KR20100053150A - Pharmaceutical composition containing aryloxyacetyl derivatives as an active ingredient for inhibiting dna methyltransferase - Google Patents
Pharmaceutical composition containing aryloxyacetyl derivatives as an active ingredient for inhibiting dna methyltransferase Download PDFInfo
- Publication number
- KR20100053150A KR20100053150A KR1020080112141A KR20080112141A KR20100053150A KR 20100053150 A KR20100053150 A KR 20100053150A KR 1020080112141 A KR1020080112141 A KR 1020080112141A KR 20080112141 A KR20080112141 A KR 20080112141A KR 20100053150 A KR20100053150 A KR 20100053150A
- Authority
- KR
- South Korea
- Prior art keywords
- pharmaceutical composition
- dna
- dna methyltransferase
- syndrome
- aryloxyacetyl
- Prior art date
Links
Images
Classifications
-
- 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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
- C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/18—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides
- C07C235/24—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/56—Ring systems containing bridged rings
- C07C2603/58—Ring systems containing bridged rings containing three rings
- C07C2603/70—Ring systems containing bridged rings containing three rings containing only six-membered rings
- C07C2603/74—Adamantanes
Abstract
Description
본 발명은 아릴옥시아세틸 유도체인 LW6를 유효성분으로 함유하는 DNA methyltransferase 억제용 약학적 조성물, DNA 메틸레이션 억제용 약학적 조성물, 또는 DNA hypermethylation에 의해 발생하는 질환들을 치료하기 위한 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for inhibiting DNA methyltransferase containing LW6, an aryloxyacetyl derivative, as an active ingredient, a pharmaceutical composition for inhibiting DNA methylation, or a pharmaceutical composition for treating diseases caused by DNA hypermethylation. .
DNA methylation은 DNA 염기서열의 CpG dinucleotide의 시토신 잔기에 methyl기를 전달하는 효소인 DNA methyltransferase에 의해 시토신에 화학변화를 일으키는 형태이다. 배아 줄기 세포 (embryonic stem cells)에는 상기 CpG island가 methylation 되어 있지 않은 반면, 성인의 CpG island는 그 C 잔기에 methylation 되어 있어 이는 후생학적(epigenetics) 요인에 근거하는 것으로 여겨진다. DNA methylation is a form of chemical change in cytosine by DNA methyltransferase, an enzyme that transfers methyl groups to cytosine residues of CpG dinucleotides in DNA sequences. Embryonic stem cells do not have methylated CpG islands, whereas adult CpG islands are methylated at their C residues, which is believed to be based on epigenetics factors.
대체로, 유전자의 발현을 조절하는 부위인 유전자의 5’-부위에 CpG islands가 많이 존재하고 있으며 그 중 60-90%의 CpG island가 methylation되어 있다. 일 반적으로 암과 같은 질병 관련 유전자의 프로모터의 CpG islands에 비정상적인 과다 메틸화 (hypermethylation)가 나타나 있으며 이것이 유전자의 유전적 전사 발현 침묵 (heritable transcriptional silencing) 현상을 유도한다 (M. Esteller, Cancer epigenetics: DNA methylation and chromatin alterations in human cancer. Adv Exp Med Biol 532 (2003) 39-49). In general, many CpG islands are present at the 5′-site of genes, which regulate the expression of genes, of which 60-90% of CpG islands are methylated. In general, abnormal hypermethylation is present on the CpG islands of promoters of disease-related genes such as cancer, which leads to hereditary transcriptional silencing of genes (M. Esteller, Cancer epigenetics: DNA methylation and chromatin alterations in human cancer.Adv Exp Med Biol 532 (2003) 39-49).
DNA methylation은, 전사 관련 단백질이 methylation된 유전자에 결합하는 것을 방해하거나 methylated DNA가 methyl-CpG-binding domain proteins (MBDs)에 결합하고, MBD는 다시 히스톤을 변형시키는 histone deacetylases, chromatin remodelling proteins 등 다른 단백질들을 유인하고, 그 결과 단단하고 불활성의 침묵 크로마틴 (silent chromatin)을 형성하게 된다. 이러한 DNA methylation과 chromatin 구조의 상관 관계는 매우 중요한데 특히 methyl-CpG-binding protein 2 (MeCP2)는 Rett syndrome과, methyl-CpG binding domain protein 2 (MBD2)는 암의 hypermethylated genes의 전사적 침묵(transcriptional silencing)과 관계가 있다 (X. Zhao, C. Pak, R.D. Smrt, and P. Jin, Epigenetics and Neural developmental disorders: Washington DC, Epigenetics 2 (2007) 126-34). DNA methylation is another protein, such as histone deacetylases and chromatin remodeling proteins that prevent transcription-related proteins from binding to methylated genes or methylated DNA binds to methyl-CpG-binding domain proteins (MBDs). Attracting them, resulting in a hard, inert, silent chromatin. The correlation between DNA methylation and chromatin structure is very important, especially methyl-CpG-binding protein 2 (MeCP2) for Rett syndrome and methyl-CpG binding domain protein 2 (MBD2) for transcriptional silencing of hypermethylated genes in cancer. (X. Zhao, C. Pak, RD Smrt, and P. Jin, Epigenetics and Neural developmental disorders: Washington DC, Epigenetics 2 (2007) 126-34).
DNA methyltransferase는 S-adenosyl methionine (SAM)을 methyl donor로 사용하여 DNA의 시토신 잔기에 methyl기를 전달하는 효소이다. 인간의 DNA methylation은 종의 효소 DNA methyltransferase 1, 3a, 및 3b (DNMT1, DNMT3a, DNMT3b)에 의해 조절되는데, DNMT3a 및 DNMT3b는 발생 초기의 DNA methylation 패턴과 관련 있어 de novo methyltransferases라고 불린다. DNMT1은 DNA replication 동안 DNA methylation patterns을 복사하는 것으로 알려진 유지 methyltransferase 이다. DNMT3L은 다른 DNMT3s와 homologous하지만 효소 촉매 활성이 없다. DNMT3L은 DNA에 결합하여 de novo methyltransferases의 활성화를 촉진시킨다. DNMT2(TRDMT1)는 DNA methyltransferases에 공통으로 있는 10 sequence motifs를 가진 DNA methyltransferase homolog이지만 DNA를 methylate하지 못하고 aspartic acid transfer RNA의 anticodon loop에 있는 cytosine-38을 methylate 시킨다 (D. Vallbohmer, J. Brabender, D. Yang, P.M. Schneider, R. Metzger, K.D. Danenberg, A.H. Holscher, and P.V. Danenberg, DNA methyltransferases messenger RNA expression and aberrant methylation of CpG islands in non-small-cell lung cancer: association and prognostic value. Clin Lung Cancer 8 (2006) 39-44). DNA methyltransferase is an enzyme that transfers methyl groups to cytosine residues in DNA using S-adenosyl methionine (SAM) as a methyl donor. Human DNA methylation is regulated by the species
DNA region의 cytosine methylation을 정량하는 간단하고 정확한 방법은 bisulfite sequencing이다. Bisulfite는 DNA에 있는 methylated cytosine은 그대로 있으나 unmethylate된 cytosine을 모두 uracil로 전환시키고 변형된 DNA는 PCR로 증폭시킬 때 uracil은 thymidine으로 읽혀지고 methylated cytosine은 그대로 cytosine로 남아 있게 된다. PCR 후 각 DNA strand는 클로닝한 후 염기서열을 결정하여 unmethylated C site를 확인할 수 있다. A simple and accurate way to quantify cytosine methylation in DNA regions is bisulfite sequencing. Bisulfite retains the methylated cytosine in the DNA but converts all the unmethylated cytosine to uracil, and when the modified DNA is amplified by PCR, the uracil is read as thymidine and the methylated cytosine remains as cytosine. After PCR, each DNA strand can be cloned and the nucleotide sequence can be determined to identify the unmethylated C site.
DNA 메틸화는 epigenetic modification에 의한 유전자 발현에 큰 축을 이루고 있는데 암 억제 유전자들의 발현이 메틸화에 의해 조절됨이 점차로 많은 유전자에서 보고되고 있는데 tumor suppressor 유전자들의 불활성화또는 침묵으로 암이 발생하게 되므로 DNMTs의 작용을 저해하여 이들 유전자들의 발현을 유도하는 시도를 진행하고 있다. DNA methylation plays a major role in gene expression by epigenetic modification. The expression of cancer suppressor genes is regulated by methylation, and many genes have been reported. Cancer is caused by inactivation or silencing of tumor suppressor genes. Attempts have been made to inhibit and induce the expression of these genes.
또한, DNA methyltransferase은 유전적 질병, stem cell 분화 조절, 후생적 조절 장애로 인한 질병, 노화 등을 대상으로 하는 다양한 질병의 치료제로 활용 가능하며 특히 맞춤의료에 유용한 약물이다. DNA methyltransferase 3b 에 돌연변이가 있으면 ICF증을 나타내며 (immunodeficiency, centromere instability and facial anomalies) 노화 기간에도 비정상적인 DNA methylation 이 나타나며 다양한 세포 내 기능에 영향을 미친다. In addition, DNA methyltransferase can be used as a therapeutic agent for various diseases targeting genetic diseases, stem cell differentiation regulation, diseases caused by epigenetic regulation disorders, and aging, and is particularly useful for personalized medicine. Mutations in DNA methyltransferase 3b indicate ICF (immunodeficiency, centromere instability and facial anomalies), abnormal DNA methylation during aging, and affect various cellular functions.
유전적 변이보다는 암발생에 중요한 후생적 변이(epigenetic alterations)가 약물학적으로 가역적이므로 혈액학 또는 종양 분야에서 더욱 많은 관심을 가지고 있으나 epigenetic 유전자 변이의 정확한 기전이 규명되어 있지 않고, 암 치료의 영역에서 epigenetics를 대상으로 하는 신약개발연구도 이제 시작단계에 있다. 최근, 저분자 화합물 inhibitors에 의하여 DNA methyltransferases의 methylation이 제어될 수 있음이 밝혀져 저분자 화합물 개발이 큰 주목을 받고 있다. 그러나 아직, 세포독성이 많은 azacytidine류 외에는 아직 특별히 이용되는 물질이 없다. Nucleoside analog인 5-aza-2'-deoxycytidine (decitabine)은 DNA methyltransferase효소의 β-elimination step을 저해하여 DNA에 covalent complex를 형성하게 함으로써 DNMTs 작용을 저해하여 효소의 분해를 유도한다. Decitabine이 활성이 있기 위해서는 세포의 genome에 끼어 들어가야 하는데 이것이 돌연변이를 유도하여 decitabine은 bone marrow에 독성이 있어 치료범위가 적다. Vidaza(R) (5-azacytidine, Pharmion)는 첫 DNA methyltransferase inhibitor 로 bone marrow 이상에서 유래되는 골수이형성증 (myelodysplastic syndrome, MDS) 치료제로 미국 FDA 승인받아 시판되는데 항암제로의 활용 가능성이 높다. Epigenetic alterations, which are important for cancer development rather than genetic variation, are pharmacologically reversible, and thus are of increasing interest in the field of hematology or oncology, but the exact mechanism of epigenetic gene mutation is not elucidated and epigenetics in the area of cancer treatment. Drug development research targeting humans is also in its infancy. Recently, it has been found that methylation of DNA methyltransferases can be controlled by inhibitors of low molecular weight compounds. However, there is no specific substance yet, except for azacytidine, which is highly cytotoxic. Nucleoside analogue 5-aza-2'-deoxycytidine (decitabine) inhibits the β-elimination step of the DNA methyltransferase enzyme to form covalent complexes in DNA, inhibiting the action of DNMTs and inducing the degradation of enzymes. In order for decitabine to be active, it must be inserted into the genome of the cell, which induces mutations and decitabine is toxic to bone marrow and thus has little therapeutic range. Vidaza (R) (5-azacytidine, Pharmion) is the first DNA methyltransferase inhibitor and is marketed under the US FDA approval for the treatment of myelodysplastic syndrome (MDS) that is derived from abnormal bone marrow.
현재, RNA 수준에서 mRNAs를 분해하여 DNMTs를 표적으로 하는 치료제가 개발 중이며 HTS화 할 수 있는 새로운 기술에 의한 저해제 개발을 기대할 수 있으며 기업체에서도 저해제 개발에 박차를 가하고 있다. Currently, a therapeutic agent targeting DNMTs by decomposing mRNAs at the RNA level is being developed, and inhibitors can be expected to be developed by a new technology capable of HTS formation, and companies are accelerating the development of inhibitors.
본 발명자들은 HIF-1α의 발현을 저해하여 항암 효과를 나타내는 약물인 아릴옥시아세틸계 유도체(실시예 18, 대한민국 특허등록 제0787131호, PCT 출원 제PCT/KR07/03216호, (Aryloxyacetylamino)benzoic acid analogues: a new class of hypoxia-inducible factor-1 inhibitors. Journal of Medicinal Chemistry 2007, 50, 1675-1684) LW6의 작용 기전을 연구하던 중 LW6는 VHL(von Hippel-Lindau)의 발현양을 증가시켜 HIF-1α(hypoxia inducible factor-1 α)의 ubiquitination 후 프로테아좀을 경유하는 HIF-1α의 분해를 촉진시킴을 알게 되었다. VHL 발현 증가 연구를 위해 VHL 프로모터 활성화와 DNA methylation의 상관 관계가 있음을 관찰하고, LW6 약물이 VHL 프로모터의 hypermethylation에 미치는 효과를 조사하였고, LW6 약물 존재 하에 VHL 프로모터 CpG island의 C site가 demethylation 되어 T로 전환됨을 확인하였다. 또한, DNA methylation을 매개하는 효소인 DNMT1의 단백질양이 감소되어 있으므로 LW6는 DNMT1의 발현을 억제함으로 VHL promoter의 methylation 억제된 것으로 추정된다. 따라서 LW6는 tumor suppressor 등 발현 억제되어 있는 유전자의 hypomethylation을 유도하는 DNA methylation 억제제 용도로 사용될 수 있음을 확인하여 본 발명에 이르렀다.The present inventors inhibited the expression of HIF-1α, an aryloxyacetyl derivative which is a drug exhibiting an anticancer effect (Example 18, Korean Patent Registration No. 0787131, PCT Application No. PCT / KR07 / 03216, (Aryloxyacetylamino) benzoic acid analogues : A new class of hypoxia-inducible factor-1 inhibitors.Journal of Medicinal Chemistry 2007, 50, 1675-1684) While studying the mechanism of action of LW6, LW6 increased the expression of VHL (von Hippel-Lindau) to increase HIF- It was found that after ubiquitination of 1x (hypoxia inducible factor-1α), it promotes the degradation of HIF-1α via the proteasome. In order to study the increased expression of VHL, we observed the correlation between VHL promoter activation and DNA methylation, and investigated the effect of LW6 drug on hypermethylation of VHL promoter. The C site of VHL promoter CpG island was demethylated in the presence of LW6 drug. Confirmed that the conversion. In addition, since the protein amount of DNMT1, an enzyme that mediates DNA methylation, is reduced, LW6 is thought to inhibit the methylation of the VHL promoter by inhibiting the expression of DNMT1. Therefore, it was confirmed that LW6 can be used as a DNA methylation inhibitor for inducing hypomethylation of genes whose expression is suppressed, such as tumor suppressor.
본 발명에서는 HCT-116 세포에 LW6를 처리한 후 염색체를 분리한 후 chromosomal DNA를 Bisulfite로 처리하고, VHL 프로모터를 PCR 한 후 DNA sequencing을 통해 염기 C가 T로 전환된 것을 확인함으로써 LW6의 DNA methytransferase 저해제의 용도로의 활용성을 제시하며 본 발명을 완성하였다.In the present invention, after treating LW6 to HCT-116 cells, chromosomes are isolated, chromosomal DNA is treated with Bisulfite, and VHL promoter is PCR and DNA C sequencing confirms that the conversion of base C to T was performed by DNA methytransferase. The present invention has been completed showing the utility of the inhibitor.
본 발명의 목적은, 약물 LW6의 작용 기전 연구를 바탕으로 hypermethylation된 VHL 프로모터를 활성화시킴으로써 VHL 발현을 증가시키는 약물 LW6의 DNA methyltransferase 억제제로서의 신규한 용도를 제공하고자 한다. It is an object of the present invention to provide a novel use of drug LW6 as a DNA methyltransferase inhibitor to increase VHL expression by activating a hypermethylated VHL promoter based on a study of the mechanism of action of drug LW6.
본 발명은 하기 화학식 1로 표시되는 아릴옥시아세틸계 화합물 (LW6) 또는 이의 약학적으로 허용되는 염을 유효성분으로 포함하는 DNA methyltransferase 억제제로서의 용도 및 DNA 메틸트렌스퍼레이즈 억제활성에 기인하는 유전자 hypermethylation 관련 질환을 치료하기 위한 약학적 조성물을 제공한다. The present invention relates to the use of an aryloxyacetyl-based compound represented by the following formula (LW6) or a pharmaceutically acceptable salt thereof as a DNA methyltransferase inhibitor, and DNA hypermethylation due to DNA methyltransferase inhibitory activity Provided are pharmaceutical compositions for treating a disease.
[화학식 1][Formula 1]
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 상기 화학식 1로 표시되는 아릴옥시아세틸계 유도체는 화학적인 합성 방법으로 제조할 수 있으며, 약학적으로 허용가능한 염의 형태로도 사용할 수 있다. The aryloxyacetyl derivative represented by Chemical Formula 1 in the present invention may be prepared by a chemical synthesis method, and may also be used in the form of a pharmaceutically acceptable salt.
약학적으로 허용가능한 염으로는 허용 가능한 유리산(free acid)에 의해 형 성된 부가염이 유용하다. 적합한 유리산으로는 유기산과 무기산을 사용할 수 있으며, 무기산으로는 염산, 브롬산, 황산 및 인산 등을 사용할 수 있고 유기산으로는 구연산(citric acid), 초산, 젖산, 주석산(tartariac acid), 말레인산, 푸마르산(fumaric acid), 포름산, 프로피온산(propionic acid), 옥살산, 트리플루오로아세트산, 벤조산, 글루콘산, 메탄술폰산, 글리콜산, 숙신산, 4-톨루엔술폰산, 갈룩투론산, 엠본산, 글루탐산 또는 아스파르트산 등을 사용할 수 있다.As pharmaceutically acceptable salts, addition salts formed with acceptable free acids are useful. Suitable free acids may be organic and inorganic acids, inorganic acids may be hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid, and organic acids may be citric acid, acetic acid, lactic acid, tartariac acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Etc. can be used.
나아가, 본 발명에 따른 상기 화학식 1로 표시되는 아릴옥시아세틸계 화합물은 약학적으로 허용가능한 염 뿐만 아니라, 통상의 방법에 의해 조제될 수 있는 모든 염, 수화물 및 용매화물을 모두 포함할 수 있다.Furthermore, the aryloxyacetyl compound represented by Chemical Formula 1 according to the present invention may include all salts, hydrates, and solvates that can be prepared by conventional methods, as well as pharmaceutically acceptable salts.
본 발명에 따른 용도에 사용되는 상기 화학식 1의 화합물은 하기 반응식 1에 나타난 바와 같이 합성될 수 있다.The compound of
[반응식 1]
구체적으로는 페녹시 아세트산(phenoxyacetic acid)(2)과 아민 화합물(3)을 출발 물질로 하여 후니그(Hunig) 염기와 축합 시약(coupling agent)을 넣고 유기용매 하에 반응시켜 화학식 (1) 화합물을 제조한다. 상기 후니그 염기로는 디이소프로필에틸아민(diisopropylamine, DIPEA) 또는 트리에틸아민(triethylamine, TEA)을 사용할 수 있다. 상기 축합시약으로는 1-[3-(디메틸아미노)프로필]-3-에틸카보디이미드 염산염(1-[3-(dimethyl amino)propyl]-3-ethylcarbodiimide hydrochloride, EDC), O-벤조트리아졸-N,N,N',N'-테트라메틸-유로니움-헥사플로로-포스페이트(HBTU), 1-히드록시벤조트리아졸(1-hydroxybenzotriazole, HOBt), 벤조트리아졸-1-일-옥시-트리스-피롤리디노-포스포늄 헥사플로로포스페이트(benzotriazol-1-yl-oxy- tris-pyrrolidino-phosphonium hexafluorophosphate, PyBOP) 및 1-히드록시-7-아자벤조트리아졸(1-hydroxy-7-azabenzotriazole, HOAt)로 이루어진 군으로부터 선택되는 하나 이상을 사용할 수 있다. 상기 유기용매로는 디메틸포름아미드(DMF)나 메틸렌클로라이드(CH2Cl2)가 바람직하다. Specifically, a phenoxyacetic acid (2) and an amine compound (3) are used as starting materials, and a Hunig base and a condensing reagent are added thereto, followed by reaction under an organic solvent. Manufacture. As the Hunig base, diisopropylethylamine (diisopropylamine, DIPEA) or triethylamine (TEA) may be used. As the condensation reagent, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (1- [3- (dimethyl amino) propyl] -3-ethylcarbodiimide hydrochloride (EDC), O-benzotriazole -N, N, N ', N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU), 1-hydroxybenzotriazole (HOBt), benzotriazol-1-yl-oxy Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and 1-hydroxy-7-azabenzotriazole (1-hydroxy-7- one or more selected from the group consisting of azabenzotriazole, HOAt). As the organic solvent, dimethylformamide (DMF) or methylene chloride (CH 2 Cl 2 ) is preferable.
이하, 상술한 화학식 1의 유도체 또는 이의 약학적으로 허용가능한 염의 용도를 구체적으로 설명한다.Hereinafter, the use of the derivative of Formula 1 or a pharmaceutically acceptable salt thereof will be described in detail.
먼저, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 DNA methytransferase 억제용 약학적 조성물을 제공한다.First, the present invention provides a pharmaceutical composition for inhibiting DNA methytransferase comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명은 또한 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 DNA 메틸레이션 억제용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for inhibiting DNA methylation comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에 의한 상기 화학식 1로 표시되는 아릴옥시아세틸계 유도체 (LW6)는 in vitro 실험에서 Hct-116 암세포주의 HIF-1α 억제에 의해 항암 효과를 나타내는 약물이다. LW6를 Hct-116 세포주에 처리한 경우 VHL의 발현양이 증가함이 확인되었다. The aryloxyacetyl derivative (LW6) represented by Formula 1 according to the present invention is a drug that exhibits anticancer effect by HIF-1α inhibition of Hct-116 cancer cell line in vitro experiments. When LW6 was treated with Hct-116 cell line, it was confirmed that the expression level of VHL was increased.
즉, LW6 처리 후 시간에 따라 VHL의 mRNA의 양이 증가되어 있음을 RT-PCR을 통해 확인하였으며, 이러한 VHL의 양적 증가는 VHL 유전자의 전사 조절에 의한 것으로 추정된다. In other words, it was confirmed through RT-PCR that the amount of mRNA of VHL increased with time after LW6 treatment, and this increase in VHL is presumed to be due to transcriptional regulation of the VHL gene.
Tumor suppressor인 VHL의 promoter에는 많은 CpG island가 존재하며 hypermethylation에 의해 발현이 억제되어 있는 것으로 보고되어 있다. Many CpG islands are present in the promoter of VHL, a Tumor Suppressor, and its expression is suppressed by hypermethylation.
본 발명에서는 LW6 약물을 hypoxia에서 HCT-116 세포에 처리한 후 염색체를 분리하였다. 분리된 chromosomal DNA를 Bisulfite로 처리하고, PCR로 VHL 프로모터를 증폭한 후 DNA sequencing을 수행하여 VHL 프로모터의 CpG island의 염기 전환을 관찰하였다. 프로모터의 -83에서 +195 부위에 있는 CpG island의 염기 C가 T로 일부 전환된 것을 확인하였다. LW6의 DNMT1 활성 저해 실험을 수행한 결과 직접적인 효소의 활성 저해는 일어나지 않았다. 세포 내에서 주요 DNA methytransferase인 DNMT1, DNMT3a의 RT-PCR 결과 LW6 처리에 의해 mRNA가 감소하는 것을 관찰하였다. 또한, 웨스턴블럿 실험을 통해 DNMT1 단백질의 양이 LW6의 농도에 의존적으로 감소하였다. 따라서 LW6는 DNMT1의 전사량을 조절함으로써 DNA methytransferase 활성을 저해하는 것을 확인하였다.In the present invention, LW6 drug was treated on HCT-116 cells in hypoxia and chromosomes were isolated. The isolated chromosomal DNA was treated with Bisulfite, amplified the VHL promoter by PCR, and DNA sequencing was performed to observe the base conversion of the CpG island of the VHL promoter. Partial conversion of the base C of the CpG island at -1 to +195 of the promoter was confirmed. Inhibition of DNMT1 activity of LW6 showed no direct inhibition of enzyme activity. RT-PCR of DNMT1 and DNMT3a, the major DNA methytransferases, was found to decrease mRNA by LW6 treatment. In addition, Western blot experiments showed that the amount of DNMT1 protein decreased depending on the concentration of LW6. Therefore, it was confirmed that LW6 inhibits DNA methytransferase activity by regulating the amount of transcription of DNMT1.
본 발명에 의한 아릴옥시아세틸계 유도체는 LW6의 DNA methyltransferase 억제제, 또는 DNA 메틸레이션 억제제로서 다양한 용도를 나타내는 데, 그 중에서도 특히 상기 살핀 바와 같이 종양 치료를 위한 활성을 나타내므로, 본 발명은 화학식(1)의 아릴옥시아세틸계 유도체의 DNA 메틸트렌스퍼레이즈 억제에 기인하는 종양을 치료하기 위한 약학적 조성물을 제공한다. The aryloxyacetyl derivatives according to the present invention show a variety of uses as DNA methyltransferase inhibitors or DNA methylation inhibitors of LW6. Among them, the aryloxyacetyl derivatives exhibit activity for the treatment of tumors, in particular as salping. Provided is a pharmaceutical composition for treating tumors caused by inhibition of DNA methyltransferase of an aryloxyacetyl derivative.
본 발명 의한 조성물에서의 상기 종양은, 폐암, 비소세포성 폐암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관 암, 신장세포 암종, 신장골반 암종, 중추신경계 (CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종, 뇌하수체 선종 등을 들 수 있다. The tumor in the composition according to the present invention, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, anal muscle cancer, colon cancer, Breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis Cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem nerve Glioma, pituitary adenoma, and the like.
본 발명은 또한 DNA 메틸트렌스퍼레이즈 억제에 기인하는 뇌신경 질환을 치료하기 위한 약학적 조성물을 제공하며, 본 발명에 있어서 신경 질환은 Rett syndrome 또는 Angelman syndrome 등을 들 수 있다. The present invention also provides a pharmaceutical composition for treating cerebral neurological diseases caused by DNA methyltransferase inhibition, wherein the neurological diseases include Rett syndrome or Angelman syndrome.
본 발명은 또한 DNA 메틸트렌스퍼레이즈 억제에 기인하는 혈액질환을 치료하기 위한 약학적 조성물을 제공하며, 본 발명에 있어서 혈액 질환은 골수이형성증 (myelodysplastic syndrome), 급성 골수성 백혈병(acute myeloid leukemia), 만성 골수성 백혈병(chronic myelogenous leukemia) 또는 만성 골수단핵구성 백혈병(chronic myelomonocytic leukemia) 등을 들 수 있다.The present invention also provides a pharmaceutical composition for the treatment of blood diseases due to DNA methyltransferase inhibition, wherein the blood diseases are myelodysplastic syndrome, acute myeloid leukemia, chronic Myelogenous leukemia or chronic myelomonocytic leukemia.
본 발명은 또한 DNA 메틸트렌스퍼레이즈 억제에 기인하는 후생유전 질환을 치료하기 위한 약학적 조성물을 제공하며, 본 발명에 있어서 상기 후생유전 질환은 ATR-X (Alpha-thalassemia X-linked mental retardation syndrome), 유약증후군 (Fragile X syndrome), 또는 ICF 증후군(immunodeficiency, centromere instability and facial anomalies) 등 일 수 있다.The present invention also provides a pharmaceutical composition for treating epigenetic disease caused by DNA methyltransferase inhibition, wherein the epigenetic disease in the present invention is ATR-X (Alpha-thalassemia X-linked mental retardation syndrome) , Fragile X syndrome, or ICF syndrome (immunodeficiency, centromere instability and facial anomalies).
본 발명은 나아가 DNA 메틸트렌스퍼레이즈 억제에 기인하는 후생학적(epigenetic) 조절제, 줄기세포 분화 조절제, 또는 체세포 융합 조절제를 제공한다. 본 발명에서 후생학적 조절제란 메틸화에 의한 염색체 구조 이상으로 인한 ATR-X (Alpha-thalassemia X-linked mental retardation syndrome), 유약증후군 (Fragile X syndrome), ICF 증후군 (immunodeficiency, centromere instability and facial anomalies) 등의 후생유전 질환 치료에 사용하는 약물을 의미한다. 줄기세포 분화 조절제는 줄기세포에서 전구 자연 살해 세포로의 분화 조절을 유도하는 약물을 의미한다. 본 발명에서 체세포 융합이란 해내고 서 핵을 제거한 성숙한 난자에 일반체세포에서 분리한 핵을 이식하고 정자를 주입한 후 수정란으로 만드는 과정을 의미한다. 핵이식란이 일반적인 수정란처럼 분할 및 발육에 적응하는 과정을 재프로그래밍이라고 하는데 이 과정을 통해 핵이식란이 탈메틸화 및 재메틸화 과정을 거쳐야 정상적인 발달 및 분화단계의 발생이 이루어진다. The present invention further provides epigenetic regulators, stem cell differentiation regulators, or somatic cell fusion regulators due to DNA methyltransferase inhibition. In the present invention, the epigenetic modulator refers to alpha-thalassemia X-linked mental retardation syndrome (ATR-X), fragile X syndrome, and ICF syndrome (immunodeficiency, centromere instability and facial anomalies) due to chromosomal abnormalities caused by methylation. Means the drug used in the treatment of epigenetic diseases. Stem cell differentiation regulator means a drug that induces differentiation of stem cells from progenitor natural killer cells. In the present invention, the somatic cell fusion refers to a process of making a fertilized egg after transplanting a nucleus isolated from normal somatic cells into a mature egg which has been removed and nucleated. Reprogramming is the process in which a nuclear transfer egg adapts to division and development like a normal fertilized egg. This process requires that the nuclear transfer egg undergoes demethylation and remethylation before normal development and differentiation occurs.
본 발명에 따른 약학적 조성물은 통상의 방법에 따른 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오 스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention may further comprise a suitable carrier, excipient or diluent according to conventional methods. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undetermined. Vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명에 따른 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 상세하게는, 제형화할 경우 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트(calcium carbonate), 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 포함되며, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성 용제 및 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical compositions according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, or sterile injectable solutions according to conventional methods. Can be. Specifically, when formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose, lactose in the compound. , Gelatin and the like can be mixed. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Oral liquid preparations include suspending agents, solvents, emulsions, syrups, and the like, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명에 따른 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명의 화합물의 일일 투여량은 0.0001 내지 100 ㎎/㎏으로, 바람직하게는 0.001~100 ㎎/㎏의 양을 일일 1회 내지 수회로 나누어 투여할 수 있다.The pharmaceutical composition according to the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the patient's condition and weight, disease Depending on the degree, drug form, route of administration, and duration, it may be appropriately selected by those skilled in the art. However, for the desired effect, the daily dosage of the compound of the present invention may be administered in 0.0001 to 100 mg / kg, preferably in an amount of 0.001 to 100 mg / kg, divided once or several times daily.
본 발명에 따른 약학적 조성물은 암 질환을 포함한 각종 질환의 치료를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다. The pharmaceutical composition according to the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the treatment of various diseases including cancer diseases.
본 발명은 아릴옥시아세틸계 유도체 LW6 약물이 VHL의 프로모터의 hypermethylation에 미치는 효과를 조사하여 LW6 약물 존재하에 VHL 프로모터의 CpG island의 C가 T로 변화되어 되어 있고 DNA methylation을 매개하는 효소인 DNMT1의 단백질양이 감소되어 있으므로, 본 발명에 의한 LW6는 DNMT1의 발현을 저해함으로 VHL promoter의 methylation을 억제하는 것으로 보인다. 따라서, LW6는 tumor suppressor 등 발현이 억제되어 있는 유전자에서 hypomethylation을 유도하여 DNA methylation 억제제로의 활용 및 치료제로서 유용하게 사용될 수 있음을 확인하였다. 본 발명은 DNA의 과다메틸화에 의해 유전자 발현이 억제되어 질병을 일으키는 암, 혈액질환, 노화 관련 질환, 뇌신경 질환 치료 등의 stem cell 분화 조 절제로 유용하게 사용될 수 있다. In the present invention, the effect of the aryloxyacetyl derivative LW6 drug on the hypermethylation of the VHL promoter was investigated. In the presence of the LW6 drug, C was changed to C in the CpG island of the VHL promoter. Since the amount is reduced, LW6 according to the present invention seems to inhibit methylation of the VHL promoter by inhibiting the expression of DNMT1. Therefore, LW6 induces hypomethylation in genes whose expression is suppressed such as tumor suppressor, and can be used as a DNA methylation inhibitor and as a therapeutic agent. The present invention can be usefully used as stem cell differentiation control for the treatment of cancer, blood diseases, aging-related diseases, cranial nerve diseases, etc., where gene expression is inhibited by overmethylation of DNA.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.
[실시예]EXAMPLE
본 발명의 실시예에서 사용한 HCT-116 cell(ATCC Cat. No. CCL2)은 RPMI 배지(Gibco, U.S.A.), 5% CO2에서 37 °C에서 배양하였으며 hypoxia는 1% O2 조건에서의 배양을 의미한다. HCT-116 cells (ATCC Cat.No. CCL2) used in the examples of the present invention were cultured at 37 ° C in RPMI medium (Gibco, USA), 5% CO 2 and hypoxia was cultured in 1% O 2 conditions it means.
실시예 1 : LW6에 의한 VHL 발현 실험Example 1: VHL expression experiment by LW6
HCT-116 cell(ATCC Cat. No. CCL2)항암 타겟인 HIF-1α의 발현 저해를 관찰하기 위해 HCT-116 cell(ATCC Cat. No. CCL2)을 60mm dish에 plating 한 후에 각각 4시간 동안 normoxia, hypoxia condition에 노출시킨 후, LW6를 각각 30 μM를 처리하고 Hypoxia (1% O2)에서 12시간 배양하면서 세포 내 존재하는 HIF-1α와 VHL 단 백질의 양을 0, 2, 4, 8, 12시간째에 각 시간대 별로 측정하였다. 시간 경과에 따라 LW6 약물에 의해 HIF-1α의 발현은 감소되는 반면 VHL protein 양은 증가됨을 웨스턴 블럿 방법에 의하여 확인 할 수 있었다(도 1). HCT-116 cells (ATCC Cat. No. CCL2) In order to observe the inhibition of expression of HIF-1α, an anticancer target, HCT-116 cells (ATCC Cat. After exposure to hypoxia conditions, LW6 was treated with 30 μM each and incubated for 12 hours in Hypoxia (1% O 2 ) to reduce the amount of HIF-1α and VHL proteins present in
VHL의 발현 증가가 mRNA 발현 조절과 관련이 있는지 조사하기 위해 HCT-116 cell(ATCC Cat. No. CCL2)을 60mm dish에 plating 한 후에 각각 4시간 동안 normoxia, hypoxia condition에 노출 후 LW6을 30 μM 처리하여 12시간 incubation 한 다음 RNA를 추출하여 RT-PCR을 시행하였다. total RNA는 Qiagen RNeasy Mini kit (cat.# 74104)을 사용하여 추출하였고 RT-PCR은 One-step RT-PCR PreMix kit (cat.# 25101, iNtRON Biotechnology, Inc., Republic of Korea)을 사용하여 매뉴얼에 지시한 대로 RNA template (1 μg/reaction)과 specific primers (표 1)를 사용하여 수행하였다. RT-PCR은 1 cycle (45 °C, 30 min, 94 °C, 5 min), 30 cycles (94 °C, 30 sec, 52 °C, 30 sec, 72 °C, 1 min), 마지막으로 1 cycle (72 °C, 5 min) 하였다. 상기 PCR을 위한 올리고 primer 서열은 각각 표1.에 나타내었다. RT-PCR 결과, LW6 약물을 처리함에 따라 VHL의 mRNA양은 현격히 증가하였으며 이러한 VHL의 발현 증가는 normoxia 또는 hypoxia에 관계 없음을 확인하였다 (도 2). To investigate whether increased expression of VHL was related to mRNA expression regulation, HCT-116 cells (ATCC Cat. No. CCL2) were plated in 60 mm dish and exposed to normoxia and hypoxia conditions for 4 hours, respectively, and treated with 30 μM of LW6. After incubation for 12 hours, RNA was extracted and RT-PCR was performed. Total RNA was extracted using the Qiagen RNeasy Mini kit (cat. # 74104) and RT-PCR was analyzed using the One-step RT-PCR PreMix kit (cat. # 25101, iNtRON Biotechnology, Inc., Republic of Korea) RNA template (1 μg / reaction) and specific primers (Table 1) was performed as directed in. RT-PCR is 1 cycle (45 ° C, 30 min, 94 ° C, 5 min), 30 cycles (94 ° C, 30 sec, 52 ° C, 30 sec, 72 ° C, 1 min), finally 1 cycle (72 ° C, 5 min). Oligo primer sequences for the PCR are shown in Table 1. As a result of RT-PCR, the amount of mRNA of VHL was significantly increased by treatment with LW6 drug, and it was confirmed that the expression of VHL was not related to normoxia or hypoxia (FIG. 2).
[표 1]TABLE 1
Tumor suppressor인 VHL의 프로모터에는 CpG island가 많이 존재하며 renal cancer인 경우 hypermethylation 되어 있다고 알려져 있으나, 대장암의 경우는 hypermethylation이 보고된 바가 없었다. 실시예 1에서는 대장암 세포주인 HCT-116 세포에서 VHL의 mRNA 양이 증가된 것 확인하였으며, 다음으로는 LW6가 DNA methylation 정도를 변화시키는 것인지, 이에 기인하여 epigenetic 조절을 하는지 조사하게 되었다. There are many CpG islands in the promoter of the Tumor Suppressor, VHL, and hypermethylation has been reported in renal cancer, but hypermethylation has not been reported in colorectal cancer. In Example 1, it was confirmed that the amount of VHL mRNA was increased in HCT-116 cells, which is a colorectal cancer cell line. Next, it was investigated whether LW6 changed the degree of DNA methylation and thereby caused epigenetic regulation.
실시예 2 : LW6에 의한 CpG island의 탈메틸화 관찰 Example 2 Observation of Demethylation of CpG Island by LW6
상기 실시예 1에서의는 LW6가 VHL 프로모터의 활성화를 통해 VHL의 발현을 증가시키는 것을 확인하였다. 이에, 세포에 LW6를 각각 1, 50, 및 100 μM 농도로 처리한 후 chromosomal DNA를 추출하였다 (프로메가 제품, Chromosomal DNA Purification Kit, CAT#. a1120). 추출한 chromosomal DNA에 bisulfite 처리하여 C를 T로 전환시킨 chromosomal DNA 얻고 (Zymo Research, Methylation kit, CAT# d5001) 이렇게 Bisulfite modified DNA를 template로 하여 MSP (Metylation-specific PCR)을 수행하였다. PCR 조건은 다음과 같다. Initial activation step : 15min at 95℃, Denaturation step : 30sec at 94℃, Annealing step : 30sec at 60℃, Extension step : 30sec at 72℃ , 총 40 사이클. Final extension step : 10min at 72℃ 이다. In Example 1, it was confirmed that LW6 increases the expression of VHL through activation of the VHL promoter. Thus, the cells were treated with LW6 at concentrations of 1, 50, and 100 μM, respectively, and then chromosomal DNA was extracted (promega product, Chromosomal DNA® Purification Kit, CAT #. A1120). Bisulfite treatment was performed on the extracted chromosomal DNA to obtain chromosomal DNA converted to T (Zymo Research, Methylation kit, CAT # d5001). Thus, MSP (Metylation-specific PCR) was performed using Bisulfite modified DNA as a template. PCR conditions are as follows. Initial activation step: 15min at 95 ℃, Denaturation step: 30sec at 94 ℃, Annealing step: 30sec at 60 ℃, Extension step: 30sec at 72 ℃, total 40 cycles. Final extension step: 10min at 72 ℃
실험 결과, LW6를 50 μM 처리하였을 때는 W4와 W5에서 일부 C염기의 T전환이 일어났으며, 100 μM 처리시에는 -185에서 +195부위에 있는 CpG island 더 많은 C염기의 T염기로 전환된 것으로 관찰되었다(도 3). 따라서, LW6가 VHL 프로모터 region의 CpG island를 탈메틸화 함을 확인하였다.As a result of the experiment, 50 μM of LW6 resulted in some C-based T-conversion at W4 and W5, and 100 μM-treated C-G island at -185 to +195, converted to more C-based T-base. Was observed (FIG. 3). Therefore, it was confirmed that LW6 demethylates the CpG island of the VHL promoter region.
실시예 3 : LW6에 의한 DNBT1 효소활성 영향 측정 (Enzyme-based DNMT assay)Example 3 Measurement of the Effect of DNBT1 Enzyme Activity by LW6 (Enzyme-based DNMT assay)
LW6에 의해 VHL 프로모터의 CpG island에 있는 C가 T로 변화된 것으로부터 DNA methyltransferase 활성이 저해되었음을 추정할 수 있었다. 나아가, 상기 DNA methyltransferase 효소 활성 저해가 그 양적인 변화에 의한 것인지 확인하기 위해 LW6에 의한 DNA methyltransferase 효소 활성 저해 실험을 수행하였다. Rhee 등 (Rhee I, Jair KW, Yen RW, Lengauer C, Herman JG, Kinzler KW, Vogelstein B, Baylin SB and Schuebel KE. (2000), Nature, 404, 1003~1007)의 방법에 따라 CpG methylase를 사용하여 LW6에 의한 DNA 메틸화를 분석하였다. 반응조건은 다음과 같았다. DMSO에 희석한 화합물을 0.5μg poly-d(I-C),5μCi adenosyl-methionine S-[methyl-H3], CpG methylase 1unit, Reaction buffer (50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM Dithiothreitol, 5% Glycerol )과 혼합하여 총 20 μl가 되도록 하였다. 이 혼합물을 두벌씩 준비하여 37 ℃에서 1시간 동안 항온 유지하여 메틸화반 응이 진행되도록 하였다. 반응이 끝난 혼합물을 Whatman P81 이온교환 Paper에 옮긴 다음, 0.2M Ammonium bicarbonate 로 5분 동안 3번 씻어 주었다. 건조된 필터를 vial에 넣고 5 ml scintillant를 첨가하여 scintillation counter로 방사능을 측정하여 DNA의 메틸화수준을 분석하였다. 각 시료의 blank는 CpG methylase를 제외한 나머지 성분을 위와 동일하게 처리하여 준비하였다. LW6는 각각 50μM 및 100 μM을, 양성 대조군으로 EGCG (epigallocatechin-3-gallate, 녹차의 주요 폴리페놀) 0.1 μM 및 1.0 μM을 사용하였다.It was estimated that DNA methyltransferase activity was inhibited by the change of C in the CpG island of VHL promoter to T by LW6. Furthermore, in order to confirm whether the inhibition of the DNA methyltransferase enzyme activity was caused by the quantitative change, the experiment of DNA methyltransferase enzyme activity inhibition by LW6 was performed. Use CpG methylase according to the method of Rhee et al. (Rhee I, Jair KW, Yen RW, Lengauer C, Herman JG, Kinzler KW, Vogelstein B, Baylin SB and Schuebel KE. (2000), Nature, 404, 1003-1007). DNA methylation by LW6 was analyzed. The reaction conditions were as follows. 0.5 μg poly-d (IC), 5 μCi adenosyl-methionine S- [methyl-H3], CpG methylase 1 unit, Reaction buffer (50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM Dithiothreitol) , 5% Glycerol) was mixed to make a total of 20 μl. The mixture was prepared in duplicate and kept at 37 ° C. for 1 hour to allow methylation reaction to proceed. The reaction mixture was transferred to Whatman P81 ion exchange paper, and then washed three times for 5 minutes with 0.2 M Ammonium bicarbonate. The dried filter was put into the vial, and 5 ml scintillant was added to measure the radioactivity using a scintillation counter to analyze the methylation level of DNA. Blanks for each sample were prepared by treating the same components as above except for CpG methylase. LW6 used 50 μM and 100 μM, respectively, and 0.1 μM and 1.0 μM of EGCG (epigallocatechin-3-gallate, the main polyphenol of green tea) as positive controls.
실험 결과, 양성 대조군인 EGCG는 낮은 농도에서도 농도 의존적으로 DNMT1 활성 저해 효과를 나타낸 반면, LW6은 높은 농도에서도 DNMT1 저해 활성에 큰 차이가 없는 것으로 미루어 LW6는 DNMT1의 효소 활성을 직접적으로 저해하지는 않는 것으로 보인다(도 4). Experimental results showed that EGCG, a positive control, showed a concentration-dependent inhibitory effect on DNMT1 activity at low concentrations, whereas LW6 did not directly inhibit DNMT1 activity at high concentrations. Therefore, LW6 does not directly inhibit the enzyme activity of DNMT1. Visible (FIG. 4).
실시예 4. LW6의 DNBT1의 발현에 미치는 영향Example 4. Effect of LW6 on Expression of DNBT1
다음으로, DNMT1의 mRNA 및 단백질 양적 측면에서의 변화를 조사하였다. Next, changes in the mRNA and protein quantitative aspects of DNMT1 were investigated.
HCT-116 (ATCC Cat. No. CCL2)을 LW6(30 μM)로 처리한 후(RPMI, 16시간, 37 °C) 세포 내 실시예 1에서 설명한 대로 DNMT 효소(DNMT 1 및 DBMT 3b)들의 RT-PCR을 수행하였다. DNMT1의 mRNA 양은 약물의 농도에 따라 점차 감소함을 확인하였다(도 5). DNMT3b는 hypoxia에서 전체적으로 mRNA 수준이 많이 감소되었고 LW6 존재하에서 더욱 감소하였다. Treatment of HCT-116 (ATCC Cat. No. CCL2) with LW6 (30 μM) (RPMI, 16 hours, 37 ° C) RT of DNMT enzymes (
HCT-116 (ATCC Cat. No. CCL2) 암세포주에 LW6 (10, 30, 50, 100 μM) 처리 후 웨 스턴 블럿 방법으로 DNMT1의 단백질 양을 측정하였을 때, DNMT1의 단백질 양은 LW6 농도에 의존적으로 감소하였다(도 6). 따라서, LW6는 DNMT1의 전사를 조절하여 DNMT1 단백질의 발현양을 감소시킴으로써 VHL 프로모터의 demethylation 효과를 나타내는 것으로 보인다. When the amount of protein of DNMT1 was measured by western blot method after treatment with LW6 (10, 30, 50, 100 μM) in HCT-116 (ATCC Cat. No. CCL2) cancer cell line, the protein amount of DNMT1 was dependent on LW6 concentration. Decreased (FIG. 6). Therefore, LW6 seems to exhibit the demethylation effect of the VHL promoter by regulating the transcription of DNMT1 to reduce the expression of DNMT1 protein.
도 1은 화학식 1로 표시되는 아릴옥시아세틸계 유도체에 의한 VHL 단백질의 양적 증가를 나타낸다.Figure 1 shows the quantitative increase of the VHL protein by the aryloxyacetyl derivative represented by the formula (1).
도 2는 화학식 1로 표시되는 아릴옥시아세틸계 유도체에 의한 VHL mRNA의 양적 증가를 나타낸다.Figure 2 shows the increase in the amount of VHL mRNA by the aryloxyacetyl derivative represented by the formula (1).
도 3은 화학식 1로 표시되는 아릴옥시아세틸계 유도체에 의한 탈메틸화를 나타낸다. 짧은 막대는 VHL 프로모터 부위에 존재하는 CpG island를 나타내고 * 표시된 부분은 아릴옥시아세틸계 유도체에 의해 탈메틸화가 일어나 염기 C가 염기 T로 전환된 site를 의미한다.3 shows demethylation by an aryloxyacetyl derivative represented by
도 4는 화학식 1로 표시되는 아릴옥시아세틸계 유도체의 DNMT1 효소 활성 저해를 나타낸다. EGCG는 양성 대조군이다. Figure 4 shows the inhibition of DNMT1 enzyme activity of the aryloxyacetyl derivative represented by the formula (1). EGCG is a positive control.
도 5는 화학식 1로 표시되는 아릴옥시아세틸계 유도체 처리 후 세포내에 존재하는 DNMT1, DNMT3b의 mRNA 발현을 나타낸다.Figure 5 shows the mRNA expression of DNMT1, DNMT3b present in the cell after the aryloxyacetyl derivative derivative represented by the formula (1).
도 6은 화학식 1로 표시되는 아릴옥시아세틸계 유도체 처리 후 세포내에 존재하는 DNMT1 단백질의 양을 나타낸다.Figure 6 shows the amount of DNMT1 protein present in the cells after the aryloxyacetyl derivatives represented by the formula (1).
Claims (10)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080112141A KR20100053150A (en) | 2008-11-12 | 2008-11-12 | Pharmaceutical composition containing aryloxyacetyl derivatives as an active ingredient for inhibiting dna methyltransferase |
| PCT/KR2009/006643 WO2010056048A2 (en) | 2008-11-12 | 2009-11-12 | Pharmaceutical composition for suppressing dna methyltransferase, containing aryloxyacetyl-based compounds as active ingredients |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020080112141A KR20100053150A (en) | 2008-11-12 | 2008-11-12 | Pharmaceutical composition containing aryloxyacetyl derivatives as an active ingredient for inhibiting dna methyltransferase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| KR20100053150A true KR20100053150A (en) | 2010-05-20 |
Family
ID=42170522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020080112141A KR20100053150A (en) | 2008-11-12 | 2008-11-12 | Pharmaceutical composition containing aryloxyacetyl derivatives as an active ingredient for inhibiting dna methyltransferase |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20100053150A (en) |
| WO (1) | WO2010056048A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101380466B1 (en) * | 2011-09-27 | 2014-04-02 | 한국생명공학연구원 | Novel compounds as HIF-1α inhibitors and manufacturing process thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE9904505D0 (en) * | 1999-12-09 | 1999-12-09 | Astra Pharma Prod | Novel compounds |
| SE0300480D0 (en) * | 2003-02-21 | 2003-02-21 | Astrazeneca Ab | Novel compounds |
| KR100787131B1 (en) * | 2006-07-04 | 2007-12-21 | 한국생명공학연구원 | Compounds that inhibit hif-1 activity the method for preparation thereof and the pharmaceutical composition containing them as an effective component |
-
2008
- 2008-11-12 KR KR1020080112141A patent/KR20100053150A/en not_active Application Discontinuation
-
2009
- 2009-11-12 WO PCT/KR2009/006643 patent/WO2010056048A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2010056048A3 (en) | 2010-08-26 |
| WO2010056048A2 (en) | 2010-05-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Weih et al. | Analysis of CpG methylation and genomic footprinting at the tyrosine aminotransferase gene: DNA methylation alone is not sufficient to prevent protein binding in vivo. | |
| EP0734436B1 (en) | Inhibition of dna methyltransferase | |
| JP7416852B2 (en) | Antisense oligonucleotides for regulating HTRA1 expression | |
| JP5416660B2 (en) | Inhibitors of DNA methylation | |
| NZ583923A (en) | Azacytidine analogues and uses thereof | |
| WO2020245233A1 (en) | Antisense oligonucleotides targeting atxn3 | |
| US20080138329A1 (en) | Inhibitors of Dna Methylation in Tumor Cells | |
| Peedicayil | The role of DNA methylation in the pathogenesis and treatment of cancer | |
| Fang et al. | Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1 | |
| EP3207932A1 (en) | Dna methyltransferase inhibitors for rett syndrome therapy | |
| AU2002322805A1 (en) | Inhibitor of DNA methylation | |
| CN110691849A (en) | Antisense oligonucleotides for modulating expression of HTRA1 | |
| KR20100053150A (en) | Pharmaceutical composition containing aryloxyacetyl derivatives as an active ingredient for inhibiting dna methyltransferase | |
| EP3355883A1 (en) | Trilobine and its natural analogs for use as a drug | |
| Roatsch et al. | DNA-modifying Enzymes | |
| JP2001514862A (en) | Modulator of DNA cytosine-5 methyltransferase and use thereof | |
| JP2021511022A (en) | Oligonucleotides for regulating PIAS4 expression | |
| Vakhitova et al. | Cytosine demethylation in the tyrosine hydroxylase gene promoter in hypothalamus cells of rat brain under the action of 2-aminoadamantane compound Ladasten | |
| WO2019145386A1 (en) | Oligonucleotides for modulating csnk1d expression | |
| AU2008200601B2 (en) | Inhibitor of DNA Methylation | |
| WO2019115416A2 (en) | Oligonucleotides for modulating fndc3b expression | |
| WO2015020194A1 (en) | Antisense oligonucleotide against acsl1 | |
| JP2015082971A (en) | Antisense oligonucleotide for acsl1 | |
| EP3740574A1 (en) | Oligonucleotides for modulating erc1 expression | |
| Buontempo et al. | MBD4/MED1 protein in DNA repair and demethylation, cancer, and other diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E601 | Decision to refuse application |