WO2023059148A1 - Use of 2-chloro-n,n-diethylethylamine hydrochloride for improving anti-cancer treatment - Google Patents

Abstract

The present invention relates to use of 2-chloro-N,N-diethylethylamine hydrochloride for improving anti-cancer treatment, and has the benefits of killing cancer cells by alkylating a DNA base using a nitrogen mustard-based compound of chemical formula 1, and exhibiting a synergistic effect when used in combination with existing PARP inhibitors.

Description

Use of 2-chloro-N,N-diethylethylamine hydrochloride for improving anti-cancer treatment

The present invention relates to the use of 2-chloro-N,N-diethylethylamine hydrochloride for improving cancer treatment, and more particularly, to kill cancer cells by alkylating DNA bases using a nitrogen mustard-based compound of Formula 1. It relates to a pharmaceutical composition for preventing or treating cancer that exhibits a synergistic effect by using in combination with an existing PARP inhibitor.

Poly (ADP-ribose) polymerase (hereinafter referred to as PARP) promotes DNA repair by binding to DNA damage and forming ADP-ribose polymers that attract repair enzymes, and base excision repair It is the major enzyme in single-strand cleavage by the pathway. When left unrepaired, single-strand breaks convert to double-strand breaks that are essentially repaired by homologous recombination during replication. Thus, inhibiting PARP is lethal in cells deficient in homologous recombination. These observations have already led to the development of PARP inhibitors to treat cancers with mutations that preclude the use of homologous recombination capacity.

As PARP is involved in a number of cellular processes, the mechanism of action of PARP inhibitors in tumor cells is not fully elucidated. The patient currently has a specific tumor type (e.g., high-grade serous ovarian cancer or triple negative brain cancer) or whose cancer is a subtype of a related molecule (e.g., BRCA1/2-mutated breast cancer, ovarian cancer, pancreatic cancer, or prostate cancer). ) is considered as a PARP inhibitor test.

PARP inhibitor (PARPi) monotherapy has shown promising efficacy and safety profiles in the clinic, but their major limitations are the need for HR deficiency and the rapid emergence of resistance. Many tumors that initially respond to PARPi treatment eventually relapse through compensatory mutations that either restore HR activity or stimulate the activation of alternative repair pathways. Therefore, the use of PARP inhibitors is limited to specific tumor types and has a problem in that they cannot be used in any cancer treatment.

In order to solve this problem, various compositions used in combination with PARP inhibitors are currently being used.

Korean Patent Publication No. 10-2018-0051500 uses a combination of a debate molecule and a PARP inhibitor for cancer treatment, and Korean Patent Publication No. 10-2018-0037210 discloses a combination using liposomal irinotecan and a PARP inhibitor for cancer treatment. therapy is initiated. However, when such combination therapy is used for a long period of time, there is a problem in that cancer cells resistant to various mutations do not die even after chemotherapy.

Therefore, in order to solve this problem, the present inventors reduced the toxicity by removing one halogen group from the existing highly toxic bis-(2-chloroethyl)ethylamine, and alkylated the DNA base using a compound with significantly reduced toxicity. By doing so, it was confirmed that it is possible to provide a pharmaceutical composition for preventing or treating cancer that can kill cancer cells and produce a synergistic effect by using it in combination with an existing PARP inhibitor, thereby completing the present invention.

[Prior art literature]

[Patent Literature]

Republic of Korea Patent Publication No. 2007-0120/163

[Non-Patent Literature]

R.K. Singh et al., European Journal of Medicinal Chemistry doi: 10.1016/j.ejmech.2018.04.001.

Subhendu Karmakar et al., Dalton Transactions DOI: 10.1039/c8dt04503h

Fredrik Lehmann et al., Processes 2021, 9, 377.

Summary of Invention

An object of the present invention is to reduce toxicity by removing one halogen group from bis-(2-chloroethyl)ethylamine, which is highly toxic, and to kill cancer cells by alkylating DNA bases using the compound with significantly reduced toxicity. It is to provide a pharmaceutical composition for preventing or treating cancer that exhibits an effect.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer that produces a synergistic effect by using the compound together with an existing PARP inhibitor.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R is a halogen group.

The present invention also provides a method for preventing or treating cancer disease comprising the step of administering to a subject a pharmaceutical composition for preventing or treating cancer comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. provides

The present invention also provides a use of a pharmaceutical composition for preventing or treating cancer comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

The present invention also provides a use in the manufacture of a drug for preventing or treating cancer comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

1A is a diagram showing the structure of UNI21.

Figure 1b is a graph in which UNI21 is screened as a drug that increases stress through a Luciferase-ATAD5 assay and an assay capable of measuring DNA replication and repair stress (DNA replication & repair stress) according to an embodiment of the present invention. 5-FUrd is a positive control.

1c is a diagram showing the results of measuring cell viability in various KO HAP1 cell lines according to an embodiment of the present invention.

Figure 1d is a diagram showing the results of Western blotting of the cell lines used in Figure 1c according to an embodiment of the present invention.

Figure 1e is a view showing the results of measuring the viability of KO cells with increasing concentrations of UNI21 in HAP1, HCT116, and XP2OS cell lines based on the data measured with 20 μM of UNI21 in Figure 1c according to an embodiment of the present invention.

Figure 1f is a diagram showing the results of confirming the synergistic effect (synergistic effect) with the PARP inhibitor (inhibitor) up parip (Olaparib) in PARP1 KO cells according to an embodiment of the present invention.

Figure 1g is a diagram showing the results of experiments performed under different conditions for oliparib and UNI21 in Figure 1f.

Figure 2a is a schematic diagram showing the in vitro reaction scheme of UNI21 and DNA base according to an embodiment of the present invention.

Figure 2b is a diagram showing the results of representative UPLC-HRAM-PRM tracking of DEAE-purine in UNI21-treated CTDNA. After incubation with UNI21 for 24 h, alkylated purines were released by thermal hydrolysis and concentrated for analysis as described in Materials and Methods. Black traces show UPLC-HRAM-PRM analysis of synthesized DEAE-guanine (panel 1, m/z = 251.1615) and DEAE-adenine (panel 3, m/z = 235.1662) standards. Red traces show UPLC-HRAM-PRM of DEAE-guanine (panel 2) and DEAE-adenine (panel 4) detected in 1.2 μg depurinated CTDNA.

Figure 2c shows representative UPLC-HRAM-PRM traces of enzymatically released DEAE-pyrimidine from 5 μg UNI21-treated CTDNA. The top panel shows DEAE-dC ( m/z = 327.2027) and the bottom panel shows DEAE-dT ( m/z = 342.2023).

3A is a diagram showing the cell cycle of HCT116 after UNI21 treatment according to an embodiment of the present invention. HCT116 wild-type and PARP1 deficient cells were incubated with different doses of UNI21 for 24 hours and the relative percentages of cell cycle phases were calculated with Flow-Jo software.

Figure 3b is a diagram showing the result of confirming the occurrence of DSB due to UNI21 processing with g-H2AX. HCT116 wild-type or PARP1 deficient cells were incubated with 80 μM UNI21 for 24 hours and the indicated protein levels were determined in whole cell extracts.

Figure 3c is a view confirming the result that UNI21 treatment improves more DNA damage in PARP1-deficient cells. The tail moment of the CometChip® analysis was calculated using Comet analysis software (Trevigen).

3D is a view confirming the result that UNI21 treatment induces more frequent SCE in HCT116 parp1 KO cells.

FIG. 3E is a diagram showing the BX53 distribution of FIG. 3D with SCEs imaged.

[Correction under Rule 91 28.11.2022]
3f is a diagram confirming that UNI21 treatment induces abnormal chromosomes in HCT116 parp1 KO cells. Figure 3g is a diagram of chromosomal breaks imaged by the BX53 G distribution in Figure 3F.

Figure 3h is a diagram confirming that the percentage of cells with 25 or more breaks per metaphase increased in HCT116 parp1 KO cells.

Figure 3i is a diagram confirming that UNI21 treatment induces more apoptosis in PARP1-deficient cells. Apoptotic cell death was quantified using Annexin V Alexa Fluor™ 488 conjugate and analyzed by flow cytometry. Data are presented as mean ± SEM.

Figure 4a is a schematic diagram showing a mouse xenograft experiment for confirming the in vivo effect of UNNI21 that UNI21 inhibits the growth of PARP1-deficient xenograft tumors in nude mice according to an embodiment of the present invention. Four million cells of either WT HCT116 or PARP1 deficient HCT116 cells were injected subcutaneously into 7-week-old male nude mice. When tumor size reached approximately 200 mm ³ , vehicle (PBS) or UNI21 (6 mg/kg) was injected intratumorally every 3 days for 16 days. The indicated assays were performed after mice were euthanized.

Figure 4b is a photograph of a PARP1 KO tumor harvested 16 days after treatment with UNI21 according to an embodiment of the present invention.

Figure 4c is a graph showing the tumor volume (tumor volume) of Figure 4b.

Figure 4d is a graph showing the results of tracing UNI21 in tumors injected every 3 days according to an embodiment of the present invention.

Figure 4e is a view showing the results of H&E staining, TUNEL assay, and γ-H2AX IHC after sectioning the harvested tumor according to an embodiment of the present invention.

5 is a diagram showing the results of an experiment using a structure in which bromine is bonded instead of chlorine according to another embodiment of the present invention.

6A to 6D are diagrams illustrating results of mass spectrometry performed based on the schematic diagram shown in FIG. 2A.

7 to 7d are diagrams illustrating the results of mass spectrometry of the red graph shown in FIG. 2b.

DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, the pharmaceutical composition for treating cancer comprising the compound of Formula 1 and a pharmaceutically acceptable salt thereof can be applied to various cancer cells resistant to PARP inhibitors to selectively and effectively kill them, and moreover, conventional PARP inhibitors It was confirmed that it exhibits a synergistic effect by using in combination with

Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R is a halogen group.

Hereinafter, the present invention will be described in more detail.

The terms used to define the compounds according to the present invention have the following meanings.

Specific examples of the term “halogen” include fluorine (F), chlorine (Cl), bromine (Br), and iodine (I), and in particular, chlorine (Cl) or bromine (Br).

The term "treatment", when used on a subject exhibiting symptoms of disease, means stopping or delaying the progression of a disease.

The term "pharmaceutical composition" may include a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof together with the compound of the present invention, if necessary.

The term "pharmaceutically acceptable" means a property that does not impair the biological activity and physical properties of a compound.

The term "carrier" refers to a substance that facilitates the addition of a compound into a cell or tissue.

The term "diluent" is defined as a substance that is diluted in water that not only stabilizes the biologically active form of the subject compound, but also dissolves the compound.

The term “excipients” refers to substances that are added for the purpose of giving a drug an appropriate hardness or shape, or to give a certain volume or weight to a size that is easy to handle when the amount of the main agent is small. it means.

Other terms and abbreviations used in this specification may be interpreted as meanings commonly understood by those skilled in the art to which the present invention belongs unless otherwise defined.

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 represented by Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R is a halogen group.

According to a more specific embodiment of the present invention, the compound of Formula 1 may be a compound represented by Formula 2 or Formula 3 below.

[Formula 2]

[Formula 3]

In the present invention, the compound of Formula 1 may form a pharmaceutically acceptable salt thereof. In the present invention, a pharmaceutically acceptable salt thereof may be used with an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and the like. organic acids such as tartaric acid, formic acid, citric acid, acetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, lactic acid, malonic acid, malic acid, salicylic acid, succinic acid, oxalic acid, propionic acid, aspartic acid, glutamic acid, citric acid, and the like; It may be an acid addition salt formed with sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and naphthalenesulfonic acid. Preferably, the pharmaceutically acceptable salt thereof may be selected from the group consisting of HCl salt, HBr salt, HI salt, H ₂ SO ₄ salt, HNO ₃ salt, and combinations thereof.

The compound of Formula 1 according to the present invention may be converted into a salt thereof by a conventional method, and the preparation of the salt may be easily performed by a person skilled in the art based on the structure of Formula 1 without a separate explanation.

Unless otherwise stated below, the compound of Formula 1 includes pharmaceutically acceptable salts thereof, and all of them should be construed as being included in the scope of the present invention. For convenience of explanation, in the present specification, they are simply expressed as compounds of Formula 1.

The cancer is squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral cancer, osteosarcoma, gallbladder cancer, kidney cancer, leukemia, bladder cancer, melanoma , brain cancer, glioma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, esophageal cancer, colorectal cancer, stomach cancer, cervical cancer, prostate cancer, ovarian cancer, head and neck cancer, and rectal cancer.

In addition, the present invention can obtain a synergistic effect by using a composition that further includes a PARP inhibitor in combination therapy.

In the present invention, the PARP inhibitor is 1 from the group consisting of olaparib, talazoparib, niraparib, rucaparib, veliparib and pamiparib More than one species can be selected.

In another aspect, the present invention treats cancer disease comprising the step of administering to a subject a pharmaceutical composition for preventing or treating cancer disease comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. or prevention methods.

In another aspect, the present invention relates to the use of a pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, and a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. It provides use in the manufacture of a medicament for preventing or treating cancer containing a salt.

Specifically, the present invention relates to a pharmaceutical composition comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, capable of treating or preventing cancer. Specifically, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof of the present invention can be used as a cancer treatment agent.

In the present invention, the quinoline derivative of Chemical Formula 1 or a pharmaceutically acceptable salt thereof has an activity of inhibiting tumor growth by alkylating DNA.

The excellent anticancer effect of the compound of Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention is determined by screening through a luciferase-ATAD5 assay (DNA replication & repair stress assay), repair gene KO HAP1, Measurement of cell viability in HCT116 and U2OS cell lines, synergistic effect in combination therapy with Olaparib, and relative amount of alkylated DNA bases through mass spectrometry It was demonstrated through in vitro experiments such as comparative analysis of alkylation priority (dG>>dA>dC~dT) and in vivo experiments with nude mouse HCT116 PARP1 KO cell xenotransplantation. This will be described later in the following embodiments.

According to one embodiment of the present invention, a salt dissolved in a buffer solution is used as a diluent, and a commonly used buffer solution may be a phosphate buffered saline solution that mimics the salt form of a human solution. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents do not modify the biological activity of the compound.

A compound of the present invention may be formulated for use in a pharmaceutical or veterinary composition that also contains a pharmaceutically or veterinarily acceptable carrier or diluent. The composition according to the present invention can be generally prepared according to a conventional method and administered in a pharmaceutically or veterinarily appropriate form.

The pharmaceutical composition of the present invention is administered orally in the form of tablets, capsules, sugar-coated, film-coated tablets, liquid solutions or suspensions, or parenterally by way of injection or infusion subcutaneously, intramuscularly or intravenously. can be administered.

The dosage may be determined according to various factors including the age, weight and condition of the patient and the route of administration. The daily dose can vary within wide limits and can be adjusted to the individual requirements in each individual case. However, in general, when the present compound is administered alone to an adult, the dosage adopted for each route of administration is 0.0001 to 50 mg/kg body weight, for example 0.01 to 1 mg/kg in the range of 0.001 to 10 mg/kg body weight. You can do it by weight.

Such administration doses may be given, for example, from 1 to 5 times per day. For intravenous injection, a suitable daily dose is 0.0001 to 1 mg/kg body weight, preferably 0.0001 to 0.1 mg/kg body weight. The daily dose may be administered as a single dose or according to a divided dose schedule.

The compounds according to the present invention can specifically alkylate cancer cells. In normal cells, PARP1, which is important for Base Excision Repair (BER), or xeroderma pigmentosum group A protein (XPA), which is important for Nuclear Excision Repair (NER), repairs these alkylated bases, whereas PARP1 or XPA, which is important for Nuclear Excision Repair (NER), repairs these alkylated bases. Cancer cells do not properly recover these alkylated bases, and thus have the effect of specifically killing them.

Hereinafter, in order to help the understanding of the present invention, it will be described in more detail through examples, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, and it is natural that such variations and modifications fall within the scope of the appended claims.

[Example]

cell line

HCT116 (ATCC), XP2OS (XPA mutation c.390-1G>C (IVS3-1G>C) to create a splicing acceptor in exon 3), XP2OS expressing WT XPA (XPA complementary, this example [17] U2OS (ATCC ^® HTB-96), HEK293T ATAD5-LUC [15] Cells were cultured in 10% fetal bovine serum (FBS, Merk), 1% antibiotic-antimycotic (penicillin 10000 units/mL, streptomycin 10000 μg/mL, Fungizone ^® (Amphotericin B) 25 μg/mL, Gibco ^® ) were incubated at 37° C. in the presence of 5% CO ₂ . HAP1 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum and 1% antibiotic-antimycotic at 37°C in the presence of 5% CO ₂ . U2OS cells containing DR-GFP (homologous recombination), SA-GFP (single-stranded annealing), EJ2-GFP (micro-homology mediated end junctions), or EJ5-GFP (non-homologous end junctions) reporters were cultured in 10% fetal bovine serum. , DMEM containing 1% penicillin/streptomycin (penicillin 10000 units/mL, streptomycin 10000 μg/mL, ^Gibco® ) and 2 μg/ml puromycin.

Transduction of lentiviral cell lines to create XP2OS cells complementary to WT XPA

SV40-transformed human fibroblasts XP2OS (XPA mutant) were cultured in DMEM (Cytiva) supplemented with 10% fetal bovine serum (FBS, millipore) and 1% penicillin/streptomycin at 37°C in the presence of 5% CO ₂ . It became. For lentiviral transduction, 0.75 μg of the pWPXL expression vector containing the XPA cDNA, 2.25 μg of the pMD2.G envelope plasmid and 2.25 μg of the psPAX2 packaging plasmid were established using Lipofectamine 3000 (L3000001, Invitrogen). HEK293T cells were transfected following the protocol and harvested one day later (Salmon, P. et al., Curr Protoc Neurosci, 2006. Chapter 4: p. Unit 4 21). One day before transfection, XP2OS cells were seeded in 6-well plates at 50% confluency and incubated with lentivirus at a multiplicity of infection of 2 for 24 hours and then grown as described above.

Plasmids, Chemicals and Antibodies

I -SceI (pCAGGS-I-SceI, referred to as pCBASce), empty vector (pCAGGS-BSKX) and dsRED vector were prepared as previously described (Bennardo, N., et al., PLoS Genet, 2008. 4(6) ): p. e1000110; Bennardo, N., et al., PLoS Genet, 2009. 5(10): p. e1000683; : p. 12411-6). Bromo- N,N -diethylethanamine hydrobromide, 2-chloro-N,N-diethylethanamine hydrochloride, 2,2,2-trifluoroethanol, 5-FUrd, Adenine, Cytosine, Guanine, Methyl methanesulfonate (MMS), sodium acetate, thymidine were prepared by Sigma - Purchased from Aldrich. Olaparib was purchased from Selleckchem (AZD2281). Anti-XPA (sc-853) and anti-alpha-tubulin (DM1A, sc-32293) were purchased from Santacruz. Anti-γH2AX (Ser139, 05-636) was purchased from Merck Millipore. Anti-PARP1 (ALX-210-302-R100) was purchased from Enzo Life Sciences.

ATAD5-luciferase assay

HEK293T ATAD5-LUC cells (Fox, J.T., et al., Proc Natl Acad Sci USA, 2012. 109(14): p. 5423-8) were plated in 96-well white black plates (Costar) at a density of 15,000 cells per well. was plated with After 24 hours, cells were treated with 5-FUrd and UNI21 and incubated for an additional 24 hours. For luciferase activity, One-Glo luciferase reagent (Promega) was added to each well, and the luminescence intensity was measured using a Synergy NEO2 Hybrid Multi-Mode Reader (BioTek).

Immunoblot analysis

Whole cell extracts were isolated and immunoblot analysis was performed as previously described (Lee, KY, et al., J Cell Biol, 2013. 200(1): p. 31-44). Briefly, RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, Halt's Protease & Phosphatase The whole cell extract was isolated by incubating the cells on ice for 40 min with Benzonase ^® nuclease in Single-Use Inhibitor Cocktail), sonicated and centrifuged. For immunoblot analysis, proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated for 20 minutes in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) supplemented with 5% skim milk for blocking, followed by overnight incubation with primary antibodies. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Enzo Life Sciences) for 1 hour in 1:5,000 diluted TBS-T. Signals were detected using enhanced chemiluminescent reagents (Thermo Fisher Scientific) by an automated imaging system (ChemiDoc™; Bio-Rad Laboratories).

flow cytometry

Cells were washed with PBS and fixed overnight with 70% ethanol in PBS. The fixed cells were then washed with PBS and incubated with 0.2 mg/mL RNase A in PBS at 37°C for 1 hour. DNA was stained with 10 μg/mL propidium iodide in PBS. Flow cytometry was performed on a FACSVerse™ flow cytometer using BD FACSuite™ software (BD Biosciences). Data analysis was performed using FlowJo software.

Apoptosis assay

Cell death was quantified using a BD FACSVerse instrument with Annexin V Alexa Fluor™ 488 conjugate (A13201, Thermo Fisher Scientific) and Flow-Jo software (version 10) according to the manufacturer's instructions.

COMET analysis

COMET analysis was performed using the CometChip® (Trevigen, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, single cell suspensions were prepared in 6 ml medium with a density of 1.0 X 10 ⁵ cells/ml. Aliquots of 100 μl cells per well were applied to the CometChip and incubated in a tissue culture incubator for 10 minutes with gentle shaking 3 times at 10 minute intervals to spread the cells evenly. The medium was removed and each CometChip in the 96-well CometChip® system was gently washed twice with 5 mL PBS. The CometChip was then covered with 6 mL of 1% 45°C low-melting agarose in PBS. After solidification of the agarose, the slides were immersed in a lysing solution (Trevigen) overnight at 4°C. The CometChip was equilibrated twice in alkaline solution at 4°C for 20 minutes, electrophoresed in alkaline solution at 22V for 50 minutes at 4°C, and neutralized in fresh 0.4M Tris (pH 7.4) buffer for 15 minutes at 4°C. Then, it was equilibrated in 20 mM Tris (pH 7.4) buffer at 4° C. for 30 minutes. The CometChip's DNA was stained with 0.2X SYBR® Gold in 20 mM Tris (pH 7.4) buffer for 2 hours at room temperature. Images were acquired with a fluorescence microscope (BX53; Olympus, Tokyo, Japan) and tail moments were calculated using Comet analysis software (Trevigen).

Cell viability assay

Cells were plated in white hard-bottom 96-well plates at a final density of 5,000 cells per well and incubated for one day before treatment with the indicated compounds. Cell viability was determined 48 hours after treatment using Cell Titer-Glo (Promega) according to the manufacturer's protocol. Viability was quantified on a Synergy NEO2 Hybrid Multi-Mode Reader (BioTek).

Preparation: Alkylation of nucleobases with 2-chloro- N,N -diethylethane-1-amine hydrochloride (UNI21)

The compound of Formula 2 (referred to as UNI21) shown in Figure 1a is 2-chloro-N, N-diethylethanamine hydrochloride (2-chloro-N, N-diethylethanamine hydrochloride, SIGMA-ALDRICH) is purchased and used did

Reaction of adenine with UNI21

2-Chloro- N,N -diethylethaneamine hydrochloride (UNI21) (28.8 mg, 0.2 mmol, 1.0 eq) was dissolved in adenine (27.0 mg, 0.2 mL) in TFE (2,2,2-trifluoroethanol) (2.0 mL). mmol, 1.0 eq) was added to the solution. The pH was adjusted to neutral by adding sodium acetate (24.6 mg, 0.3 mmol, 1.5 equiv). The reaction mixture was stirred at 37 °C or 60 °C for 3 days. The crude reaction was filtered through a syringe filter. Filtered chemicals were characterized by UPLC-HRAM-PRM as described below.

Reaction of Guanine with UNI21

2-Chloro- N,N -diethylethaneamine hydrochloride (UNI21) (28.8 mg, 0.2 mmol, 1.0 equiv) was added to a solution of guanine (30.2 mg, 0.2 mmol, 1.0 equiv) in TFE (2.0 mL). did The pH was adjusted to neutral by adding sodium acetate (24.6 mg, 0.3 mmol, 1.5 equiv). The reaction mixture was stirred at 37 °C or 60 °C for 3 days. The crude reaction was filtered through a syringe filter. Filtered chemicals were characterized by UPLC-HRAM-PRM as described below.

The reaction of thymine and UNI21

2-Chloro- N,N -diethylethaneamine hydrochloride (UNI21) (28.8 mg, 0.2 mmol, 1.0 equiv) was added to a solution of thymine (25.2 mg, 0.2 mmol, 1.0 equiv) in TFE (2.0 mL). did The pH was adjusted to neutral by adding sodium acetate (24.6 mg, 0.3 mmol, 1.5 equiv). The reaction mixture was stirred at 37 °C or 60 °C for 3 days. The crude reaction was filtered through a syringe filter. Filtered chemicals were characterized by UPLC-HRAM-PRM as described below.

Reaction of cytosine with UNI21

2-Chloro- N,N -diethylethaneamine hydrochloride (UNI21) (28.8 mg, 0.2 mmol, 1.0 equiv) was added to a solution of cytosine (22.2 mg, 0.2 mmol, 1.0 equiv) in TFE (2.0 mL). did Sodium acetate (24.6 mg, 0.3 mmol, 1.5 equiv) was added to adjust pH neutral. The reaction mixture was stirred at 37 °C or 60 °C for 3 days. The crude reaction was filtered through a syringe filter. Filtered chemicals were characterized by UPLC-HRAM-PRM as described below.

Solid-phase extraction (SPE) purification of alkyl nucleobases

Aliquots of each N- (diethylamino-ethyl)-nucleobase (DEAE-nucleobase) reaction were reconstituted in 1 mL of 5% methanol in water and sonicated for 30 minutes. After sonication, the solution was centrifuged at 14,000 rcf for 10 min at room temperature to pellet the solid precipitate. An Oasis ^® HLB 30 mg extraction cartridge (Waters, Milford, MA) was placed on the vacuum manifold and conditioned by adding 1 mL of water twice followed by 1 mL of methanol with gentle vacuum applied. Then, the sample solution was loaded onto the column and washed twice with 2 mL of 5% methanol in water. DEAE-purine nucleobases and DEAE-pyrimidine nucleobases were eluted by adding 500 μL 100% methanol twice. Collected eluates were concentrated by centrifugal vacuum and stored at -20°C for future analysis.

Alkylation of calf thymus DNA (CTDNA) with 2-chloro- N,N -diethylethanamine hydrochloride (UNI21)

An aliquot of 100 μg CTDNA in water or PBS was diluted with 190 μL of water or PBS, then 10 μL of 2 mM 2-chloro- N,N -diethylethanamine hydrochloride was added to a final concentration of 100 μM. The solution was then incubated at 37°C for 16 hours to allow alkylation of nucleotides followed by depurination of DEAE-purine bases by heating at 70°C for 1 hour. The released DEAE-purine nucleobases were separated from the DNA backbone by centrifugation at 14,000 rcf for 10 min at 4° C. through a Nanosep ^® centrifugal separator with an Omega ^™ 10 kDa membrane. The filter was further washed with equal volumes of de-ionized water twice and once with 100 μL 50:50 Acetonitrile (ACN):DI water. All collected solutions (depurine solutions) were concentrated to dryness by centrifugal vacuum and stored at -20 °C for future experiments. The DNA backbone of the filter was resuspended in 100 μL water (DNA backbone solution) and recovered from the filter and stored at -20 °C for future experiments.

Characterization of Alkyl-Nucleobase Standards by Ultra-Performance Liquid Chromatography-High Resolution Accurate Mass Parallel Reaction Monitoring (UPLC-HRAM-PRM)

Alkylation of purine and pyrimidine nucleobases with 2-chloro- N,N -diethylethaneamine hydrochloride was solid-phase extraction (SPE)-purified by UPLC-HRAM-PRM analysis in positive mode as follows. was confirmed by analyzing the reaction product; A Hypersil GOLD 1.9 mm C18 column (100 x 1.0 mm) was run with a gradient of buffer A (15 mM ammonium acetate, pH 7.0) and buffer B (100% acetonitrile), starting at 2% buffer B for 2 minutes and running for 8 minutes. linearly increased to 25% Buffer B over 20 minutes, then to 80% Buffer B over 2 minutes, held constant at 80% Buffer B for 2 minutes, followed by 2 minutes to 2% buffer, and finally re-equilibrated in 2% buffer B for 9 minutes. The mass spectrometry (MS) settings were as follows. Electrospray voltage (3500 V), capillary temperature 320 ° C, full scan AGC (1e ⁶ ), full scan resolution 70,000, HESI temperature 150 ° C, sheath gas, auxiliary gas and sweep gas flow rates of 35 each; 10 and 1 arbitrary units. Parallel reaction monitoring (PRM) MS settings were as follows: PRM AGC (5e ⁴ ) and PRM resolution at 35,000. The DEAE-purine and DEAE-pyrimidine nucleobase PRM settings are as follows. ESI ⁺ -PRM N7-DEAE-guanine and N9-DEAE-guanine: m/z (+1) = 251.1615 at 9-14.0 min; ESI ⁺ -PRM N1-DEAE-adenine, N3-DEAE-adenine and N7-DEAE-adenine (N9-DEAE-adenine potential): m/z (+1) = 235.1661 at 12-15 min; ESI+-PRM N1-DEAE-cytosine and N3-DEAE-cytosine: m/z (+1) = 211.1550 at 4-7 min and 8-13 min; and ESI+-PRM N1-DEAE-thymine and N3-DEAE-thymine: m/z (+1) = 226.1547 at 10-13 min.

Analysis of CTDNA Alkylation with 2-Chloro- N,N -diethylethanamine Hydrochloride (UNI21) by UPLC-HRAM-PRM

The above depurine solution was reconstituted in 50 μL water and measured with a microvolume UV spectrophotometer (Thermo Scientific™ NanoDrop) using the extinction coefficient for guanosine to confirm the presence of nucleobases. An equivalent of 1.2 μg of CTDNA in depurine solution was analyzed by the DEAE-purine UPLC-HRAM-PRM method described above.

The above DNA backbone solution was measured by microvolume UV spectrophotometry and a 25 μg CTDNA aliquot was diluted in 150 μL 1X NEB Nucleoside Digestion Mix Reaction Buffer and 2.5 μL NEB Nucleoside Digestion Mix (1 μL per 10 μg CTDNA). mix) for 4 hours at 37°C. After incubation, digestive enzymes were removed by centrifugation at 14,000 rcf for 10 minutes at 4° C. through a Nanosep ^® centrifuge with an Omega ^™ 10 kDa membrane. The filter was further washed once more with the same volume of deionized water and twice more with 100 mL 50:50 ACN:DI water. All collected solutions were concentrated to dryness by centrifugal vacuum. The resulting lysate was reconstituted in 25 μL LC-MS water and a 5 μg aliquot of CTDNA was analyzed by a modified DEAE-pyrimidine nucleoside UPLC-HRAM-PRM assay as described. ESI ⁺ -PRM DEAE-2'-deoxycytidine: m/z (+1) = 327.20270 at 11-14.0 min and ESI ⁺ -PRM DEAE-thymidine: m/z (+1) = at 11-14 342.20230 respectively. The UPLC-PRM MS setup was exactly the same as described for the DEAE-purine base analysis described above.

mouse xenograft

Animal experiments were performed in accordance with the guidelines of the UNIST Institute for Animal Testing (IACUC). 7-week-old male BALB/c nude mice were purchased from Orient Bio (Gyeonggi, Korea). Four million cells of WT or PARP1 KO HCT116 were suspended in 150 μL of sterile HBSS (Welgene, Gyeongbuk, Korea) and injected subcutaneously into the left flank. Intratumoral injections of vehicle (PBS) or UNI21 were performed every 3 days for 16 days after each tumor reached approximately 200 mm ³ in size. Tumor size was measured using calipers every 3 days for 16 days after drug treatment. Tumor volume was measured by the formula:

All mice were euthanized and tumors were harvested for immunostaining.

immunohistochemistry

Hematoxylin and Eosin (H&E) staining and TUNEL and γ-H2AX immunostaining were commercially performed by Histoire (Seoul, Korea). Collected tumors were fixed in formalin and picked up in Histoire. Detailed immunostaining procedures can be found on the Historia website (http://www.histoire.co.kr/).

statistical analysis

Statistical analysis was performed using GraphPad Prism (version 9.0.0). Significance is expressed as a p value: p >0.5 (ns), p <0.05 (*), p <0.01 (**), p <0.001 (***), p <0.0001 (****)). Groups were compared using normal one-way analysis of variance with single integrated variance, Dunnett's multiple comparison test (Figure 1B-C). Groups were compared using ordinary two-way ANOVA, Sidak's multiple comparison test, single integrated variance (Figures 1E-G, 3C, F, H, I). Groups were compared using an unpaired two-tailed t -test (Fig. 4C, D).

Example 1: UNI21 selectively kills XPA or PARP1 deficient cells.

We previously developed a high-throughput genotoxicity screening assay to identify genotoxic compounds using ATAD5 expression as a biomarker (Fox, JT, et al., Proc Natl Acad Sci USA, 2012. 109(14): p. 5423 -8). Using this assay, it was found that 2-chloro- N,N -diethylethanamine hydrochloride (UNI21) (FIG. 1A) increased ATAD5-luciferase expression (FIG. 1B). Tumors often have defective DNA damage repair pathways and become susceptible to specific DNA damaging agents. For example, BRCA1-deficient tumors unable to undergo homologous recombination are pathways required for homology-based DNA double-strand break (DSB) repair and are susceptible to DNA DSB-inducing agents such as cisplatin or ionizing radiation. do. To find the DNA damage repair pathways associated with UNI21, another HAP1 cell line defective in other DNA damage repair pathways was cultured with UNI21 for 48 h and then cell viability was measured using the Cell Titer-Glo Luminescent Cell Viability Assay. Compared to the wild type, xpa KO and parp1 KO HAP1 cell lines showed significant sensitivity to 20 mM UNI21 treatment (Fig. 1c and Fig. 1d). A similar sensitivity to UNI21 was observed in a dose-dependent manner in the parp1 KO HCT116 cell line (Fig. 1D, E). In addition, cell viability was measured in XP2OS cells and XPA mutant XP patient cells. Compared to XP2OS cells complemented with wild-type XPA, XP2OS cells showed significant sensitivity to UNI21 in a dose-dependent manner (FIGS. 1D and 1E).

To independently confirm the selective sensitivity of UNI21 against the parp1 KO cell line, the viability of HAP1, HCT116 and U2OS cell lines was assessed after co-treatment with a fixed concentration of Olaparib and increasing concentrations of UNI21. Similar to the parp1 KO cell line, co-treatment with Olaparib induced sensitivity to UNI21 in all cell lines tested in a dose-dependent manner (Fig. 1f). Simultaneous treatment with increasing doses of olaparib and fixed concentrations of UNI21 induced sensitivity in a dose-dependent manner in all HAP1, HCT116 and U2OS cell lines (Fig. 1g). Taken together, UNI21 can cause selective lethal effects in cells defective in nucleotide excision repair (NER) or PARP1-dependent repair pathways.

Example 2: Characterization of alkylated nucleobases with 2-chloro- N,N -diethylethanamine hydrochloride (UNI21)

UNI21 is an antinitrogen mustard and has electrophilic properties when Cl ligands are displaced by an intramolecular ring closure reaction to generate aziridinium ions. These highly reactive electrophiles can alkylate the nucleophilic positions of DNA nucleobases. To test this potential mechanism, we investigated the alkylation of purine and pyrimidine nucleobases by UNI21 (Fig. 2a). 0.2 mmol of each nucleobase and half of 0.2 mmol of UNI21 were carried out according to the conditions described previously (Balcome, S., et al., Chem Res Toxicol, 2004. 17(7): p. 950-62). To confirm the alkylation of purine and pyrimidine nucleobases by UNI21, the reaction mixture was purified by solid phase extraction and analyzed by high resolution accurate mass spectrometry parallel reaction monitoring (UPLC-HRAM-PRM). Using this assay, the desired diethylethenaminium (DEAE)-adenine ( m/z = 235.1671), DEAE-guanine ( m/z = 251.1611), DEAE-thymine (m/z = 226.0972) and DEAE-cytosine (m/z = 211.1551) was found to elute at 13.1 and 14.3 min (weak peak), 10.2 and 12.1 min, 11.0 and 11.5 min, and 4.8 and 9.2 min, respectively (Fig. 6a). Parallel Reaction Monitoring (PRM) fragmentation shows that all four DEAE-nucleobases share the same major cleavage fragment in the diethyl linker so that N,N -diethylethenaminium [M - base + H]+, m/z = 100.1124 and N-vinyl. nucleobase ion [M - NC ₄ H ₁₁ ] ⁺ ; DEAE-adenine m/z = 235.1671 to 162.0772, DEAE-guanine m/z = 251.1611 to 178.0721, DEAE-thymine m/z = 226.0972 to 153.0656, DEAE-thymine m/z = 226.0972 to 153.0656. A similar reaction with 2-bromo-N,N-diethietan-1-amine gave the same product, but in very reduced yield, 2-(diethylamino)ethyl acetate quenched to acetate was used as the main product. . Based on the UPLC-HRAM-PRM results, the observation of multiple peaks with similar fragmentation patterns is the N1, N3, N7, N9 or O ⁶ positions of guanine, adenine, N1, O ² or N3 of cytosine, N1, O of thymine ² , N3 or O ⁴ positions.

Example 3: Identification and characterization of calf thymus DNA (CTDNA) alkylated by 2-chloro- N,N -diethylethanamine hydrochloride (UNI21)

To further investigate UNI21 alkylation of nucleotides in double-stranded DNA, CTDNA was incubated with UNI21 for 16 hours. Alkylated purines were released from the DNA backbone by thermal hydrolysis and analyzed by UPLC-HRAM-PRM analysis. When 240 ng of CTDNA was analyzed, the presence of DEAE-guanine was confirmed at 10.3 minutes (FIG. 2b). Since the N9 position of guanine in double-stranded DNA is not accessible, the observed peak at 10.3 min is expected to be N7-DEAE-guanine and the standard peak at 12.1 min is expected to be N9-DEAE-guanine. DEAE-adenine could not be detected while analyzing 240 nanograms of CTDNA. A weak signal for DEAE-adenine could be detected at both 13.1 and 14.3 min when the depurination substrate was increased 5-fold with 1.2 μg of CTDNA (Fig. 2b). Analysis of the PRM data showed that both peaks had the expected fragmentation pattern. Considering that the N3 position of 2'-deoxyadenosine is the most reactive position with other nitrogen mustards in DNA (Balcome, S., et al., Chem Res Toxicol, 2004. 17(7): p. 950-62 Povirk, L.F. et al., Mutat Res, 1994. 318(3): p. 205-26), a weak signal at 13.1 min corresponds to N1-DEAE-adenine or N7-DEAE-adenine and a stronger signal at 14.3 min. The signal corresponds to N3-DEAE-adenine.

The remaining alkylated DNA backbone was digested with NEB nucleoside digestion mix to generate DEAE-2'-deoxypyrimidines. When the degradation of 5 μg of CTDNA was analyzed by the modified DEAE-pyrimidine nucleoside UPLC-HRAM-PRM analysis, two DEAE-dC peaks at 11.9 and 13.3 minutes and one DEAE-dT peak at 12.3 minutes were detected. could (Fig. 2c). The DEAE-dC peak at 11.9 min yields fragments of m/z = 211.1511, 138.0661 and 100.1124 corresponding to cleavage of the 2'-deoxysugar [M - dR + H ^] + N-vinyl-cytosine at the diethyl linker. ions [M - NC ₄ H ₁₁ - dR + H] ⁺ and N,N -diethylethenaminium [M - dC + H] ⁺ (FIG. 7). The DEAE-dC peak at 13.3 min yielded indistinguishable fragments of m/z = 283.17488, 177.11195, 133.08587 and 89.06019. The DEAE-dT peak at 12.3 min yielded fragments of m/z = 226.1547, 153.0656 and 100.1124, corresponding to cleavage of the 2'-deoxy sugar [M - dR + H] ⁺ and N -vinyl in the diethyl linker. -cytosine ion [M - NC ₄ H ₁₁ - dR + H]+ and N,N -diethylethenaminium [M - dT + H] ⁺ (Fig. 7). Given the inaccessibility of the N1 position of pyrimidines in double-stranded DNA, the peak observed at 11.9 min is expected to correspond to N ³ -DEAE-dC or O ² -DEAE-dC, whereas the peak at 12.1 min corresponds to O2 It is expected to correspond to -DEAE-dT, N ³ -DEAE-dT or O ⁴ -DEAE-dT. Taken together, UNI21 (2-chloro- N,N -diethylethanamine hydrochloride) is most reactive with 2'-deoxyguanosine, followed by 2'-deoxyadenosine and 2'-deoxypyrimidine. (dG >> dA > dC ~ dT).

To investigate the reactivity and alkylation potential, the mutant HAP1 cell line was treated with a UNI21 derivative in which the chloride leaving group was replaced with a bromide leaving group (FIGS. 5d and 5e). However, compared to UNI21, the sensitivity of bromide-substituted derivatives did not increase significantly.

Example 4: UNI21 induces more DNA cleavage in PARP1 deficient cells

Because UNI21 induces alkylation of nucleobases, UNI21 treatment should prevent S-phase progression. The effect of UNI21 on cell cycle progression in HCT116 WT and parp1 KO cell lines was investigated. Both wild-type and parp1 KO cells were arrested in S phase upon treatment with increasing concentrations of UNI21 (Fig. 3a). Since parp1 KO cells were selectively killed by UNI21, DNA damage markers were compared in wild-type cells treated with 80 μM UNI21 and parp1 KO cells for 24 hours. Consistent with the cell viability results, higher γH2AX induction was found in UNI21-treated parp1 KO cells (Fig. 3b). In addition, the actual DNA damage measured by alkaline comet assay showed more DNA breakage induced in parp1 KO cells than in wild-type cells upon UNI21 treatment (Fig. 3c). To investigate genomic instability, sister chromatid exchange (SCE) and chromosome breaks were tested in HCT116 WT and parp1 KO cells after 24 h treatment with 80 μM UNI21. In previous studies, PARP1-deficient cells increased SCE without damage (Hochegger, H., et al., EMBO J, 2006. 25(6): p. 1305-14; Wang, ZQ, et al., Genes Dev, 1997. 11(18): p.2347-58;de Murcia, JM, et al., Proc Natl Acad Sci USA, 1997.94(14): p.7303-7; Biochem Biophys Res Commun, 1980. 97(4): p.1311-6). More frequent SCEs and disruptions were found in UNI21-treated parp1 KO cells (Figures 3d, e, f and g). In particular, a high number of 25 or more disruptions per metaphase was observed in UNI21-treated parp1 KO cells (Fig. 3H). Finally, apoptotic cell death was quantified using Annexin V Alexa Fluor ^™ 488 conjugate. Compared to the wild type, the parp1 KO cell line showed significantly increased apoptosis upon treatment with 20 mM UNI21 (FIG. 3i). These results show that UNI21 induces more DNA damage, chromosomal aberrations and apoptosis in PARP1-deficient cells.

Example 5: Growth of parp1 KO xenograft tumors is selectively inhibited by UNI21 treatment

To confirm the effect of UNI21 in vivo, xenotransplantation was performed using nude mice (Fig. 4a). Four million cells of wild-type or parp1 KO HCT116 were injected subcutaneously into the left flank to form xenograft tumors. When tumors reached approximately 200 mm ³ , vehicle or UNI21 was injected intratumorally. Vehicle-treated wild-type and parp1 KO tumors continued to grow. In contrast to the continued growth of wild-type tumors, HCT116 parp1 KO engraftment showed significant delay in tumor growth by UNI21 treatment (FIGS. 4B and 4C). Regularly tracked xenograft tumor volume in each group demonstrated that UNI21 selectively delayed the proliferation of PARP1 deficient HCT116 xenograft tumors (FIG. 4D). The histology of apoptosis and DNA damage was investigated by TUNEL assay and γ-H2AX immunostaining (Fig. 4e). Consistent with the in vitro data, UNI21 treatment induced apoptosis and induced γ-H2AX. Overall, UNI21 specifically inhibits the growth of PARP1-deficient tumors in vivo.

The pharmaceutical composition for treating cancer comprising the compound of Formula 1 and a pharmaceutically acceptable salt thereof according to the present invention can be applied to various cancer cells resistant to PARP inhibitors to selectively and effectively kill them, thereby treating or preventing cancer diseases. can

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the claims and their equivalents.

Claims (6)

1. A pharmaceutical composition for preventing or treating cancer comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

   [Formula 1]

   

   In Formula 1, R is a halogen group.
2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the compound represented by Formula 1 is a compound represented by Formula 2 or Formula 3.

   [Formula 2]

   

   [Formula 3]

   
3. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the salt is selected from the group consisting of HCl salt, HBr salt, HI salt, H2SO4 salt, HNO3 salt, and combinations thereof.
4. The method of claim 1, wherein the cancer is squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung cancer, peritoneal cancer, colon cancer, biliary tract tumor, nasopharyngeal cancer, laryngeal cancer, bronchial cancer, oral cancer, osteosarcoma, gallbladder cancer, kidney cancer, selected from the group consisting of leukemia, bladder cancer, melanoma, brain cancer, glioma, brain tumor, skin cancer, pancreatic cancer, breast cancer, liver cancer, bone marrow cancer, esophageal cancer, colon cancer, stomach cancer, cervical cancer, prostate cancer, ovarian cancer, head and neck cancer, and rectal cancer A pharmaceutical composition for preventing or treating cancer, characterized in that.
5. The pharmaceutical composition for preventing or treating cancer according to claim 1, further comprising a PARP inhibitor.
6. The method of claim 5, wherein the PARP inhibitor is from the group consisting of olaparib, talazoparib, niraparib, rucaparib, veliparib and pamiparib A pharmaceutical composition for preventing or treating cancer, characterized in that at least one selected.

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