WO2008044903A1

WO2008044903A1 - Anti-cancer agent comprising 3,4-dihydroisoquinolinium salt derivative

Info

Publication number: WO2008044903A1
Application number: PCT/KR2007/005018
Authority: WO
Inventors: Kiwon Song
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2006-10-13
Filing date: 2007-10-12
Publication date: 2008-04-17
Also published as: KR100812032B1

Abstract

The present invention relates to an ant i -cancer agent comprising a 3,4- dihydroisoquinolinium salt derivative. The 3,4-dihydroisoquinolinium salt derivative of the present invention selectively induces cytotoxicity and cell death in the cancer cells with a loss of p53 function due to p53 gene deletions and mutations, thereby being used to treat cancer caused by defects in the p53 gene, while minimizing the side effects to normal cells.

Description

[DESCRIPTION] [Invention Title]

ANTI-CANCER AGENT COMPRISING 3,4-DIHYDROISOQUINOLINIUM SALT DERIVATIVE [Technical Field]

<i> The present invention relates to an anti-cancer agent comprising a 3,4- dihydroisoquinoliniura salt derivative. [Background Art]

<2> Normal tissue homeostasis is achieved by a balance between the rate of cell proliferation and cell death. Aged cells are removed by programmed cell death (apoptosis) in the body, and signals are transduced to trigger cell death by various factors including DNA damage, hypoxia, signaling imbalance provoked by oncogene activation, depletion of survival factors, and alterations in surrounding tissue. If cell death is suppressed, normal cell homeostasis is disrupted, which may lead to cancer.

<3> Mutations of p53 tumor suppressor gene are the most common genetic changes found in cancer caused by suppression of cell death. Normal p53 protein activates a checkpoint that senses abnormalities in cell cycle progression and inhibits cell division, and blocks abnormal cell proliferation to prevent cells from undergoing carcinogenesis. When cancer is induced, p53 protein binds to a promoter of target gene to induce its transcription, thus cell death is induced. Further, ρ53 tumor suppressor gene inhibits cell transformation, and suppresses tumorigenesis of cancer cells (Hinds P. W. et al., J. Viol. 68, 739 (1989)). p53 protein recognizes a specific consensus DNA sequence of p53 promoter region and activates the expression of tumor suppressor genes including p21. Most of mutated p53 proteins found in human tumor cells have loss of transcriptional activation function, which indicates that the transcriptional activation function of p53 plays an essential role in suppressing tumor (Wiman K. G., Exp. Cell Res. 237, 14 (1997); Lane, D. P., 358, 15-16 (1992); Kastan M. B et al . , Cancer Res. 51, 6304-6311 (1991); Kuerbitz S. J. et al . , Proc. Natl. Acad. Sci. USA, 89, 7491-7495 (1992)). <4> Mutated p53 genes are frequently observed in human tumors by 50-60% or more. An oncogenic virus degrades p53 protein or interferes with its function to cause tumor proliferation. Therefore, loss-of-function p53 genes are found in most of cancer cells, and in particular, frequently in liver cancer, lung cancer, and colorectal cancer.

<5> Further, in normal cells, p53 gene plays a pivotal role in preventing DNA from damages by various carcinogens such as radiation and chemicals. When normal cells are treated with γ-ray radiation or DNA damaging agents such as etoposide, cells operate a checkpoint halting cell cycle progression. If the DNA damage proves to be irreparable, apoptosis is initiated. However, in the cells expressing deleted or mutated p53 gene, cell cycle arrest and cell death caused by _Y -ray radiation or the like do not occur. Thus, genome damage and transformation into cancer cells are induced. That is, it can be said that p53 actively regulates cell death. It was found that if normal p53 genes are allowed to be expressed in cancer cells with p53 gene mutations, the cells undergo cell death, which also implies that p53 protein has a function of regulating cell death (Ko et al . , Hum. Gene Ther., 7, 1683-1691, (1996)). Therefore, gene therapy and anti-cancer agents using p53 gene have been actively developed.

<6> In this context, a method for regressing tumor by recombinant adenovirus constructs with normal p53 gene in US patent No. 6,143,290; a method for inhibiting the growth of p53 deficient tumor cells by administering normal p53 gene in US patent No. 6,017,524; and a method for activating p53 protein in WO 94/12202 are disclosed. Further, a pharmaceutical composition for treating tumor cells with p53 gene deletions and mutations, comprising recombinant virus with p53 gene and a pharmaceutically acceptable carrier in US patent No. 5,693,522; and a pharmaceutical composition containing an actin inhibitor for the treatment of cancer lacking functional p53 or p21 gene in Korean patent publication No. 10-2003-85421 are disclosed.

<7> As described above, in the development of gene therapy and anti-cancer agents using p53 gene, p53 genes are inserted into tumor cells with p53 gene deletions and mutations, so as to recover the expression of normal p53 proteins and inhibit abnormal proliferation of tumor cells, thereby treating tumor. That is, for the development of anti-cancer agents, studies have been made on a method of directly introducing normal p53 gene into tumor via adenovirus (Frank et al . , Clin. Cancer Res., 4, 2521-2528 (1998); Yung et al., Proc. Am. Assoc. Cancer Res., 36, 423 (1995); Liu et al . , Cancer Res., 54, 3662-3667 (1994)).

<8> On the other hand, most of anti-cancer agents that have been currently used are cytotoxic substances inhibiting cell proliferation, and affect not only tumor cells, but also normal cells, so as to generate serious side effects. Accordingly, there has been a strong demand for the development of anti-cancer agents, which affect tumor cells, but not normal cells.

[Disclosure]

[Technical Problem]

<9> The present inventors have investigated chemical library of about eighty isoquinoline derivatives exhibiting antifungal activity against Candida albicans in order to identify an anti-cancer agent that targets cancer cells without affecting normal cells, and screened the derivatives displaying selective toxicity to cancer cell lines. They found that among them, a 3,4-dihydroisoquinolinium salt derivative specifically suppresses cancer cells containing deletions and mutations in the p53 gene, so as to induce cell death, thereby completing the present invention.

[Technical Solution]

<io> An object of the present invention is to provide an anti-cancer agent comprising a 3,4-dihydroisoquinolinium salt derivative.

[Description of Drawings]

<π> Fig. 1 is a drawing of cell morphology illustrating cytotoxicity and induction of cell death in cancer cell lines with p53 gene deletions (p53-/- ), a cervical cancer cell line (HeLa) and colon cancer-derived cell line (HCT116/E6), and in cell lines with normal p53 gene (p53+/+) such as a human embryonic kidney cell line (HEK293) and wild type colon cancer-derived cell line (HCTl16), by the treatment with the 3,4-dihydroisoquinolinium salt derivative of the present invention!

<i2> Fig. 2 is a drawing illustrating the result of an MTT assay, in which the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on cell proliferation is examined in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and the human embryonic kidney cell with normal p53 gene (HEK293) (p53+/+);

<13> Fig. 3 is a drawing illustrating the result of DAPI staining method, in which the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on DNA aggregation or the formation of multi-nucleated cell is examined by using a fluorescence microscope in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and the human embryonic kidney cell with normal p53 gene (HEK293) (p53+/+);

<14> Fig. 4 is a drawing illustrating the result of electrophoresis, in which the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on DNA fragmentation is examined by the detection of DNA fragmentation in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and the human embryonic kidney cell with normal p53 gene (HEK293) (p53+/+);

<i5> Fig. 5 is a drawing illustrating the result of flow cytometry, in which the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on cell cycle distribution is examined in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and the human embryonic kidney cell with normal p53 gene (HEK293) (ρ53+/+);

<16> Fig. 6 is a drawing illustrating the result of karyotype analysis, in which the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and the human embryonic kidney cell with normal p53 gene (HEK293) (p53+/+) are treated with 3,4-dihydroisoquinolinium salt derivative of the present invention (1, 6, 9, and 12 μM) and examined by flow cytometry after 24 hours; <17> Fig. 7 is a drawing illustrating the result of karyotype analysis, in which the colon cancer-derived cell lines, wild type cell line with normal ρ53 gene (HCT116) (p53+/+) and cell line with an inactivated p53 by viral protein E6 (HCT116/E6) (p53-/-) were treated with the 3,4- dihydroisoquinolinium salt derivative of the present invention (1, 3, 6, 9, 12, and 15 μM) and the sub-Gl peak was measured by flow cytometry after 24 hours;

<i8> Fig. 8 is a graph illustrating the result of Fig. 7;

<19> Fig. 9 is a drawing illustrating the result of measuring caspase-3 activity in the cancer cells with p53 gene deletions and mutations and cells with normal p53 gene treated with the 3,4-dihydroisoquinolinium salt derivative of the present invention; and

<20> Fig. 10 is a drawing illustrating the result of measuring the activity of a pro-apoptotic protein, bax and an anti-apoptotic protein, be1-2 in the cancer cells with p53 gene deletions and mutations and cells with normal p53 gene treated with the 3,4-dihydroisoquinolinium salt derivative of the present invention. [Best Mode]

<2i> The present invention provides an anti-cancer agent comprising a 3,4- dihydroisoquinolinium salt derivative represented by the following Formula 1- 1.

<22> <Formula 1-1>

F

<24> The compound of Formula 1-1 is a compound that is disclosed in Korean

Patent Application No. 10-2005-46749, and supplied by Hanwha Co. to be used.

Further, the compound can be prepared by using a method disclosed in the patent document to be used. <25> In a human embryonic kidney cell line (HEK293) and wild type colon cancer-derived cell line (HCT116) with normal p53 genes, which are treated with the 3,4-dihydroisoquinolinium salt derivative of the present invention, the cells display a flattened morphology by the expression of p53 gene and cell death is not induced. Further, in the cells, the normal p53 gene is expressed to suppress DNA synthesis and karyokinesis, thus multi-nucleated cells are not increased, most of the cells have one nucleus, the cell cycle is arrested in the G2/M phase, and cell growth is temporarily arrested. In addition, the expression of bcl-2, which is an anti-apoptotic gene, is increased.

<26> In contrast, in a cervical cancer cell line (HeLa) and colon cancer- derived cell line (HCT116/E6) with p53 gene deletions and mutations, which are treated with the 3,4-dihydroisoquinolinium salt derivative of the present invention, the p53 gene cannot be normally controlled. Thus, the DNA synthesis and karyokinesis continuously occur, two or more of multi-nucleated cell are observed, and DNA fragments are detected. Further, the population of 'sub-Gl' cells with less than 2N DNA increases, the activity of caspase-3 increases depending on the time, and the expression of pro-apoptotic bax gene increases depending on the time, resulting in dramatic cell death.

<27> Accordingly, the 3,4-dihydroisoquinolinium salt derivative of the present invention selectively induces cytotoxicity and cell death in the cancer cells with a loss of p53 function due to p53 gene deletions and mutations, thereby being used to treat cancer caused by defects in the p53 gene, while minimizing the side effects to normal cells.

<28> Examples of cancer with defects in the p53 gene include various types of cancer, gynecological tumors, endocrine gland cancer, central nervous system tumor, and ureter cancer, specifically lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft-tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, childhood solid tumor, differentiated lymphoma, bladder cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal tumor, brain stem glioma or pituitary adenoma, but are not limited thereto.

<29> The term 'cancer cell with a loss of p53 function due to p53 deletions and mutations' as used herein refers to a cancer cell, in which p53 dysfunction due to mutations by DNA damages or the like impairs cell cycle regulation or normal regulation ability in response to various internal/external influences, that is, a cancer cell that produces abnormal (dominant negative) proteins due to deletion or mutation of the p53 gene on chromosome.

<30> The composition of the present invention may contain one or more known active ingredients having an anticancer effect, in addition to the 3,4- dihydroisoquinolinium salt derivative of Formula 1-1.

<3i> For administration, the composition of the invention can be prepared including at least one pharmaceutically acceptable carrier, in addition to the active ingredients as described above. Examples of the pharmaceutically acceptable carrier include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and a mixture of two or more thereof. If necessary, the composition may also contain other conventional additives, such as antioxidants, buffers, and bacteriostatic agents. Moreover, the composition may additionally contain diluents, dispersants, surfactants, binders, and lubricants in order to formulate it into injectable formulations such as aqueous solution, suspension, and emulsion, pills, capsules, granules or tablets. Furthermore, the composition may preferably be formulated depending on particular diseases and its components, using the method described in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA., which is a suitable method in the relevant field of art.

<32> The composition of the invention may be administered orally or parenteral Iy (for example, intravein, subcutaneous, intraperitoneal, or o

topical application), and the dosage can vary depending on various factors, including patient's weight, age, sex, health condition, diet, and administration time, administration route, secretion rate, and disease severity, etc.. The compound of Formula 1-1 is administered at a daily dosage of about 0.01-100 mg/kg, and more preferably one time or several times.

<33> The composition of the invention may be used alone or in combination with surgical operations, hormone therapies, chemical therapies, and other methods using biological reaction regulators in order to prevent and treat cancer . [Mode for Invention]

<34> Hereinafter, preferred Examples are provided for the purpose of illustrating the present invention. However, these Examples are for the illustrative purpose only, and the invention is not intended to be limited by these Examples.

<35> Example 1: Morphological change of cancer cells with p53 gene deletions and mutations and cells with normal p53 genes by treatment with 3,4- dihydroisoquinolinium salt derivative

<36> In order to evaluate the cytotoxicity of 3,4-dihydroisoquinolinium salt derivative of the present invention on cancer cells with p53 gene deletions and mutations and cells with normal ρ53 genes, cell morphology was examined using a phase contrast microscope.

<37> As cells with p53 gene deletions (p53-/-), a cervical cancer cell line (HeLa) and colon cancer-derived cell line (HCT116/E6) were used, and as cells with normal p53 genes (p53+/+), a human embryonic kidney cell line (HEK293) and wild type colon cancer-derived cell line (HCT116) were used.

<38> The cervical cancer cell line (HeLa) (p53-/-) and human embryonic kidney cell line (HEK293) (p53+/+) were cultured in DMEM media (Dulbeccos Modification of Eagles Medium) containing 10% FBS (Fetal bovine serum), and the wild type colon cancer-derived cell line (HCTl16) (p53+/+) and colon cancer-derived cell line with mutated p53 (HCT116/E6) (p53-/-) were cultured in RPMI media 1640 (Rosewell Park Memorial Institute Medium) containing 10% FBS for 24 hour or more, and then 1, 6, and 12 μM of 3,4-dihydro-2-(2,6- difluorobenzyl)-6,7-dimethoxy-l-pentadecylisoquinolinium chloride of Formula 1-1 were added thereto. After 24 hours, each cell was magnified 100 times and 200 times, and examined under a phase contrast microscope.

<39> The result is shown in Fig. 1.

<40> As shown in Fig. 1, in the human embryonic kidney cell line (HEK293) and wild type colon cancer-derived cell line (HCT116) with normal p53 genes, which had been treated with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy- 1-pentadecylisoquinolinium chloride of Formula 1-1, there was no dramatic change in cell morphology by the p53 expression. In contrast, in the cervical cancer cell line (HeLa) and colon cancer-derived cell line (HCT116/E6) with p53 gene deletions and mutations, cell death was extremely induced. If cell death occurs, the cell morphology changes to round up and its size is reduced. The morphological change by normal p53 expression includes a flattened morphology, which is different from that of cell death. As a result, it is indicated that in the cells with normal p53 genes, cell death is not induced due to the p53 expression.

<41>

<42> Example 2: Effect of 3,4-dihydroisoquinolinium salt derivative on cell proliferation of cancer cells with p53 gene deletions and mutations and cells with normal p53 genes (MTT assay)

<43> In order to confirm the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on cell proliferation of cancer cells with p53 gene deletions and mutations and cells with normal p53 genes, an MTT assay was performed.

<44> The cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) were cultured in DMEM media containing 10% FBS for 24 hours

₄ or more. Then, each cell was placed in a 96-well plate at 2X10 cells/well in a final volume of 200 μ&, and further cultured for 24 hours. Subsequently, the 96-well plates containing each cell were treated with 3,4-dihydro-2-(2,6- difluorobenzyl)-6,7-dimethoxy-l-pentadecylisoquinolinium chloride of Formula 1-1 to give a final concentration of 1, 3, 6, 9, and 12 μM. At 24 hours after treating with 3,4-dihydro-2-(2,6~difluorobenzyl)-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1, an MTT (Tetrazoliunrbased colorimetric) assay was performed to indirectly measure cell viability.

<45> The MTT assay is an assay using the ability of mitochondria that reduces a yellow water-soluble substrate, MTT tetrazolium into a purple water-insoluble MTT formazan [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide] by the action of dehydrogenase.

<46> The MTT-formazan shows a maximum absorbance at 540-570 nm, and the absorbance measured at this wavelength reflects the metabolic activity of the living cell. Therefore, 20 μi of MTT (δmg/mi¹, in PBS) were added to each cell group treated with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1. While observing the cells under a microscope, the media and MTT solution were removed between one hour and two and a half hours after formation of MTT-formazan, and 200 μi of DMSO were added to each well to measure the absorbance at 570 nm.

<47> The result is shown in Fig. 2.

<48> As shown in Fig. 2, cell death rapidly increased in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-), which had been treated with 3,4-dihydro-2-(2,6~difluorobenzyl)-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1, as compared to the human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) , which had been treated with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1. The result is the same as the result of cell morphology shown in Fig. 1, and it was found that an IC50 value (defined as the concentration of drug required for 50% reduction in cell viability) of the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) was 4 μM, which is much lower than that of the human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) being 6 μM. <49>

<50> Example 3: Effect of 3,4-dihydroisoquinolinium salt derivative on DNA aggregation or formation of multi-nucleated cell in cancer cells with p53 gene deletions and mutations (DAPI staining method)

<5i> In order to confirm the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on DNA aggregation or formation of multi^¬ nucleated cell in cancer cells with p53 gene deletions and mutations, the cells were stained using a DAPI staining method, and then observed with a fluorescence microscope.

<52> The cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) were cultured in DMEM media containing 10% FBS for 24 hours or more. Then, 1, 3, and 6 μM of 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7- dimethoxy-1-pentadecylisoquinolinium chloride of Formula 1-1 were added to the cells, and cultured for 24 hours. Subsequently, each cell was stained by the DAPI staining method (4,6-diamidine-2-ρhenylindole, dihydrochloride) to stain its nucleus, and then observed with a fluorescence microscope. As a negative control group, the cells were treated with only a solvent, DMSO (dimethyl sulfoxide), and as a positive control group, the cells were treated with 30 and 50 μM of etoposide, which has been well known as a chemical agent causing the DNA damage-induced cell death.

<53> The result is shown in Fig.3.

<54> As shown in Fig. 3, in the human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+), which had been treated with 3,4-dihydro-2-(2,6- difluorobenzyl)-6,7-dimethoxy-l-pentadecylisoquinolinium chloride of Formula 1-1, it was found that most of the cells had one nucleus, and only a small number of cells had two nuclei. In contrast, in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-), which had been treated with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1, it was found that multi^¬ nucleated cells having two or more of nuclei increased. From the result, it can be seen that when the human embryonic kidney cell line with normal p53 gene (HEK293) was treated with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7- dimethoxy-1-pentadecylisoquinolinium chloride of Formula 1-1, cytokinesis was inhibited, and cells having two nuclei occurred, but DNA synthesis and karyokinesis were suppressed by the expression of normal p53 gene, and no more multi-nucleated cells increased. However, in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-), two or more multi-nucleated cells were observed, which was caused by the continuous DNA synthesis and karyokinesis due to the abnormal regulation of the gene. It can be thought that among the cells, a large number of cells may undergo cell death.

<55>

<56> Example 4: Effect of 3,4-dihydroisoquinolinium salt derivative on DNA fragmentation and cell cycle distribution in cancer cells with p53 gene deletions and mutations

<57> 1. DNA fragmentation

<58> In order to confirm the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on DNA fragmentation in cancer cells with p53 gene deletions and mutations, an electrophoresis was performed to detect the DNA fragmentation.

<59> Each of the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) was aliquotted in a 60 mm culture dish at a seeding concentration of 5X10

and cultured in DMEM media containing 10% FBS for 24 hours or more. The cells were treated with 25 μM of 3,4-dihydro-2-(2,6-difluorobenzy1 )-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1, and cultured for 6 hours. After completing the culture, DNA was extracted from each cell, and the electrophoresis was performed to detect the DNA fragmentation.

<60> The result is shown in Fig.4.

<6i> As shown in Fig. 4, in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-), which had been treated with 3,4- dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l-pentadecylisoquinolinium chloride of Formula 1-1, the DNA fragmentation was detected, and thus it can be seen that the cells undergo cell death.

<62> 2. Cell cycle distribution

<63> In order to confirm the effect of 3,4-dihydroisoquinolinium salt derivative of the present invention on cell cycle distribution in cancer cells with p53 gene deletions and mutations, the cells were analyzed using a flow cytometry.

<64> Each of the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-) and human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) was aliquotted in a 60 mm culture dish at a seeding concentration of 5X10 cells/mP, and cultured in DMEM media containing 10% FBS for 24 hours or more. Each cell was treated with 3 μM and 6 μM of 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1, and cultured for 12 hours and 24 hours. After completing the culture, the cells were recovered to be fixed in 70% ethanol, and stained with 100 //I of 1 mg/mi PI (propodium iodide). Then, the amounts of DNA in the cells were quantified using a FACS (Fluorescence activated cell sorter, Becton Dickinson, San Jose, CA), and cell cycle distribution was analyzed. The result of flow cytometry was read with cell quest software.

<65> The result is shown in Fig. 5.

<66> As shown in Fig. 5, in the human embryonic kidney cell (HEK293) (p53+/+), it was found that most of the cells were arrested in G2/M phase according to the treatment with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7- dimethoxy-1-pentadecylisoquinolinium chloride of Formula 1-1 for 24 hours. In contrast, in cancer cells with p53 gene deletions and mutations, which had been treated with 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l- pentadecyl isoquinol inium chloride of Formula 1-1, the population of 'sub-Gl' cells with less than 2N DNA increased. In the karyotype analysis by flow cytometry, the 'sub-Gl' cell with less than 2N DNA is a cell that has a fragmented nuclear DNA by endonuclease cleavage, and undergoes cell death.

<67>

<68> Example 5' Effect of 3,4-dihydroisoquinolinium salt derivative on cell cycle distribution of colon cancer cells with p53 gene deletions and mutations

<69> In Example 4, it was found that in cancer cells with p53 gene deletions and mutations, which had been treated with 3,4-dihydro-2-(2,6- difluorobenzyl)-6,7-dimethoxy-l-pentadecyl isoquinolinium chloride of Formula 1-1, the population of 'sub-Gl' cells with less than 2N DNA increased and the cells were induced to cell death.

<70> In order to confirm the result, the same colon cancer-derived cell line, a wild type cell line with normal p53 gene (HCT116) (p53+/+) and a cell line with an inactivated p53 by viral protein E6 (HCT116/E6) (ρ53-/-) were analyzed using a flow cytometry under the same conditions as Example 4.

<7i> Each of the cervical cancer cell line with ρ53 gene deletions and mutations (HeLa) (p53-/-) and human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+) was treated with 1, 6, 9, and 12 μM of 3,4- dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy-l-pentadecyl isoquinolinium chloride of Formula 1-1, and after 24 hours, the karyotype analysis was performed using a flow cytometry. The result is shown in Fig. 6.

<72> The colon cancer-derived cell lines, wild type cell line with normal p53 gene (HCT116) (p53+/+) and cell line with an inactivated p53 by viral protein E6 (HCT116/E6) (p53-/-) were treated with 1, 3, 6, 9, 12, and 15 μM of 3,4-dihydro-2-(2,6—difluorobenzy1 )-6,7-dimethoxy-l- pentadecyl isoquinolinium chloride of Formula 1-1, and after 24 hours, the karyotype analysis was performed using a flow cytometry. The result of measuring the sub-Gl peak is shown in Fig. 7. The result of Fig. 7 is represented by the graph shown in Fig.8.

<73> As shown in Figs. 7 and 8, when the colon cancer-derived cell line with p53 gene deletions and mutations (HCT116/E6) (p53-/-) was treated with 3 μM and 6 μM of 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-diraethoxy-l- pentadecylisoquinolinium chloride of Formula 1-1, the population of 'sutrGl' cells increased by 50%, which is the same as the result in Example 4. In contrast, even though the wild type cell line with normal p53 gene (HCTl16) (p53+/+) was treated with increasing concentrations of 3,4-dihydro-2-(2,6- difluorobenzyl)-6,7-dimethoxy-l-pentadecyl isoquinolinium chloride of Formula 1-1, there was no change in the population of 'sub-Gl' cells, and the population of 'G2/M' cells greatly increased.

<74>

<75> Example 6^* Effect of 3,4-dihydroisoquinolinium salt derivative on caspase-3 activity in cancer cells with p53 gene deletions and mutations (western-blotting)

<76> In order to confirm whether the 3,4-dihydroisoquinolinium salt derivative of the present invention induces cell death in cancer cells with p53 gene deletions and mutations, caspase-3 activity was measured.

<77> Cell death is generally initiated as follows. The interaction of anti- apoptotic protein bcl-2 and pro-apoptotic protein bax as mitochondrial outer membrane proteins reduces the membrane potential of mitochondrial outer membrane, so as to induce the release of cytochrome C into the cytosol, which sequentially activates proteases, called caspase.

<78> Accordingly, the induction of cell death can be confirmed by an assay for caspase activation. That is because the enzyme is present in an inactivated form, and activated by apoptosis-inducing signals to be involved in the destruction of cells ultimately led to death.

<79> Each of the cervical cancer cell line (HeLa) (p53-/-) and colon cancer- derived cell line (HCT116/E6) (p53-/-) with ρ53 gene deletions and mutations, and human embryonic kidney cell line (HEK293) (p53+/+) and colon cancer- derived cell line wild type (HCT116) (p53+/+) with normal p53 gene was aliquotted in a 60 mm culture dish at a seeding concentration of 5X10 cells/ nύ, and cultured in DMEM media containing 10% FBS for 24 hours. The cells were treated with 9 μM of 3,4-dihydro-2-(2,6-difluorobenzyl)-6,7-dimethoxy- 1-pentadecylisoquinolinium chloride of Formula 1-1 and reacted for 24 hours. Then, the cells were recovered, and caspase-3 activity was measured by western-blotting.

<80> The result is shown in Fig. 9.

<8i> As shown in Fig. 9, the cell death by the addition of 3,4-dihydro~2- (2,6—difluorobenzyl)-6,7-dimethoxy-l-pentadecylisoquinolinium chloride of Formula 1-1 was selectively found in the cervical cancer cell line (HeLa) (p53-/-) and colon cancer-derived cell line (HCT116) (p53-/-) with p53 gene deletions and mutations, which was confirmed by the increase in caspase-3 activity depending on the time.

<82>

<83> Example 7: Effect of 3,4-dihydroisoquinolinium salt derivative on cell death in cancer cells with p53 gene deletions and mutations (western- blotting)

<84> In order to confirm whether the 3,4-dihydroisoquinolinium salt derivative of the present invention promotes cell death in cancer cells with p53 gene deletions and mutations, the following experiment was performed.

<85> Apoptosis inducing factors vary depending on the type of cells, and examples thereof include physiologically active substances such as tumor necrosis factor (TNF), Fas ligand, metastatic growth factors, neurotransmitters, removal of growth factor, calcium, and glucocorticoid and cell damage induced factors such as thermal sensitization, viral infection, bacterial toxins, oncogenes, tumor suppressors, cytotoxic T cell, nutrient limitation, and antimetabolites. Among the genes involved in cell death, factors having a function of inhibiting cell death had been found in the cell, and examples thereof include bcl-2, bclX-L pseudogenes. In contrast, bax has been known as a gene inducing cell death. In this Example, the experiment was performed in the same manner as in Example 6.

<86> The result is shown in Fig. 10.

<87> As shown in Fig. 10, in the cervical cancer cell line with p53 gene deletions and mutations (HeLa) (p53-/-), which had been treated with 3,4- dihydro-2-(2,6~difluorobenzyl)-6,7-dimethoxy-l-pentadecylisoquinolinium chloride of Formula 1-1, the expression of pro-apoptotic gene, bax increased depending on the time, whereas in human embryonic kidney cell line with normal p53 gene (HEK293) (p53+/+), the expression of anti-apoptotic gene, bcl-2 increased.

<88>

<89> Hereinafter, Formulation Examples for the composition of the present invention will be illustrated.

<90> Formulation Example 1: Preparation of liquid injectable formulation <9i> An injectable liquid formulation containing 10 mg of the active ingredient was prepared as the following method.

<92> 1 g of the compound of Formula 1-1, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to be 100 ml. The solution was put into a bottle, and heated to be sterilized at 20"C for 30 minutes. <93> The composition of the injectable liquid formulation is as follows. <94> Compound of Formula 1-1 1 g

<95> Sodium chloride 0.6 g

<96> Ascorbic acid 0.1 g

<97> Distilled water predetermined amount

<98> Formulation Example 2- Preparation of syrup formulation <99> A syrup formulation containing the compound of Formula 1-1 as an active ingredient (2%, weight/volume) was prepared as the following method. <ioo> The compound of Formula 1-1, saccharin, and sugar were dissolved in 80 g of warm water. The solution was cooled, and a solution containing glycerin, saccharin, flavor, ethanol, sorbic acid, and distilled water was added thereto. Water was added to the mixture to be 100 ml. <ioi> The composition of the syrup formulation is as follows. <i02> Compound of Formula 1-1 2 g

<i03> Saccharin 0.8 g

<iO4> Sugar 25.4 g

<iO5> Glycerin 8.Og <iO6> Flavor 0.04 g

<i07> Ethanol 4.0 g

<iO8> Sorbic acid 0.4 g

<iO9> Distilled water Predetermined amount

<iio> Formulation Example 3: Preparation of tablet formulation <πi> A tablet formulation containing 15 mg of the active ingredient was prepared as the following method.

<ii2> 250 g of the compound of Formula 1-1 was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% Gelatin solution was added to the mixture, and then pulverized to pass through a 14- mesh sieve. The mixture was dried. Then, 160 g of potato starch, 50 g of talc, and 5 g of magnesium stearate were added thereto to prepare a tablet. <ii3> The composition of the tablet formulation is as follows. <ii4> Compound of Formula 1-1 250 g

<ii5> Lactose 175.9 g

<ii6> Potato starch 180 g

<ii7> Colloidal silicic acid 32 g

<ii8> 10% Gelatin solution

<ii9> Potato starch 160 g

<12O> Talc 50g

<i2i> Magnesium stearate 5 g

[Industrial Applicability]

<122> The 3,4-dihydroisoquinolinium salt derivative of the present invention selectively induces cytotoxicity and cell death in the cancer cells with a loss of p53 function due to p53 gene deletions and mutations, thereby being used to treat cancer caused by defects in the p53 gene, while minimizing the side effects to normal cells.

Claims

[CLAIMS] [Claim 1]

An anti-cancer agent comprising a compound represented by the following Formula 1-1.

[Claim 2]

The anti-cancer agent according to claim 1, wherein the cancer is caused by defects in a p53 gene. [Claim 3]

The anti-cancer agent according to claim 2, wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, thyroid cancer, parathyroid cancer, soft-tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, childhood solid tumor, differentiated lymphoma, bladder cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal tumor, brain stem glioma and pituitary adenoma. [Claim 4]

The anti-cancer agent according to claim 3, wherein the cancer is colorectal cancer or cervical carcinoma.