Composition for inhibiting cancer cell proliferation

Abstract

To provide a cancer cell proliferation inhibitory composition that contains a specific cyclic anthraquinone derivative.SOLUTION: A cancer cell proliferation inhibitory composition contains a cyclic anthraquinone derivative represented by the following structural formula.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition containing a cyclic anthraquinone derivative that suppresses the proliferation of cancer cells. [Background technology]

[0002] In recent years, treatment using molecularly targeted drugs (molecular targeted therapy) has become widely used as a cancer treatment method. While conventional chemotherapy mainly involves administering a combination of anticancer drugs that are effective for each cancer site (e.g., lung cancer), molecular targeted therapy involves administering drugs that target and attack molecular-level abnormalities that are characteristic of the patient's cancer cells.

[0003] Because this molecular targeted therapy targets cancer cells, it is thought to have relatively little adverse effect on normal cells and few side effects. Molecular targeted therapy also has the advantage that it is possible to predict to some extent whether the drug will be effective before administration.

[0004] In molecular targeted cancer therapy, apart from those targeting well-known mutant kinases or specific cancer microenvironments, it has been reported that abnormally expressed DNA of specific genes, such as the c-myc oncogene, which promotes increased cell proliferation, and hTERT, which accelerates telomerase action in telomere elongation, is highly correlated with cancer development (see, for example, Non-Patent Document 1). Therefore, it is expected that abnormally expressed DNA of these specific genes will be promising targets in molecular targeted cancer therapy.

[0005] As such, various studies on molecular targeted cancer therapy are being conducted, and new therapeutic agents useful for molecular targeted cancer therapy are needed.

[0006] On the other hand, anthraquinone, known as a DNA threading intercalator, preferentially binds to GC base pairs and inhibits DNA replication and transcription. Its derivatives, doxorubicin and nogalamycin, have been used as classical chemotherapeutic agents for cancer, but are known to cause side effects such as cardiomyopathy (see, for example, Non-Patent Documents 2 and 3).

[0007] Anthraquinones also function as binding agents for guanine quadruplexes (G-quadruplexes; G4s), and are known to effectively inhibit telomerase by recognizing and stabilizing the G4 structure (see, for example, Non-Patent Documents 4 and 5). Thus, small molecules that bind strongly to G4s and have telomerase inhibitory effects are expected to be therapeutic agents for cancer. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Fumi Nagatsugi, Kazumitsu Onizuka, Functional G-Quadruplex Binding Molecules, Chem. Lett. 2020, Vol.49, No.7,771-780 [Non-patent document 2] Suhail A. Islam, SN, Bijukumar M. Gandecha, Malcolm Partridge, Laurence H.Patterson, & Brown, aJR Comparative Computer Graphics and Solution Studies of the DNA Interaction of Substituted Anthraquinones Based on Doxorubicin and Mitoxantrone. Journal of Medicinal Chemistry 28, 857-864 (1985) [Non-patent document 3] Singal, P.K. & Iliskovic, N. Doxorubicin-induced cardiomyopathy. N Engl J Med 339, 900-905 (1998).

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present invention has been made in view of the above background, and an object of the present invention is to provide a composition containing a specific cyclic anthraquinone derivative that inhibits the proliferation of cancer cells and is also to provide a composition that is useful for molecular-targeted cancer therapy. [Means for solving the problem]

[0010] The present inventors have discovered that cyclic anthraquinone derivatives having a planar benzene ring in the cyclic portion effectively and selectively inhibit the proliferation of cancer cells, and have completed the present invention.

[0011] That is, the present invention is as follows. [1] A composition for inhibiting the proliferation of cancer cells, comprising a cyclic anthraquinone derivative represented by the following formula (I):

[0012] [ka] [In formula (I), R 1 is any one of the linking groups represented by the following formulas (a-1) to (a-5) (* represents a bonding position, and NH-* is R 2 ) and R 2 is any one of the linking groups represented by the following formulas (b-1) to (b-5) (* indicates the bonding position).

[0013] [ka]

[0014] [ka]

[0015] [2] The composition for inhibiting cancer cell proliferation according to [1] above, wherein the cyclic anthraquinone derivative is represented by the following structural formula:

[0016] [ka]

[0017] [3] A composition for inhibiting cancer cell proliferation according to [1] or [2] above, characterized in that it inhibits the telomerase activity of the chromosomes of cancer cells and suppresses the proliferation of cancer cells. [4] A composition for inhibiting the proliferation of cancer cells according to any one of [1] to [3] above, which induces apoptosis in cancer cells and inhibits the proliferation of cancer cells. [5] A composition for inhibiting the proliferation of cancer cells according to any one of [1] to [4] above, characterized in that it has less effect on normal cells other than cancer cells than chemical cancer treatment drugs. [6] A composition for inhibiting cancer cell proliferation according to any one of [1] to [5] above, which is a molecular targeted cancer therapeutic drug. [Effects of the Invention]

[0018] The composition containing the cyclic anthraquinone derivative of the present invention effectively and selectively inhibits the proliferation of cancer cells and can be used in molecular targeted cancer therapy. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows an outline of the evaluation of the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 2] FIG. 1 shows an electrophoretic image of the TRAP assay of the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 3] FIG. 1 shows the results of measuring the telomerase inhibitory activity (IC50 value) of the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 4] FIG. 1 shows the results of TERT mRNA expression levels in cancer cell lines (Ca9-22, 3T3-E1) and normal cell lines (NHEK, BMC) treated with the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 5] FIG. 1 shows the results of measuring the cell viability of cancer cell lines (Ca9-22) and normal cell lines (NHEK, BMC) treated with the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 6] FIG. 1 shows the results of hTERT mRNA expression levels in human cancer cell lines (Ca9-22, Hela, HEK293, HSC-2) treated with the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 7] FIG. 1 shows the results of measuring the cell viability of human cancer cell lines (Ca9-22, Hela, HEK293, HSC-2) treated with the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 8] FIG. 1 shows the results of Annexin V / PI staining assay of the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 9] FIG. 1 shows the results of Western blot analysis of the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 10] FIG. 1 shows the change in tumor volume over time following administration of the cyclic anthraquinone derivative cAQ-mBen of the present invention in an animal test using mice. [Figure 11] 1 shows photographs of tumor cells collected from mice after five doses (10 days) of the cyclic anthraquinone derivative cAQ-mBen of the present invention (2 days after the final dose). [Figure 12] FIG. 1 shows changes in mouse body weight over time following administration of the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 13] 1 shows hematoxylin-eosin stained images of the liver and kidney taken from a mouse administered with the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 14] 1 is a graph showing the size of glomeruli from the liver and kidney collected from mice administered with the cyclic anthraquinone derivative cAQ-mBen of the present invention. [Figure 15] FIG. 1 shows the results of measuring physiological indices (BUN, UA, AST, ALT) in biochemical analysis of serum collected from mice administered with the cyclic anthraquinone derivative cAQ-mBen of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] [Cyclic anthraquinone derivative contained in the composition of the present invention] The cyclic anthraquinone derivative contained in the composition of the present invention is a compound represented by the following formula (I). The cyclic anthraquinone derivative may be in the form of a free form or a pharmacologically acceptable salt. Furthermore, the anthraquinone structure may have a substituent as long as the effects of the present invention are achieved.

[0021] [ka]

[0022] In formula (I), R 1 is a divalent organic linking group represented by the following formulas (a-1) to (a-5), and the linking group represented by formula (a-1) is particularly preferred. In formulas (a-1) to (a-5), * represents a bonding position, and NH-* is R 2 is linked to.

[0023] [ka]

[0024] Also, R 2 is an organic linking group represented by the following formulas (b-1) to (b-5), and the linking group represented by formula (b-1) is particularly preferred. In formulas (b-1) to (b-5), * represents a bonding position.

[0025] [ka]

[0026] Specifically, the following can be exemplified as particularly preferred cyclic anthraquinone derivatives (I):

[0027] [ka]

[0028] [Method of producing the cyclic anthraquinone derivative contained in the composition of the present invention] The cyclic anthraquinone derivative contained in the composition of the present invention can be produced using 1,5-dihaloanthraquinone represented by the following structural formula as a starting material. Examples of the halogen atom (X) in 1,5-dihaloanthraquinone include chlorine, bromine, and iodine atoms, with chlorine being preferred. In other words, 1,5-dichloroanthraquinone is preferred.

[0029] [ka]

[0030] The starting material, 1,5-dihaloanthraquinone, is reacted with an amine compound represented by the following formulas (a'-1) to (a'-5).

[0031] [ka]

[0032] As a result, for example, when an amine compound represented by formula (a'-1) is reacted, the following compound is obtained (specifically, see the production of compound 1 in the Examples).

[0033] [ka]

[0034] Subsequently, the above compound is reacted with a dicarboxylic acid represented by the following formulas (b'-1) to (b'-5).

[0035] [ka]

[0036] When an amine compound represented by formula (b'-1) is reacted with this, a cyclic anthraquinone derivative of the present invention (compound cAQ-mBen) represented by the following structural formula is obtained (for details, see the production of compound cAQ-mBen in the Examples).

[0037] [ka]

[0038] The above reactions can be carried out under general reaction conditions. For example, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), etc. can be used as solvents. The reaction temperature can be about 0 to 100°C, and preferably about 30 to 80°C.

[0039] [Composition of the present invention] The composition of the present invention is characterized by containing a cyclic anthraquinone derivative represented by the above general formula (I) and suppressing the proliferation of cancer cells. The composition of the present invention may contain, in addition to the cyclic anthraquinone derivative represented by the general formula (I), other pharmaceutically acceptable ingredients such as a solvent, an excipient, etc.

[0040] The composition of the present invention contains a cyclic anthraquinone derivative represented by the general formula (I) and can stabilize the G-quadruplex that constitutes the telomere, a part of the chromosome of a cancer cell, and inhibit the telomerase activity of the chromosome of the cancer cell. Furthermore, it can selectively induce apoptosis in the cancer cell. Therefore, the composition of the present invention is useful as a molecular-targeted cancer therapeutic agent.

[0041] The composition of the present invention can be used as an oral or parenteral agent. Examples of the oral agent form include tablets, capsules, powders, granules, rods, plates, blocks, solids, pills, caplets, chewable tablets, liquids, pastes, creams, and gels. Examples of parenteral agents include intravenous agents, intramuscular agents, subcutaneous agents, and transdermal agents.

[0042] The composition of the present invention can be used, for example, as a pharmaceutical composition, and can also be used as a food composition such as a functional food composition or a specified health food composition. [Example]

[0043] Specific examples of the present invention will be described below, but the scope of the present invention is not limited to these examples.

[0044] The cyclic anthraquinone derivative (cAQ-mBen) of the present invention was produced. As comparative examples, the acyclic anthraquinone derivative compound AQ-Ac, and the cyclic anthraquinone derivatives cAQ-C2 and cAQ-ch, which do not have a benzene ring in the cyclic portion, were produced. Hereinafter, the cyclic anthraquinone derivative will sometimes be referred to as the cAQ derivative, and the acyclic anthraquinone derivative will sometimes be referred to as the AQ derivative. The manufacturing process is outlined below.

[0045] [ka]

[0046] <Production of the cyclic anthraquinone derivative (cAQ-mBen) of the present invention> (Production of Compound 1) First, compound 1 represented by the following structural formula was prepared.

[0047] [ka]

[0048] Specifically, 7.22 g (50.7 mmol) of N,N-bis(3-aminopropylmethylamine) was added to 1.4 g (5.05 mmol) of 1,5-dichloroanthraquinone, and the mixture was refluxed in a 140 °C oil bath for 4 hours and then cooled to room temperature. The reaction mixture was poured into 500 mL of 2 mol / L sodium hydroxide and stirred for 20 minutes. The precipitate was then collected by suction filtration and vacuum drying. The resulting viscous dark-red solid was dissolved in a minimum amount of methanol and reprecipitated in 300 mL of 2 mol / L sodium hydroxide. The precipitate was then collected by suction filtration and vacuum dried again to obtain compound 1 (30%, yield, 0.76 g) as a viscous dark-red solid.

[0049] The purity of compound 1 was confirmed by RP-HPLC (Shimazu, Japan), MALDI-TOF-MS (Bruker, Billerica, MA, USA), 1 The results were confirmed by H-NMR (Bruker DRX500, USA). MALDI-TOFMS (positive mode, DHBA) m / z = 496.688 (calculated value for C28H42N6O2 + H+ = 495.680).

[0050] (Production of Compound cAQ-mBen) Subsequently, the compound cAQ-mBen represented by the following structural formula was produced using the compound 1.

[0051] [ka]

[0052] Specifically, 0.39 g of compound 1 (0.78 mmol) was added to 120 mL of DMF and stirred at room temperature. Next, 0.14 g of isophthalic acid (0.78 mmol) and 0.89 g of HATU (0.22 mmol) were dissolved in 100 mL of DMF, and the mixture was then added dropwise to compound 1 over 1.5 hours. After the entire mixture was added to compound 1, the mixture was stirred for 1 day (24 hours). After evaporation and vacuum drying, the product was recrystallized three times using a mixed solvent of acetone and ethyl acetate and separated by filtration. The filtrate was then evaporated and purified by silica chromatography (CHCl3:MeOH:DEA=1:0.05:0.03), and the fraction with an Rf value of 0.2 was collected. After evaporation and vacuum drying, compound cAQ-mBen (yield 3%, 12 mg) was obtained as a purple solid.

[0053] The purity of compound cAQ-mBen was confirmed by RP-HPLC, MALDI-TOF-MS, 1 This was confirmed by H-NMR. MALDI-TOF-MS (positive mode, DHBA) m / z = 626.939 (calculated for C36H44N6O4+H+ = 625.781); 1 H-NMR (400 MHz, CDCl3, δ ppm): 1.80 (quin, 4H, J = 6.1 Hz), 1.90 (quin, 4H, J = 5.9 Hz), 2.31 (s, 6H), 2.52 (t, 4H, J = 6.1 Hz), 2.60 (t, 4H, J = 6.4 Hz), 3.40 (quin, 4H, J = 6.1 Hz), 3.46 (quin, 4H, J = 5.9 Hz), 6.83 (dd, 2H), 7.04 (s, 2H), 7.40 (m, 4H), 7.55 (m, 1H), 7.86 (s, 1H), 9.76 (s, 2H).

[0054] <Preparation of Compound AQ-Ac as a Comparative Example> Using the above compound 1, a compound AQ-Ac represented by the following structural formula was produced.

[0055] [ka]

[0056] Specifically, 0.49 g of compound 1 (9.9 mmol) was added to 20 mL of pyridine and 1 mL of acetic acid, and the mixture was refluxed in an oil bath at 140 °C for 4 hours. After refluxing, the solvent was removed by evaporation, and 5 mL of toluene was added and azeotroped four times. The target compound was purified by silica chromatography (CHCl3:MeOH:DEA = 1:0.1:0.1), and the fraction with an Rf value of 0.2 was collected. After evaporation and vacuum drying, compound AQ-Ac (yield 22%, 122 mg) was obtained as shiny red crystals.

[0057] The purity of compound AQ-Ac was confirmed by RP-HPLC, MALDI-TOF-MS, 1 This was confirmed by H-NMR. MALDI-TOF-MS (positive mode, DHBA) m / z = 579.556 (calculated for C32H46N6O4 + H+ = 579.754)

[0058] <Preparation of Compound cAQ-C2 as a Comparative Example> Using the above compound 1, a compound cAQ-C2 represented by the following structural formula was produced.

[0059] [ka]

[0060] Specifically, 0.25 g of compound 1 (0.51 mmol) was added to 70 mL of DMF and stirred at room temperature. 0.072 g of succinic acid (0.61 mmol) and 0.57 g of HATU (1.5 mmol) were dissolved in 80 mL of DMF, and the mixture was then added dropwise to compound 1 over 1.5 hours. After the entire mixture was added to compound 1, the mixture was stirred for 17 hours. The solvent was then removed by evaporation. The target compound was purified by RP-HPLC (Shimazu, Japan) (column: Inertsil ODS-4; flow rate: 2.7 mL / min; wavelength: 210 nm; column temperature: 40 °C). The peak with a retention time of 17 minutes was collected and dried by lyophilization. Compound cAQ-C2 (yield 9%, 28 mg) was obtained as a red solid.

[0061] The purity of compound cAQ-C2 was confirmed by RP-HPLC, MALDI-TOF-MS, 1 This was confirmed by H-NMR. MALDI-TOF-MS (positive mode, DHBA) m / z = 579.077 (calculated for C32H44N6O4 + H+ = 577.738)

[0062] <Production of Compound cAQ-ch as a Comparative Example> Using the above compound 1, a compound cAQ-ch represented by the following structural formula was produced.

[0063] [ka]

[0064] Specifically, 0.25 g of compound 1 (1.5 mmol) was added to 350 mL of DMF and stirred at room temperature. Next, 0.32 g of 1,1-cyclohexane-diacetic acid (1.6 mmol) and 1.7 g of HATU (4.5 mmol) were added to compound 1 with stirring. After 2 days, the solvent was removed by evaporation, and the target compound was purified by silica chromatography (CHCl3:DEA = 1:0.07) and the fraction with an Rf value of 0.3 was collected. After evaporation and vacuum drying, compound cAQ-ch (yield 9%, 86 mg) was obtained as a red solid.

[0065] The purity of compound cAQ-ch was confirmed by RP-HPLC, MALDI-TOF-MS, 1 This was confirmed by H-NMR. MALDI-TOF-MS (positive mode, DHBA) m / z = 661.270 (calculated for C38H54N6O4 + H+ = 659.881)

[0066] The effect of the cyclic anthraquinone derivative of the present invention, compound cAQ-mBen, on cancer cells was evaluated. The outline of the results is shown in Figure 1.

[0067] [TRAP assay] The telomerase inhibitory ability of compound cAQ-mBen was confirmed by TRAP assay (Telomeric Repeat Amplification Protocol assay).

[0068] For the TRAP assay, the TRAPeze Telomerase Detection Kit (EMDMillipore, Billerica, MA) was used.

[0069] The TS forward primer was extended with telomerase (1000 ng protein extract from HeLa cells) in the TRAP reaction buffer. Freshly prepared ligand solutions (cAQ derivatives, 0-150 μM) were added to the TRAP reaction system. A treatment without ligand was used as a positive control, and a treatment without telomerase and inactivated telomerase (heated at 95°C for 12 hours) were used as negative controls.

[0070] The specific procedure was as follows: first, an extension step at 30°C for 60 min, followed by a 5-min incubation at 95°C, followed by 35 cycles of PCR (94°C, 1 min; 62°C, 1 min; 72°C, 1 min).

[0071] The resulting telomerase extension products were analyzed on a 12.5% ​​polyacrylamide gel. After electrophoresis, the gel was stained with 1x GelStar Nucleic Acid Stain (Takara Bio, Japan) in 0.7x TBE buffer for 30 minutes and photographed. 50 The value was calculated.

[0072] Figure 2 shows the electrophoresis image, and Figure 3 shows the IC 50 The result of the value is shown. As shown in Figure 2, the shift band of the compound cAQ-mBen clearly decreased with increasing concentration, indicating that the elongation of telomeric DNA was inhibited. Furthermore, as shown in Figure 3, the compound cAQ-mBen of the present invention exhibited a significantly higher IC than the other cAQ derivatives according to the comparative examples. 50 showed the strongest inhibitory effect at approximately 4.3 μm. These results suggest that the compound of the present invention, cAQ-mBen, inhibits the function of telomerase and shortens the length of telomeric DNA.

[0073] [Cell proliferation assay] To determine whether the compound of the present invention, cAQ-mBen, selectively inhibits tumor cell proliferation, cell proliferation assays were performed in several cell lines.

[0074] (cell culture) The cell lines used are as follows: Human cell lines Ca9-22, SAS, HEK293, HeLa, and HSC-2 were obtained from the RIKEN Cell Bank (Ibaraki, Japan). These cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Normal human epidermal keratinocytes (NHEK; ASF-4-4L2) were obtained from Cell Applications, Inc. (San Diego, CA) and maintained in keratinocyte growth medium (Cell Applications, Inc.).

[0075] (Preparation of bone marrow cells) Bone marrow cells were prepared as follows. Bone marrow cells (BMCs) were collected from the femurs and tibias of 8-10 week-old male mice (ddY strain: Deutschland, Denken, Yoken) and suspended in Eagle's minimum essential medium alpha modified (Kyowa Kirin, Tokyo) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 1100 μg / ml streptomycin, and 10,000 U / ml macrophage colony-stimulating factor (M-CSF).

[0076] (Cell proliferation assay) These cell suspensions were plated in 96-well tissue culture plates and subjected to cell proliferation assays. Cell proliferation was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Specifically, cells plated on a 96-well plate were cultured for 24 hours to stabilize, then treated with compound cAQ-mBen for 48 hours, and then a CCK-8 assay was performed. For comparison, compound AQ-Ac, compound cAQ-C2, compound cAQ-ch, and cisplatin (CDDP), a representative cancer therapeutic agent, were used instead of compound cAQ-mBen.

[0077] (I C 50 (measurement of values) From the results of the cell proliferation assay, IC 50 The values ​​were calculated and the results are shown in Table 1.

[0078] [Table 1]

[0079] As shown in Table 1, compound cAQ-mBen exhibited an IC 50 The IC value is generally less than 1 μm, which means that it has a high ability to suppress the growth of cancer cells, but its toxicity to normal cell lines is IC 50 The value was low, at over 6 μm. 50 The value was 0.3 μm, nearly 20 times lower than that of normal cell lines such as mouse bone marrow cells. In contrast, the comparative compound cAQ-C2, the compound cAQ-ch, and cisplatin (CDDP) exhibited growth inhibitory effects equal to or greater than those on normal cells than on cancer cell lines.

[0080] [Investigation of TERT mRNA expression levels] To investigate whether the suppression of cell proliferation by the compound cAQ-mBen correlates with the inhibition of telomerase, the expression levels of TERT mRNA, a rate-limiting determinant that controls telomerase activity, were examined in different cancer and normal cell lines.

[0081] The results are shown in Figures 4 and 5. As shown in Figure 4, in the normal cell lines using NHEK and BMC, TERT mRNA expression was barely detectable compared to the human cancer cell line Ca9-22 and the mouse cell line 3T3-E1. As shown in Figure 5, in Ca9-22, cell growth inhibition was clearly stronger than in the other two normal cell lines (NHEK, BMC).

[0082] Next, we investigated the relationship between the expression level of hTERT and the effect of the compound cAQ-mBen in four different human cancer cell lines.

[0083] The results are shown in Figures 6 and 7. As shown in Figure 6, the TERT mRNA expression levels in Ca9-22 and HeLa were approximately two-fold higher than those in HEK293 and HSC-2. Furthermore, as shown in Figure 7, the proliferation of all cell lines was inhibited by the compound cAQ-mBen in a dose-dependent manner. However, the inhibition curves for Ca9-22 and HeLa were shifted relative to those for HEK293 and HSC-2, and the inhibition pattern showed a positive correlation with the TERT expression levels of these cells. This suggests that the compound cAQ-mBen tends to exert its inhibitory effect more sensitively on cell lines with high telomerase activity.

[0084] [Detection of apoptosis] To investigate whether the cell growth inhibitory effect of compound cAQ-mBen is mediated by the induction of cell death, the tumor cell line SAS was incubated with compound cAQ-mBen or CDDP as a positive control, and then apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (Nacalai Tesque, Kyoto, Japan) or by Western blotting, which monitors the digestion of procaspase 3 and PARP.

[0085] (Annexin V / PI staining assay) SAS cells cultured for 24 hours were treated with 5 μM of the compound cAQ-mBen or 50 μM of CDDP for 24 hours. Drug-treated cells were either lysed and subjected to Western blotting or harvested by trypsinization and stained with Annexin V-FITC and propidium iodide (PI). Images of cells stained with Annexin V-FITC and PI were taken using a fluorescence microscope equipped with a digital camera (BZ9000, Keyence, Osaka, Japan).

[0086] (Western blot analysis) The drug-treated cells were washed with ice-cold phosphate-buffered saline and then lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% sodium dodecyl sulfate, 10% glycerol, 1% β-mercaptoethanol) to extract total proteins, followed by Western blotting.

[0087] Blots were developed with horseradish peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence substrate reagents, ImmunoStar LD (Fujifilm Wako) or Immobilon (Merck-Millipore, Billerica, MA, USA). Images were acquired using an LAS-3000mini (Fujifilm, Tokyo, Japan). Antibodies against caspase-3 and poly(ADP-ribose) polymerase (PARP) (Abcam, Cambridge, UK), β-actin, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies were used.

[0088] FIG. 8 shows the results of the Annexin V / PI staining assay, and FIG. 9 shows the results of the Western blot analysis. As shown in Figure 8, after 24 hours of culture in the presence of 5 μM of the compound cAQ-mBen, approximately 30% of the cells were positive for Annexin V (indicating cells in the early stage of apoptosis) and approximately 10% for PI signaling (indicating necrotic cells), both of which were at levels comparable to those observed in SAS cultured with 50 μM CDDP. Furthermore, as shown in Figure 9, the similar apoptosis-inducing effects of 5 μM cAQ-mBen and 50 μM CDDP were confirmed by Western blotting, which detected distinct digestion of PARP and caspase-3.

[0089] These results suggest that the compound cAQ-mBen induces significant apoptosis in tumor cells.

[0090] [Animal testing] The effects of the compound cAQ-mBen on tumor growth and side effects were investigated in animals. CDDP was used as a chemotherapy control.

[0091] The evaluation was carried out using mice injected with SAS cells. Specifically, 4-week-old male KSN / Slc nude mice purchased from Japan SLC Co., Ltd. (Shizuoka, Japan) were housed under a 12-hour light / dark cycle at a constant room temperature and provided with standard rodent chow and water ad libitum. After the mice were housed under these conditions for more than one week, the experiment was initiated.

[0092] SAS cells were harvested from subconfluent cultures by trypsinization and suspended in serum-free DMEM. This cell suspension (5.0 × 106 cells, 0.2 ml / mouse) was subcutaneously injected into the backs of nude mice. Seven days after cell inoculation, approximately 50 mm 3 Twenty-seven mice bearing xenograft tumors were randomly divided into three groups.

[0093] The tumor volume was calculated by measuring the length and width using a vernier caliper and using the following formula:

[0094] Tumor volume (mm 3 ) = length (mm) x width (mm)

[0095] Nine mice in each group were intraperitoneally administered 0.003 mmol / kg of the compound cAQ-mBen, 0.030 mmol / kg of CDDP, or saline as a control every two days.

[0096] (Tumor tissue analysis) Figure 10 shows the change in tumor volume over time following administration of the compound cAQ-mBen of the present invention. In Figure 10, the tumor volumes after 10 days are shown for the control, the compound cAQ-mBen of the present invention, and CDDP, from top to bottom. Figure 11 shows photographs of tumor cells collected from mice after five doses (10 days) of the compound cAQ-mBen of the present invention (two days after the final dose). Figure 12 shows the change in mouse weight over time following administration of the compound cAQ-mBen of the present invention.

[0097] As shown in Figure 10, when no chemical substance was administered, tumor volume continued to increase, but in mice administered with the compounds cAQ-mBen and CDDP, the increase in tumor volume was significantly suppressed. As shown in Figure 11, the effect is clear when comparing the size of tumor cells after 10 days. Furthermore, even though the dose of the compound cAQ-mBen was 10 times lower than that of CDDP, it achieved the same tumor growth inhibitory effect as CDDP.

[0098] On the other hand, as shown in FIG. 12, CDDP clearly reduced the body weight of the mice, but the compound cAQ-mBen had almost no effect on the body weight during the test period.

[0099] (Liver and kidney histopathological analysis) After five doses (10 days) of drug administration (2 days after the final dose), the livers and kidneys were collected from the mice, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. The sections were stained with a hematoxylin-eosin staining kit (Muto Pure Chemical Industries, Ltd., Tokyo, Japan), and images were captured using a digital camera-equipped optical microscope (BZ9000; Keyence, Osaka, Japan).

[0100] Figure 13 shows photographed images stained with hematoxylin and eosin, and Figure 14 shows a graph showing the size of glomeruli. As shown in Figures 13 and 14, mice administered CDDP showed similar pathological changes in the liver and kidney, with significantly smaller glomeruli, whereas the kidneys of mice administered the compound cAQ-mBen showed no effect on the cell arrangement or size of the glomeruli.

[0101] (Blood analysis) After administering the drug five times (for 10 days) (2 days after the final administration), whole blood was collected from the mice, allowed to clot, and then centrifuged at 2800 × g for 20 minutes at 25°C to prepare serum, which was then subjected to biochemical analysis at Oriental Yeast Co., Ltd. (Shiga Prefecture, Japan).

[0102] The results are shown in Figure 15. The left side of the graph is the control, the center is the compound of the present invention cAQ-mBen, and the right side is CDDP. As shown in Figure 15, CDDP-treated mice showed significant increases in various physiological indices (BUN, UA, AST, ALT), confirming damage to the liver and kidneys. In contrast, there was no significant difference between the cAQ-mBen-treated group and the control group, indicating that cAQ-mBen had little effect on these tissues.

[0103] The results of the tumor tissue analysis, liver and kidney histopathological analysis, and blood analysis indicated that compound cAQ-mBen exhibited a strong tumor growth inhibitory effect comparable to that of CDDP. Furthermore, it was suggested that compound cAQ-mBen was more specific to tumor tissue and had less adverse effects on normal tissues in mice. [Industrial Applicability]

[0104] The novel cyclic anthraquinone derivatives of the present invention can be used in molecular targeted cancer therapy and are therefore industrially useful.

Claims

1. A composition for inhibiting cancer cell proliferation, comprising a compound represented by the following structural formula: 【Chemistry 1】

2. 2. The composition for inhibiting cancer cell proliferation according to claim 1, which inhibits the telomerase activity of the chromosomes of cancer cells and suppresses the proliferation of cancer cells.

3. 3. The composition for inhibiting cancer cell proliferation according to claim 1 or 2, which induces apoptosis in cancer cells and inhibits the proliferation of cancer cells.

4. 4. The composition for inhibiting cancer cell proliferation according to any one of claims 1 to 3, characterized in that it has less effect on normal cells other than cancer cells than chemical cancer treatment drugs.

5. 5. The composition for inhibiting cancer cell proliferation according to any one of claims 1 to 4, which is a molecular targeted cancer therapeutic drug.

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