WO2023059148A1 - Utilisation de chlorhydrate de 2-chloro-n, n-diethyléthylamine pour améliorer le traitement anticancéreux - Google Patents

Utilisation de chlorhydrate de 2-chloro-n, n-diethyléthylamine pour améliorer le traitement anticancéreux Download PDF

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WO2023059148A1
WO2023059148A1 PCT/KR2022/015184 KR2022015184W WO2023059148A1 WO 2023059148 A1 WO2023059148 A1 WO 2023059148A1 KR 2022015184 W KR2022015184 W KR 2022015184W WO 2023059148 A1 WO2023059148 A1 WO 2023059148A1
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cancer
formula
uni21
salt
deae
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Korean (ko)
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명경재
위민우
4세아놀드 스캇 그뢸러
쉐러올란도
김건우
최장현
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기초과학연구원
울산과학기술원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

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  • 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.
  • PARP Poly (ADP-ribose) polymerase
  • 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.
  • a specific tumor type e.g., high-grade serous ovarian cancer or triple negative brain cancer
  • a subtype of a related molecule e.g., BRCA1/2-mutated breast cancer, ovarian cancer, pancreatic cancer, or prostate cancer.
  • 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.
  • Korean Patent Publication No. 10-2018-0051500 uses a combination of a debate molecule and a PARP inhibitor for cancer treatment
  • Korean Patent Publication No. 10-2018-0037210 discloses a combination using liposomal irinotecan and a PARP inhibitor for cancer treatment. therapy is initiated.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ M 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.
  • alkylated purines were released by thermal hydrolysis and concentrated for analysis as described in Materials and Methods.
  • Red traces show UPLC-HRAM-PRM of DEAE-guanine (panel 2) and DEAE-adenine (panel 4) detected in 1.2 ⁇ g depurinated CTDNA.
  • Figure 2c shows representative UPLC-HRAM-PRM traces of enzymatically released DEAE-pyrimidine from 5 ⁇ g UNI21-treated CTDNA.
  • 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 ⁇ M 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.
  • 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 FluorTM 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.
  • 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 ⁇ -H2AX IHC after sectioning the harvested tumor according to an embodiment of the present invention.
  • FIG. 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.
  • FIG. 7 to 7d are diagrams illustrating the results of mass spectrometry of the red graph shown in FIG. 2b.
  • 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
  • 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.
  • R is a halogen group.
  • halogen include fluorine (F), chlorine (Cl), bromine (Br), and iodine (I), and in particular, chlorine (Cl) or bromine (Br).
  • treatment when used on a subject exhibiting symptoms of disease, means stopping or delaying the progression of a disease.
  • composition may include a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof together with the compound of the present invention, if necessary.
  • pharmaceutically acceptable means a property that does not impair the biological activity and physical properties of a compound.
  • carrier refers to a substance that facilitates the addition of a compound into a cell or tissue.
  • diuent 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.
  • 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.
  • 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.
  • R is a halogen group.
  • the compound of Formula 1 may be a compound represented by Formula 2 or Formula 3 below.
  • the compound of Formula 1 may form a pharmaceutically acceptable salt thereof.
  • 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.
  • 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.
  • 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.
  • the present invention can obtain a synergistic effect by using a composition that further includes a PARP inhibitor in combination therapy.
  • the PARP inhibitor is 1 from the group consisting of olaparib, talazoparib, niraparib, rucaparib, veliparib and pamiparib More than one species can be selected.
  • 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.
  • 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.
  • the present invention relates to a pharmaceutical composition
  • 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.
  • the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof of the present invention can be used as a cancer treatment agent.
  • 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.
  • a salt dissolved in a buffer solution is used as a diluent
  • 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.
  • 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.
  • 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.
  • 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
  • XPA xeroderma pigmentosum group A protein
  • NER Nuclear Excision Repair
  • Cancer cells do not properly recover these alkylated bases, and thus have the effect of specifically killing them.
  • 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
  • U2OS ATCC ® HTB-96
  • HEK293T ATAD5-LUC Cells were cultured in 10% fetal bovine serum (FBS, Merk), 1% antibiotic-antimycotic (penicillin 10000 units/mL, streptomycin 10000 ⁇ g/mL, Fungizone ® (Amphotericin B) 25 ⁇ g/mL, Gibco ® ) were incubated at 37° C.
  • 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 .
  • IMDM Iscove's Modified Dulbecco's Medium
  • 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 ⁇ g/mL, Gibco® ) and 2 ⁇ g/ml puromycin.
  • 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.
  • DMEM Cytiva
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin at 37°C in the presence of 5% CO 2 . It became.
  • 0.75 ⁇ g of the pWPXL expression vector containing the XPA cDNA, 2.25 ⁇ g of the pMD2.G envelope plasmid and 2.25 ⁇ g 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).
  • 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.
  • 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).
  • 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 (ChemiDocTM; Bio-Rad Laboratories).
  • Cell death was quantified using a BD FACSVerse instrument with Annexin V Alexa FluorTM 488 conjugate (A13201, Thermo Fisher Scientific) and Flow-Jo software (version 10) according to the manufacturer's instructions.
  • 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 ⁇ l 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.
  • 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).
  • 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).
  • 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
  • DEAE-purine nucleobases and DEAE-pyrimidine nucleobases were eluted by adding 500 ⁇ L 100% methanol twice. Collected eluates were concentrated by centrifugal vacuum and stored at -20°C for future analysis.
  • CTDNA calf thymus DNA
  • UNI21 2-chloro- N,N -diethylethanamine hydrochloride
  • the filter was further washed with equal volumes of de-ionized water twice and once with 100 ⁇ L 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 ⁇ L water (DNA backbone solution) and recovered from the filter and stored at -20 °C for future experiments.
  • MS mass spectrometry
  • 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-thymine and N3-DEAE-thymine: m/z (+1) 226.1547 at 10-13 min.
  • depurine solution was reconstituted in 50 ⁇ L water and measured with a microvolume UV spectrophotometer (Thermo ScientificTM NanoDrop) using the extinction coefficient for guanosine to confirm the presence of nucleobases.
  • An equivalent of 1.2 ⁇ g 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 ⁇ g CTDNA aliquot was diluted in 150 ⁇ L 1X NEB Nucleoside Digestion Mix Reaction Buffer and 2.5 ⁇ L NEB Nucleoside Digestion Mix (1 ⁇ L per 10 ⁇ g 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 TM 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.
  • Hematoxylin and Eosin (H&E) staining and TUNEL and ⁇ -H2AX immunostaining were commercially performed by era (Seoul, Korea). Collected tumors were fixed in formalin and picked up in dozens. Detailed immunostaining procedures can be found on the H historian website (http://www. part.co.kr/).
  • Example 1 UNI21 selectively kills XPA or PARP1 deficient cells.
  • 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.
  • DSB DNA double-strand break
  • DNA DSB-inducing agents such as cisplatin or ionizing radiation.
  • HAP1, HCT116 and U2OS cell lines were 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.
  • NER nucleotide excision repair
  • 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.
  • Fig. 2a 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).
  • the reaction mixture was purified by solid phase extraction and analyzed by high resolution accurate mass spectrometry parallel reaction monitoring (UPLC-HRAM-PRM).
  • UPLC-HRAM-PRM high resolution accurate mass spectrometry parallel reaction monitoring
  • Example 3 Identification and characterization of calf thymus DNA (CTDNA) alkylated by 2-chloro- N,N -diethylethanamine hydrochloride (UNI21)
  • 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 ⁇ g of CTDNA (Fig. 2b). Analysis of the PRM data showed that both peaks had the expected fragmentation pattern.
  • 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.
  • NEB nucleoside digestion mix When the degradation of 5 ⁇ g 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 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.
  • UNI21 (2-chloro- N,N -diethylethanamine hydrochloride) is most reactive with 2'-deoxyguanosine, followed by 2'-deoxyadenosine and 2'-deoxypyrimidine. (dG >> dA > dC ⁇ dT).
  • 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).
  • the sensitivity of bromide-substituted derivatives did not increase significantly.
  • Example 4 UNI21 induces more DNA cleavage in PARP1 deficient cells
  • 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 ⁇ M UNI21 and parp1 KO cells for 24 hours. Consistent with the cell viability results, higher ⁇ H2AX induction was found in UNI21-treated parp1 KO cells (Fig. 3b).
  • 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
  • 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.
  • tumors reached approximately 200 mm 3
  • vehicle or UNI21 was injected intratumorally.
  • Vehicle-treated wild-type and parp1 KO tumors continued to grow.
  • HCT116 parp1 KO engraftment showed significant delay in tumor growth by UNI21 treatment (FIGS. 4B and 4C).
  • 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.

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Abstract

La présente invention concerne l'utilisation de chlorhydrate de 2-chloro-N, N-diéthylethylamine pour améliorer le traitement anticancéreux, et présente les avantages de tuer des cellules cancéreuses par alkylation d'une base d'ADN à l'aide d'un composé à base de moutarde azoté de formule chimique 1, et présentant un effet synergique lors d'une utilisation en combinaison avec des inhibiteurs de PARP existants.
PCT/KR2022/015184 2021-10-08 2022-10-07 Utilisation de chlorhydrate de 2-chloro-n, n-diethyléthylamine pour améliorer le traitement anticancéreux WO2023059148A1 (fr)

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KR20200131251A (ko) * 2018-02-15 2020-11-23 센화 바이오사이언시즈 인코포레이티드 퀴놀론 유사체 및 이의 염, 조성물, 및 이들의 사용 방법
KR20210006945A (ko) * 2018-05-08 2021-01-19 퀸스랜드 유니버시티 오브 테크놀로지 치료에 대한 암 반응 결정
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