JP2023086478A - Cancer cell proliferation inhibitory composition - Google Patents

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 The present invention relates to a composition containing a cyclic anthraquinone derivative and suppressing the growth of cancer cells.

In recent years, as a method of treating cancer, treatment using a molecularly targeted therapeutic drug (molecularly targeted treatment) has been widely performed. In contrast to conventional chemotherapy, which combines and administers effective anticancer drugs mainly for each cancer site (e.g., lung cancer), molecular targeted therapy uses molecules specific to the patient's cancer cells. It administers drugs that target and attack abnormal levels.

Since this molecular-targeted therapy targets cancer cells, it is believed that it has relatively little adverse effect on normal cells and causes few side effects. Molecular-targeted therapy also has the advantage of being able to predict to some extent whether the drug will be effective or not before administration.

In targeted cancer therapy, except for the well-known mutated kinases or those targeting specific cancer microenvironments, the c-myc oncogene that promotes increased cell proliferation, telomerase in telomere elongation It has been reported that abnormally expressed DNA of a specific gene such as hTERT, which accelerates the action, is highly correlated with cancer development (see, for example, Non-Patent Document 1). is expected to be a promising target for molecularly targeted cancer therapy.

As described above, various studies have been conducted on molecular target cancer therapy, and new therapeutic agents useful for molecular target cancer therapy are desired.

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 nogaramycin, have been used as classical chemical cancer therapeutic agents, but are known to cause side effects such as cardiomyopathy (see, for example, Non-Patent Documents 2 and 3).

Anthraquinone is also known to act as a binding agent to a guanine quadruplex (G-quadruplex; G4) and effectively inhibit telomerase by recognizing and stabilizing the G4 structure ( For example, see Non-Patent Documents 4 and 5). Thus, a small molecule that strongly binds to G4 and has a telomerase inhibitory effect is expected as a therapeutic agent for cancer.

Fumi Nagatsugi, Kazumitsu Onizuka, Functional G-Quadruplex Binding Molecules, Chem. Lett. 2020, Vol.49, No.7,771-780 Suhail A. Islam, S.N., Bijukumar M. Gandecha, Malcolm Partridge, Laurence H.Patterson, & Brown, a.J.R. 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) Singal, P.K. & Iliskovic, N. Doxorubicin-induced cardiomyopathy. N Engl J Med 339, 900-905 (1998). Giuseppe Zagotto, C.S., Lorena Lucatello, Claudia Pivetta, Sergio A. Cadamuro, Keith R. Fox, Stephen Neidle, and Manlio Palumbo Aminoacyl-Anthraquinone Conjugates as Telomerase Inhibitors: Synthesis, Biophysical and Biological Evaluation. J. Med. Chem. 51, 5566-5574 (2008). Collie, G. et al. Selectivity in small molecule binding to human telomeric RNA and DNA quadruplexes. Chem Commun (Camb), 7482-7484 (2009). Zambre, V.P., Murumkar, P.R., Giridhar, R. & Yadav, M.R. Development of highly predictive 3D-QSAR CoMSIA models for anthraquinone and acridone derivatives as telomerase inhibitors targeting G-quadruplex DNA telomere. J Mol Graph Model 29, 229-239 ( 2010).

The present invention has been made in view of the above background, and an object of the present invention is to provide a composition that contains a specific cyclic anthraquinone derivative and suppresses the growth of cancer cells. Another object of the present invention is to provide a composition useful for molecularly targeted cancer therapy.

The present inventors have found that a cyclic anthraquinone derivative having a planar benzene ring in the cyclic portion effectively and selectively inhibits cancer cell growth, and have completed the present invention.

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

［式（Ｉ）中、Ｒ^１は、下記式（ａ－１）～（ａ－５）で示されるいずれかの連結基（※は結合位置を表し、ＮＨ－※が、Ｒ^２に連結される。）であり、Ｒ^２が、下記式（ｂ－１）～（ｂ－５）で示されるいずれかの連結基（※は結合位置を表す。）である。］

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

[2] The composition for suppressing cancer cell growth according to [1] above, wherein the cyclic anthraquinone derivative is represented by the following structural formula.

[3] The composition for inhibiting cancer cell growth according to the above [1] or [2], which inhibits telomerase activity possessed by chromosomes of cancer cells to suppress cancer cell proliferation.
[4] The composition for suppressing cancer cell proliferation according to any one of [1] to [3] above, which induces apoptosis of cancer cells and suppresses proliferation of cancer cells.
[5] The composition for suppressing cancer cell proliferation according to any one of [1] to [4] above, which has less effect on normal cells other than cancer cells compared to chemical cancer therapeutic agents.
[6] The composition for suppressing cancer cell proliferation according to any one of [1] to [5] above, which is a molecularly targeted cancer therapeutic drug.

The composition containing the cyclic anthraquinone derivative of the present invention effectively and selectively suppresses the growth of cancer cells, and can be used for molecular target therapy of cancer.

FIG. 2 is a diagram showing an overview of the evaluation of the cyclic anthraquinone derivative cAQ-mBen of the present invention. FIG. 3 shows an electrophoresis image of the TRAP assay of the cyclic anthraquinone derivative cAQ-mBen of the present invention. FIG. 2 shows the measurement results of the telomerase inhibitory activity (IC ₅₀ value) of the cyclic anthraquinone derivative cAQ-mBen of the present invention. FIG. 2 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. FIG. 2 is a diagram showing measurement results of 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. FIG. 2 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. FIG. 2 is a diagram showing the measurement results of 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. FIG. 3 shows the results of an annexin V/PI staining assay for the cyclic anthraquinone derivative cAQ-mBen of the present invention. FIG. 2 shows the results of Western blot analysis of the cyclic anthraquinone derivative cAQ-mBen of the present invention. FIG. 2 shows changes in tumor volume over time associated with administration of the cyclic anthraquinone derivative cAQ-mBen of the present invention in animal tests using mice. Fig. 3 is a photograph of tumor cells collected from mice after 5 administrations (10 days) of the cyclic anthraquinone derivative cAQ-mBen of the present invention (2 days after the last administration). FIG. 2 is a diagram showing changes in body weight over time in mice following administration of the cyclic anthraquinone derivative cAQ-mBen of the present invention. Fig. 3 shows hematoxylin and eosin-stained images of livers and kidneys collected from mice to which the cyclic anthraquinone derivative cAQ-mBen of the present invention was administered. 1 is a graph showing the sizes of glomeruli in livers and kidneys collected from mice to which the cyclic anthraquinone derivative cAQ-mBen of the present invention was administered. FIG. 2 shows the results of measuring physiological indices (BUN, UA, AST, ALT) in biochemical analysis of serum collected from mice to which the cyclic anthraquinone derivative cAQ-mBen of the present invention was administered.

[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 form of the cyclic anthraquinone derivative may be a free form or a pharmacologically acceptable salt form. Further, the anthraquinone structure may have a substituent as long as the effects of the present invention are exhibited.

In formula (I), R ¹ is a divalent organic linking group represented by formulas (a-1) to (a-5) below, and the linking group represented by formula (a-1) is particularly preferable. In formulas (a-1) to (a-5), * represents a bonding position, and NH-* is linked to ^R2 .

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 preferable. In formulas (b-1) to (b-5), * represents a bonding position.

That is, the following can be exemplified as particularly preferred cyclic anthraquinone derivatives (I).

[Method for producing a 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 a 1,5-dihaloanthraquinone represented by the following structural formula as a starting material. The halogen atom (X) of 1,5-dihaloanthraquinone includes a chlorine atom, a bromine atom, an iodine atom and the like, with a chlorine atom being preferred. That is, 1,5-dichloroanthraquinone is preferred.

Amine compounds represented by the following formulas (a'-1) to (a'-5) are reacted with 1,5-dihaloanthraquinone as a starting material.

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

Subsequently, the compounds are reacted with dicarboxylic acids represented by the following formulas (b'-1) to (b'-5).

As a result, when the amine compound represented by formula (b'-1) is reacted, the cyclic anthraquinone derivative of the present invention (compound cAQ-mBen) represented by the following structural formula can be obtained (specifically, Examples preparation of the compound cAQ-mBen).

Each of the above reactions can be carried out under general reaction conditions. For example, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO) and the like can be used as solvents. The reaction temperature may be about 0 to 100°C, preferably about 30 to 80°C.

[Composition of the present invention]
The composition of the present invention is characterized by containing the cyclic anthraquinone derivative represented by the 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 general formula (I), other pharmaceutically acceptable ingredients such as solvents and excipients.

Since the composition of the present invention contains the cyclic anthraquinone derivative represented by the above general formula (I), it stabilizes the guanine quadruplex (G-quadruplex) that constitutes the telomere, which is a part of the chromosome of cancer cells. and inhibits the telomerase activity of cancer cell chromosomes. Furthermore, it can selectively induce apoptosis of cancer cells. Therefore, the composition of the present invention is useful as a molecularly targeted cancer therapeutic agent.

The composition of the present invention can be used as an oral agent and a parenteral agent. Forms of oral preparations include tablets, capsules, powders, granules, rods, plates, blocks, solids, rounds, caplets, chewables, liquids, pastes, creams, and gels. and the like. Parenteral agents include, for example, intravenous administration agents, intramuscular administration agents, subcutaneous administration agents, transdermal administration agents and the like.

The composition of the present invention can be used, for example, as a pharmaceutical composition, and can also be used as food compositions such as food compositions with function claims and food compositions for specified health uses.

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

The cyclic anthraquinone derivative (cAQ-mBen) of the present invention was produced. As comparative examples, the compound AQ-Ac, which is an acyclic anthraquinone derivative, and the compounds cAQ-C2 and cAQ-ch, which are cyclic anthraquinone derivatives having no benzene ring in the cyclic portion, were produced. Hereinafter, the cyclic anthraquinone derivative is sometimes referred to as cAQ derivative, and the non-cyclic anthraquinone derivative is sometimes referred to as AQ derivative.
An outline of the manufacturing process is shown below.

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

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 placed in an oil bath at 140°C. After refluxing for 4 hours, the mixture was cooled to room temperature. After the reaction solution was poured into 500 mL of 2 mol/L sodium hydroxide and stirred for 20 minutes, the precipitate was collected by suction filtration and vacuum drying. The resulting sticky black-red solid was dissolved in a minimum amount of methanol and reprecipitated in 300 mL of 2 mol/L sodium hydroxide. After that, the precipitate was collected by suction filtration and vacuum-dried again to obtain compound 1 (yield 30%, 0.76 g) as a viscous black-red solid.

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

(Production of compound cAQ-mBen)
Subsequently, using compound 1 above, a compound cAQ-mBen represented by the following structural formula was produced.

Specifically, 0.39 g of compound 1 (0.78 mmol) was added to 120 mL of DMF and stirred at room temperature. Then 0.14 g isophthalic acid (0.78 mmol), 0.89 g HATU (0.22 mmol) were dissolved in 100 mL DMF, then the mixture was added dropwise to compound 1 for 1.5 hours. All mixture was added to Compound 1 and then 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 isolated by filtration.
The filtrate was then evaporated and purified by silica chromatography (CHCl3:MeOH:DEA=1:0.05:0.03), collecting fractions with an Rf value of 0.2. After evaporation and vacuum drying, compound cAQ-mBen (3% yield, 12 mg) was obtained as a purple solid.

The purity of compound cAQ-mBen was confirmed by RP-HPLC, MALDI-TOF-MS and ¹ H-NMR.
MALDI-TOF-MS (positive mode, DHBA) m/z=626.939 (calcd for C36H44N6O4+H+=625.781); ¹ H-NMR (400 MHz, CDCl ₃ , δ ppm): 1.80 (quin, 4H, J = 6.1Hz), 1.90 (quin, 4H, J = 5.9Hz), 2.31 (s, 6H), 2.52 (t, 4H, J = 6.1Hz), 2.60 (t, 4H, J = 6.4Hz), 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).

<Production of compound AQ-Ac as a comparative example>
Compound AQ-Ac represented by the following structural formula was produced using Compound 1 above.

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 azeotroping was carried out four times by adding 5 mL of toluene. The target compound was purified by silica chromatography (CHCl3:MeOH:DEA=1:0.1:0.1), and fractions with an Rf value of 0.2 were collected. After evaporation and vacuum drying, compound AQ-Ac (22% yield, 122 mg) was obtained as shiny red crystals.

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

<Production of compound cAQ-C2 as a comparative example>
Compound cAQ-C2 represented by the following structural formula was produced using compound 1 above.

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, then the mixture was added dropwise to compound 1 in 1.5 hours. After all the mixture was added to compound 1, it was stirred for 17 hours. The solvent was then removed by evaporation. The target compound was purified by RP-HPLC (Shimazu, Japan) (column: InertsilODS-4; flow rate, 2.7 mL/min; wavelength, 210 nm; column temperature, 40°C). The 17 minute retention time peak was collected and dried by lyophilization. Compound cAQ-C2 (9% yield, 28 mg) was obtained as a red solid.

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

<Production of compound cAQ-ch as a comparative example>
Compound cAQ-ch represented by the following structural formula was produced using compound 1 above.

Specifically, 0.25 g of compound 1 (1.5 mmol) was added to 350 mL of DMF and stirred at room temperature. Then 0.32 g of 1,1-cyclohexane-diacetic acid (1.6 mmol), 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 desired compound was purified by silica chromatography (CHCl3:DEA=1:0.07), collecting fractions with an Rf value of 0.3. After evaporation and vacuum drying, compound cAQ-ch (9% yield, 86 mg) was obtained as a red solid.

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

The effects of the compound cAQ-mBen, which is a cyclic anthraquinone derivative of the present invention, on cancer cells were evaluated. The outline is shown in FIG.

[TRAP assay]
The TRAP assay (Assay Telomere Repeat Amplification Protocol Assay) confirmed the ability of compound cAQ-mBen to inhibit telomerase.

The TRAP assay used the TRAPeze Telomerase Detection Kit (EMDMillipore, Billerica, MA).

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

The specific procedure involved first an extension step at 30°C for 60 minutes, followed by incubation at 95°C for 5 minutes. PCR was then performed for 35 cycles (94°C, 1 min; 62°C, 1 min; 72°C, 1 min).

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. In addition, IC ₅₀ values were calculated.

FIG. 2 shows the electrophoresis image, and FIG. 3 shows the IC ₅₀ value results.
As shown in FIG. 2, compound cAQ-mBen clearly decreased the shift band and inhibited elongation of telomeric DNA as the concentration increased. In addition, as shown in FIG. 3, the compound cAQ-mBen of the present invention exhibited the strongest inhibitory ability with an IC ₅₀ of about 4.3 μm compared with other cAQ derivatives according to comparative examples.
These results suggest that the compound cAQ-mBen of the present invention inhibits the function of telomerase and shortens telomere DNA length.

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

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

(Preparation of bone marrow cells)
Bone marrow cell preparation was performed as follows.
Bone marrow cells (BMC) were harvested from femurs and tibias of 8-10 week old male mice (ddY strains: Deutschland, Denken, Yoken). The BMCs were added to Eagle's minimum essential medium α-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). suspended in

(cell proliferation assay)
These cell suspensions were plated in 96-well tissue culture plates and cell proliferation assays were performed.
Cell proliferation was evaluated using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto). Specifically, cells plated in 96-well plates were cultured for 24 hours and stabilized, then treated with compound cAQ-mBen for 48 hours, followed by CCK-8 assay. For comparison, instead of compound cAQ-mBen, compound AQ-Ac, compound cAQ-C2, compound cAQ-ch, and cisplatin (CDDP), which is one of the typical anticancer drugs, were used.

(Measurement of _IC50 value)
_IC50 values were calculated from the results of cell proliferation assays. Table 1 shows the results.

As shown in Table 1, the compound cAQ-mBen has an _IC50 value of approximately 1 μm or less, indicating a high ability to suppress the proliferation _of cancer cells. rice field. In particular, the IC ₅₀ value of Hela was 0.3 μm, which is nearly 20 times lower than that of normal cell lines such as mouse bone marrow cells.
In contrast, the compound cAQ-C2, compound cAQ-ch, and cisplatin (CDDP) of the comparative examples showed an equivalent or stronger growth inhibitory effect on normal cells than on cancer cell lines.

[Investigation of TERT mRNA expression level]
To investigate whether the suppression of cell proliferation of the compound cAQ-mBen correlates with the inhibition of telomerase, the expression level of TERT mRNA, the rate-limiting determinant controlling telomerase activity, was examined in different cancer cell lines and normal cells. I checked the stock.

The results are shown in FIGS. 4 and 5. FIG.
As shown in FIG. 4, in normal cell lines using NHEK and BMC, almost no TERT mRNA expression was detected compared to human cancer cell line Ca9-22 and mouse cell line 3T3-E1. As shown in , in Ca9-22, clearly stronger cell growth suppression was observed than in the other two normal cell lines (NHEK, BMC).

Next, we investigated the relationship between hTERT expression levels and the effects of the compound cAQ-mBen in four different human cancer cell lines.

The results are shown in FIGS. 6 and 7. FIG.
As shown in FIG. 6, the TERT mRNA expression levels in Ca9-22 and HeLa were about 2-fold higher than those of HEK293 and HSC-2. Also, as shown in FIG. 7, the proliferation of all cells was dose-dependently inhibited by the compound cAQ-mBen, but the inhibition curves for Ca9-22 and HeLa were higher than those for HEK293 and HSC-2. The inhibition pattern was positively correlated with the TERT expression level in these cells. From this, it is considered that the compound cAQ-mBen tends to exert its inhibitory effect more sensitively on cell lines with high telomerase activity.

[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, followed by Annexin V-FITC Apoptosis was detected using the Apoptosis Detection Kit (Nacalai Tesque, Kyoto, Japan) or Western blotting that monitors the digestion of procasese 3 and PARP.

(Annexin V/PI staining assay)
SAS cells cultured for 24 hours were treated with compound cAQ-mBen at 5 μM or CDDP at 50 μM for 24 hours.
Drug-treated cells were lysed for 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 (BZ9000, Keyence, Osaka, Japan) equipped with a digital camera.

(Western blot analysis)
Drug-treated cells were washed with ice-cold phosphate-buffered saline and lysed with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% sodium dodecyl sulfate, 10% glycerol, 1% β-mercaptoethanol). Total protein was extracted using a cytoplasmic hemocytometer and subjected to Western blotting.

Blots were developed with a secondary antibody conjugated with horseradish peroxidase and visualized using the enhanced chemiluminescent substrate reagent ImmunoStar LD (Fujifilm Wako) or Immobilon (Merck-Millipore, Billerica, Mass., USA). Images were acquired using a LAS-3000mini (Fujifilm, Tokyo, Japan). Antibodies to caspase-3 and poly (ADP-ribose) polymerase (PARP) (Abcam, Cambridge, UK), antibodies to β-actin, horse rabbit peroxidase labeled anti-mouse or anti-rabbit antibodies were used.

FIG. 8 shows the results of annexin V/PI staining assay, and FIG. 9 shows the results of Western blot analysis.
As shown in FIG. 8, when cultured for 24 hours in the presence of 5 μM of the compound cAQ-mBen, about 30% of the cells responded to AnnexinV (indicating early apoptotic cells), and about 10% of the cells responded to PI signals (necrosis). cells) were positive, both at levels comparable to SAS cultured with 50 μM CDDP. Moreover, as shown in FIG. 9, similar apoptosis-inducing effects of 5 μM cAQ-mBen and 50 μM CDDP were confirmed by detecting clear digestion of PARP and Caspase-3 by Western blotting.

These results suggested that compound cAQ-mBen induces significant apoptosis in tumor cells.

[Animal test]
We investigated the effects of the compound cAQ-mBen on tumor growth and side effects at the animal level. CDDP was used as a chemotherapy control.

Evaluation was performed using mice generated by injecting SAS cells.
Specifically, first, 4-week-old male KSN/Slc nude mice purchased from Japan SLC Co., Ltd. (Shizuoka, Japan) were housed at a constant room temperature with a 12-h light-dark cycle, and then fed with standard rodents. Chow and water were provided ad libitum. After breeding the mice under these conditions for more than one week, the experiment was started.

SAS cells were harvested from subconfluent cultures by trypsinization and suspended in serum-free DMEM. This cell suspension (5.0×10 6 cells, 0.2 ml/mouse) was injected subcutaneously into the back of nude mice. Seven days after cell inoculation, 27 mice with xenograft tumors of approximately 50 mm ³ were randomly divided into 3 groups.

The volume of the tumor was obtained by measuring the length and width using vernier calipers and using the following formula.

Tumor volume ( ^mm3 ) = length (mm) x width (mm)

Nine mice in each group were intraperitoneally injected every 2 days with compound cAQ-mBen at 0.003 mmol/kg, CDDP at 0.030 μmol/kg, and saline as a control.

(tumor tissue analysis)
FIG. 10 shows changes in tumor volume over time following administration of the compound cAQ-mBen of the present invention. In addition, in FIG. 10, the tumor volume after 10 days shows the control, the compound cAQ-mBen of the present invention, and CDDP from the top. In addition, FIG. 11 shows photographs of tumor cells collected from mice after 5 administrations (10 days) of the compound cAQ-mBen of the present invention (2 days after the last administration). Moreover, FIG. 12 shows changes in body weight of mice over time associated with the administration of the compound cAQ-mBen of the present invention.

As shown in FIG. 10, the tumor volume continued to increase in the absence of chemical administration, whereas the increase in tumor volume was significantly suppressed in mice administered with the compounds cAQ-mBen and CDDP. As shown in FIG. 11, the effect is clear when comparing the size of tumor cells after 10 days. Although the dose of the compound cAQ-mBen was 10 times lower than that of CDDP, a tumor growth inhibitory effect equivalent to that of CDDP was obtained.

On the other hand, as shown in Figure 12, CDDP clearly decreased the body weight of mice, while the compound cAQ-mBen had little effect on body weight during the test period.

(Histopathological analysis of liver and kidney)
Livers and kidneys harvested from mice after 5 doses (10 days) of drug (2 days after the last dose) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. After staining the obtained sections with a hematoxylin and eosin staining kit (Muto Pure Chemical Co., Ltd., Tokyo, Japan), images were acquired with an optical microscope (BZ9000; Keyence, Osaka, Japan) equipped with a digital camera.

FIG. 13 shows a photographed image of hematoxylin-eosin staining, and FIG. 14 shows a graph showing the size of glomeruli.
As shown in Figures 13 and 14, CDDP-administered mice exhibited similar pathological changes in the liver and kidneys, with significantly smaller glomeruli, whereas cAQ-mBen-administered mice exhibited similar pathological changes. In the kidney, there was no effect on glomerular cell arrangement or size.

(blood analysis)
Whole blood was collected from mice after 5 doses (10 days) of drug administration (2 days after the last dose), and then centrifuged at 2800 xg for 20 minutes at 25°C to prepare serum. The serum was subjected to biochemical analysis at Oriental Yeast Co., Ltd. (Shiga Prefecture).

The results are shown in FIG. The left side of the graph is the control, the middle side is the compound cAQ-mBen of the present invention, and the right side is CDDP.
As shown in FIG. 15, in CDDP-administered mice, various physiological indices (BUN, UA, AST, ALT) were significantly elevated, confirming damage to the liver and kidneys. In contrast, there was no significant difference between the compound cAQ-mBen administration group and the control group, and compound cAQ-mBen had little effect on these tissues.

From the results of the above tumor tissue analysis, liver and kidney histopathological analysis, and blood analysis, it was found that the compound cAQ-mBen exhibits a strong tumor growth inhibitory effect comparable to that of CDDP. It was also suggested that the compound cAQ-mBen is more specific to tumor tissue and has less adverse effects on normal tissue in mice.

INDUSTRIAL APPLICABILITY The novel cyclic anthraquinone derivative of the present invention is industrially useful because it can be used for molecularly targeted therapy of cancer.

Claims (6)

A composition for inhibiting cancer cell growth, comprising a cyclic anthraquinone derivative represented by the following formula (I).

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

2. The composition for inhibiting cancer cell proliferation according to claim 1, wherein the cyclic anthraquinone derivative is represented by the following structural formula.

3. The composition for suppressing cancer cell proliferation according to claim 1, which inhibits telomerase activity of cancer cell chromosomes and suppresses proliferation of cancer cells. 4. The composition for suppressing cancer cell proliferation according to any one of claims 1 to 3, which induces apoptosis of cancer cells and suppresses proliferation of cancer cells. 5. The composition for suppressing cancer cell proliferation according to any one of claims 1 to 4, which has less effect on normal cells other than cancer cells compared to chemical cancer therapeutic agents. 6. The composition for suppressing cancer cell proliferation according to any one of claims 1 to 5, which is a molecularly targeted cancer therapeutic drug.

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