as an active ingredient.

Where

R ¹ and R ² are each independently hydrogen, methoxy, difluoromethoxy, ethoxy, ethoxycarbonyl;

R ³ to R ⁵ are each independently hydrogen, methyl, methoxy, ethoxy, trifluoroethoxy.

Hereinafter, the present invention will be described in detail.

The inventors have found that cancer cells are more resistant than normal cells in acidic conditions, ie, at lower pH, through experiments at various pHs (see FIG. 1). From these facts, immunocytochemical staining with anti-proton pump antibodies assuming that the ability to disperse H ⁺ in cancer cells will be better, resulting in a more abundant proton pump in cancer cells than normal cells. It was found (see FIG. 2).

Accordingly, the present inventors have administered a benzimidazole derivative, such as pantoprazole, which is well known as a proton pump inhibitor, to normal cells and cancer cells, in the judgment that inhibiting the proton pump may selectively kill cancer cells. As a result, cell growth was not inhibited in normal cells, but cell growth was inhibited and killed in cancer cells.

Accordingly, the present invention is characterized by providing an anticancer agent containing a compound represented by the following formula (1) having a proton pump inhibitory activity as an active ingredient among benzimidazole derivatives.

(Formula 1)

Where

R ¹ and R ² are each independently hydrogen, methoxy, difluoromethoxy, ethoxy, ethoxycarbonyl;

R ³ to R ⁵ are each independently hydrogen, methyl, methoxy, ethoxy, trifluoroethoxy.

The active ingredient of the anticancer agent according to the present invention may be used without limitation as long as it is a benzimidazole derivative of Formula 1 having a proton pump inhibitory activity.

Specifically, the active ingredient of the anticancer agent according to the present invention is

5-difluoromethoxy-2 [[((3,4-dimethoxy-2-pyridinyl) methyl] sulfinyl] -1H-benzimidazole (pantoprazole),

5-methoxy-2 [[(4-methoxy-3,5-dimethyl-2-pyridinyl) methyl] sulfinyl] -1H-benzimidazole (omeprazole),

2-[[[3-methyl-4- (2,2,2-trifluoroethoxy) -2-pyridinyl] methyl] sulfinyl] -1H-benzimidazole (lansoprazole),

2-[[(2-pyridinyl) methyl] sulfinyl] -1H-benzimidazole (timomozol),

5-ethoxycarbonyl-6-methyl-2 [[(3-methyl-2-pyridinyl) methyl] sulfinyl] -1H-benzimidazole (picoprazole),

Rameprazole

It is preferable that it is and most preferable is pantoprazole.

Double pantoprazole, omeprazole and lansoprazole are represented by the following Chemical Formulas 2 to 4, respectively.

In the present invention, whether the compound of Formula 1 according to the present invention exhibits an anticancer effect was confirmed by cell growth rate inhibition and cancer cell death experiment of cancer cells.

Specifically, in one embodiment of the present invention by treating pantoprazole to normal cells and cancer cell lines, it was examined whether the growth of cancer cells is inhibited. As a result, in the normal cell line, there was no significant difference in the growth rate between the group to which the pantoprazole was administered and the group not to be administered (see FIG. Although there was no difference, it was confirmed that the pantoprazole-administered group inhibited the growth rate of cancer cells sensitive to pH reduction (see FIG. 3A).

Apoptosis is a type of genetically programmed cell death in which cells such as DNA fragmentation, phospholipid distribution, and permeability change of cell membranes are observed. In addition, apoptosis is in many cases accompanied by the activation of caspase cascade, many types of caspases involved, but in particular the cleavage of the activation of caspase-3 or its substrate (poly (ADP-ribose) polymerase) This important function is known. Therefore, caspase-3 activation is a major determinant of apoptosis and an important indicator in confirming apoptosis by drugs.

In another embodiment of the present invention, in order to confirm whether the compound of Formula 1 according to the present invention causes cancer cell death, it was confirmed whether the typical phenomenon of the above-described cell death occurs in cancer cells.

First, as a result of observing the regular DNA fragmentation phenomenon by the electrophoresis method, it was confirmed that the DNA fragmentation in cancer cells is increased by the compound of Formula 1 according to the present invention (see FIG. 4A), caspase- in cancer cells- It was confirmed that 3 is activated and its substrate PARP is cleaved (see FIG. 4b), and it is confirmed that there is a change in phospholipid distribution and membrane permeability in cancer cells (see FIG. 4c).

Meanwhile, in another embodiment of the present invention, the anticancer activity of the compound of Formula 1 according to the present invention was investigated for human gastric cancer xenograft in athymic nude mouse. As a result, it was confirmed that the tumor size was reduced by 77.4% in the intraperitoneally administered mice and 44.68% in the intratumorally administered mice compared to the control group on day 22 after xenograft (see FIGS. 5A to 5D).

Therefore, the compound according to the present invention having a proton pump inhibitory activity not only shows a selective anticancer action on cancer cells by inducing apoptosis, but also has an excellent anticancer activity that suppresses tumor growth even through animal testing, which is an effective ingredient. Anticancer agent containing as may be useful for the treatment of cancer, in particular, gastric cancer.

The anticancer agent of the present invention contains a compound having a proton pump inhibitory activity such as pantoprazole, omeprazole, or lansoprazole as an active ingredient in the benzimidazole derivative. The compound may be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical formulation. That is, the compound may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, humectants, disintegrating agents, and surfactants are commonly used. It is prepared by. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin in a mixed herbal extract. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions and syrups, and include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to the commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and 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 the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and gelatin may be used.

The effective amount of the compound of the present invention may be appropriately adjusted according to the age, weight, etc. of a person, but in general, 0.6 mg / kg may be administered once daily.

Hereinafter, the present invention will be described in more detail with reference to 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 Characterization of Cancer Cell Lines

(1-1) Cell Culture

Human gastric cancer cell lines (AGS, KatoIII, MKN-45, MKN-28), human fibroblasts (COS-1), rat normal gastric epithelial cell line (RGM-1) and rat normal intestinal epithelial cell line (IEC-6) Was purchased from the American Cell Line Bank (ATCC), and Korean gastric cancer cell lines, SNU-601 and SNU-1, were purchased from the Korea Cell Line Bank (KCLB).

AGS, KatoIII, MKN-28, MKN-45, COS-1, SNU-601 and SNU-1 were cultured in RPMI 1640 medium (Gibco BRL), and rat normal gastric mucosal cells RGM-1 were 10% FBS (fetal). bovine serum) and 100 units / ml penicillin containing DMEM-F12 medium (Gibco BRL) and IEC-6 cell line was cultured in DMEM-high glucose medium (Gibco BRL) containing 10% bovine insulin.

(1-2) Identification of Acid Resistance of Cancer Cell Lines and Immunocytochemical Staining

Cells cultured in Example (1-1) were cultured on media having different pH, such as 7.4, 6.9, 6.5, 5.9, and 5.4, and cell viability was examined by MTT asssay method. As a result, as shown in Figure 1, normal cells (RGM-1, IEC-6, and COS-1), as the pH decreases, especially since pH 5.9, the cell viability decreased rapidly, while cancer cell lines (AGS, Kato III, MKN-45, MKN-28, AGS, SNU-601 and SNU-1) were confirmed that there is no rapid change in cell viability like normal cells even if the pH is decreased.

Human gastric cancer cell lines AGS, MKN-45 and rat normal cell lines RGM-1 and IEC-6 were incubated for 24 hours in a curve slide cell culture dish, respectively. After fixing the cells with methanol, the anti-proton pump alpha subunit antibody (Santa Cruz Biotechnology) was diluted to 100 and reacted for 2 hours. Subsequently, the secondary antibody conjugated with Cy3 (Zymed) was reacted for 1 hour and then observed with a fluorescence microscope. As a result of immunocytochemical staining with an anti-proton pump antibody, it was confirmed that the proton pump was more abundant in cancer cells than normal cells (see FIG. 2). Strong expression was confirmed mainly in the cell membrane and cytoplasm of cancer cells.

Example 2 Confirmation of Cancer Cell Growth Inhibition

The inventors injected pantoprazole, a benzimidazole derivative, well known as a proton pump inhibitor, to investigate whether cancer cell growth is inhibited.

Cancer cells and normal cell lines were treated with 50mM pantoprazole (Aitana, Germany; Pacific Pharmaceuticals, Korea) and cultured for 24 hours at each pH of the cell medium, and cell viability was confirmed by MTT assay. As a result, in the normal cell line, there was no significant difference in survival rate between the group administered with pantoprazole and the group not administered (see FIG. 3B). On the other hand, the cancer cell line did not show a significant difference in the survival rate in the group not administered pantoprazole, but the survival rate of cancer cells was inhibited sensitively to the decrease in pH in the group administered with pantoprazole (see FIG. 3A). This is a prodrug in which pantoprazole is converted into an activated form under acidic conditions, indicating that the activated form at low pH significantly inhibits the growth of cancer cells.

Example 3 Confirmation of Cancer Cell Death

The present inventors have found a phenomenon such as DNA fragmentation, activation of caspase-3 or cleavage of PARP (poly (ADP-ribose) polymerase), a substrate associated with apoptosis, phospholipid distribution, and changes in cell membrane permeability. It was investigated whether this also occurs in cancer cells treated with the compound according to the present invention.

(3-1) Genomic DNA Fragmentation Observation

RGM-1 and MKN-45 cells were incubated for 24 hours in the group administered with pantoprazole and the group not administered at pH 7.4 and pH 5.4, respectively. Cells were then lysed in cell lysate (10 mM Tris, 5 mM EDTA, 1% Triton X-100) for 15 minutes. After centrifugation, the supernatant was taken, 0.1 mg / ml of proteinase K was added, and the reaction was performed at 37 ° C. for 1 hour. Thereafter, an equal amount of phenol / chloroform was added to the supernatant to purify the nucleic acid, and two times ethanol and 0.3 M acetic acid were added to precipitate the nucleic acid. The precipitated nucleic acid was dissolved in TE buffer, treated with RNase, and electrophoresed on 1.8% agarose gel to observe fragmentation of genomic DNA.

As a result, as shown in Fig. 4a, fragmentation of genomic DNA was not observed in RGM-1 cells, which are normal cell lines, regardless of pH change or administration of pantoprazole. On the other hand, genomic DNA fragmentation was not observed in pH change in human gastric cancer cell line MKN-45, but significant genomic DNA fragmentation was observed in the group administered with pantoprazole. From this it can be seen that pantoprazole induces apoptosis specifically in cancer cell lines.

(3-2) Observation of caspase-3 and PARP cleavage

Activation of caspase-3, a protease that plays an important role in cell death, and cleavage of PARP, an intracellular matrix, were confirmed by Western blot.

RGM-1 and MKN-45 cells were dosed with 0.3mM and 0.6mM pantoprazole for 16 hours, and the cells were lysed to extract cellular proteins. The extracted protein was electrophoresed at 12% and 8% SDS-PAGE, and then the developed protein was transferred to a PVDF membrane by semi-dry transfer (hoeffer). The membrane was blocked for 1 hour in 5% skim milk, and then reacted with anti-caspase-3 and PARP antibody (Santa Cruze Biotechnology) diluted at 1,1000 for 16 hours at 4 ° C. After washing the membrane with TBST, the secondary antibody diluted 1: 2000 was reacted for 1 hour and observed by enhanced chemiluminescence (ECL).

As a result, as shown in Figure 4b, procaspase-3, a proenzyme form of caspase-3, in the cancer cell line MKN-45 was specifically used in the dose of pantoprazole. Decrease, and its substrate, PARP, was cleaved. From this, it can be seen that pantoprazole activates caspase-3, an apoptosis-related enzyme in cancer cells.

(3-3) Observation of changes in phospholipid distribution and membrane permeability

Apoptosis occurs with various specific phenomena in the cell, among which changes in the permeability of the cytoplasmic membrane and the exposure of the phospholipid phosphatidylserine to the cytoplasmic outer membrane of the cell membrane are observed as an unusual phenomenon. (J Immunol Meth (1995) 184: 39-51; J Exp Med 182: 1545-1556). Therefore, we investigated whether this phenomenon is observed in apoptosis induced by pantoprazole.

RGM-1 and MKN-45 cells were divided into groups administered with and without 0.5 mM pantoprazole and incubated for 16 hours. After removing the cell culture solution and reacting with a dye solution (BD Biosciences) containing Annexin V-FITC and PI (propidium iodide) for 15 minutes, the cells were observed using a phase contrast microscope. As a result, as shown in Figure 4c, it was observed that significantly more cells are stained in the cancer cell line MKN-45 compared to the normal cell line RGM-1. Therefore, it was confirmed that pantoprazole induces a change in the phospholipid of the cell membrane which occurs early in apoptosis specifically to cancer cell lines.

Example 4 Confirmation of Anticancer Effect in Mice

When the compound according to the present invention, specifically, pantoprazole was injected into athymic nude mice, it was investigated whether it influences the growth of human gastric cancer xenograft.

First, the human gastric cancer cell line MKN-45 was suspended in PBS solution to 5 × 10 ⁷ / ml. The cells were injected subcutaneously into each mouse at 5 × 10 ⁶ / site. Thereafter, nine mice per group were randomly assigned. The first group received PBS intratumorally once daily from 14 days after the cancer cells were injected. The second group was intraperitoneally injected with 0.4 mg / kg of pantoprazole once daily from 1 day after the cancer cell injection. The third group received 0.4 mg / kg of pantoprazole intratumorally once daily from 14 days after cancer cell injection. The tumor volume was calculated by measuring the intestinal and short cancer tumors of each mouse in a clipper at two-day intervals. On day 22 after the injection of cancer cells, mice were sacrificed to isolate tumor tissues, and hematoxylin and eosin staining and TUNEL staining were performed on the tissue sections.

As a result, the tumor size decreased by 77.4% in intra-peritoneal (IP) mice and 44.68% in intra-tumoral (IT) mice compared to the control group at day 22 after xenograft. It could be confirmed (see FIGS. 5A and 5B). As a result of H & E staining, it was observed that tumor tissue was significantly reduced and cell necrosis progressed in the center of pantoprazole intraperitoneally or intratumorally (see FIG. 5C). In addition, TUNEL staining, it was confirmed that apoptosis was induced in the group in which pantoprazole was administered intraperitoneally or intratumorally (see FIG. 5D).

As described above, an anticancer agent containing a compound of the present invention as an active ingredient selectively kills only tumor cells and thus can be used as a useful anticancer agent.

1 shows cancer cell lines (Kato III, AGS, MKN-45, MKN-28, SNU-601 and SNU-1) and normal cells (RGM-1, IEC-6 and COS-1) according to the pH change of the culture medium. It is a graph showing cell viability.

Figure 2 is an immunofluorescence staining showing the intracellular expression of the alpha subunit of the proton pump in cancer cell lines (AGS, MKN-45) and normal cell lines (RGM-1, IEC-6).

Figure 3 is when pantoprazole is administered to cancer cell lines (MKN-45, MKN-28, AGS, Kato III, SNU-601 and SNU-1) (A), and normal cells (RGM-1, IEC-6) And cell viability when administered to COS-1) (B).

4A is a gel photograph of genomic DNA fragmentation when pantoprazole was administered to cancer cell lines (MKN-45) and normal cells (RGM-1).

FIG. 4b shows whether caspase-3 and its substrate, PARP (poly (ADP-ribose) polymerase), were cleaved when pantoprazole was administered to cancer cell lines (MKN-45) and normal cells (RGM-1). Western blot picture.

FIG. 4C is a diagram illustrating changes in phosphatidylserine distribution and membrane permeability when pantoprazole was administered to cancer cell lines (MKN-45) and normal cells (RGM-1).

FIG. 5A is a graph showing tumor growth rate of human gastric cancer xenograft when pantoprazole was injected intra-peritoneal (IP) or intra-tumoral (IT) into thymus-free mice.

Figure 5b is a graph showing the shape and size change of human gastric cancer xenografts when pantoprazole is injected intraperitoneally or tumorally into thymus-free mice.

FIG. 5C is a photomicrograph of a cross section of human gastric cancer xenografts with hematoxylin and eosin (H & E) staining when pantoprazole was injected intraperitoneally or intratumorally into mice without thymus.

Figure 5d is a photomicrograph of the degree of cell death by TUNEL staining cross section of human gastric cancer xenograft when pantoprazole is injected intraperitoneally or tumorally into the thymus-free mouse.

Claims (5)

An anticancer agent containing a compound represented by the following formula (1) having a proton pump inhibitory activity as an active ingredient: Formula 1 Where R ¹ and R ² are each independently hydrogen, methoxy, difluoromethoxy, ethoxy, ethoxycarbonyl; R ³ to R ⁵ are each independently hydrogen, methyl, methoxy, ethoxy, trifluoroethoxy. 2. The anticancer agent according to claim 1, wherein the compound is selected from the group consisting of pantoprazole, omeprazole, lansoprazole, timoprazole, picoprazole and lameprazole. 3. The anticancer agent according to claim 2, wherein the compound is pantoprazole. The anticancer agent according to claim 1, wherein the cancer is selected from the group consisting of gastric cancer, liver cancer, brain tumor, breast cancer and skin cancer. The anticancer agent according to claim 1, wherein the cancer is gastric cancer.