KR20130039916A - Anticancer composition containing the benzohydroxymethoxychalcone - Google Patents
Anticancer composition containing the benzohydroxymethoxychalcone Download PDFInfo
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- KR20130039916A KR20130039916A KR1020110104580A KR20110104580A KR20130039916A KR 20130039916 A KR20130039916 A KR 20130039916A KR 1020110104580 A KR1020110104580 A KR 1020110104580A KR 20110104580 A KR20110104580 A KR 20110104580A KR 20130039916 A KR20130039916 A KR 20130039916A
- Authority
- KR
- South Korea
- Prior art keywords
- cancer
- benzohydroxymethoxychalcone
- compound
- caspase
- pharmaceutical composition
- Prior art date
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Abstract
Description
본 발명은 암 질환의 예방 및 치료용 약학조성물에 관한 것으로서, 더욱 상세하게는 세포사멸 조절 단백질인 카스파제(caspase) 활성에 의해 암세포의 성장억제 및 사멸작용을 갖는 하기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물 및 이를 포함하는 암 질환의 예방 및 치료용 약학 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing and treating cancer diseases, and more particularly, to a benzohydride represented by the following Chemical Formula 1 having the effect of inhibiting and killing the growth of cancer cells by caspase activity, which is an apoptosis regulating protein. It relates to a benzohydroxymethoxychalcone compound and a pharmaceutical composition for the prevention and treatment of cancer diseases comprising the same.
[화학식 1][Formula 1]
암세포의 콜로니 형성능 측정 (Colony Forming Assay; Clonogenic assay) 방법은 세포의 증식과 생존에 대한 특정 물질의 영향을 분석하는 방법으로 항암효과를 가진 물질의 스크리닝에 사용되고 있다(Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315-9). 세포자살(apoptosis) 또는 계획된 세포사멸(programmed cell death; PCD) 현상은 세포 스스로 죽음을 유도시키는 세포 생리 현상(세포 자살; cell suicide)을 의미한다(Steller, Science 1995,267,1445-1449). 계획된 세포사멸 현상은 생체의 태아 발달 과정에서 각종 기관(organ)의 형태를 적절하게 만들어주는 등의 생리적 역할뿐만 아니라(Jacobson 등, Cell,1997,88,347-354), TNF-α 수용체나 Fas 등과 같은 수용체에 리간드가 결합되거나 자외선이나 방사능 노출, 독성물질, DNA 손상 자극 등의 각종 세포 스트레스 자극 신호에 노출되어 세포의 게놈(genome)에서 심각한 손상이 발생되면 세포 스스로 죽음을 유도시키는 생체 방어 시스템으로 작용한다(Li와 Yuan, Current Opinion in Cell Biology 1999,11,261-266). Colony Forming Assay (Clonogenic assay) is a method for analyzing the effects of certain substances on the proliferation and survival of cancer cells (Franken NA, Rodermond HM, Stap J). , Haveman J, van Bree C. Clonogenic assay of cells in vitro.Nat Protoc . 2006; 1: 2315-9). Apoptosis or programmed cell death (PCD) is a cell physiological phenomenon (cell suicide) that induces cell death itself (Steller, Science 1995, 267, 1445-1449). Planned apoptosis is not only a physiological role such as the proper formation of organs in the fetal development of a living body (Jacobson et al., Cell, 1997, 88,347-354), but also TNF-α receptors and Fas. When a ligand is bound to a receptor or exposed to various cellular stress stimulation signals such as ultraviolet rays, radiation exposure, toxic substances, or DNA damage stimulation, a serious damage in the genome of a cell serves as a biological defense system that induces cell death itself. (Li and Yuan, Current Opinion in Cell Biology 1999, 11, 261-266).
카스파제(Caspase)는 세포사멸을 유도시키는 씨스테인 단백분해 효소 (cystein protease)중의 하나로서, 통상 비활성형의 전구효소(proenzyme)인 프로카스파제(pro-Caspase) 형태로 존재하고 있다. 프로카스파제 단백질은 pro-domain과 small-subunit, large-subunit 등의 구조로 구성되어있는데, 세포사멸 자극 신호에 의해 pro-domain과 large-subunit 사이 와 small-subunit 와 large-subunit 사이가 잘려진 후 small-subunit와 large-subunit 끼리만 다시 결합하여 활성형 효소로 전환된다. DNA 손상 자극에 의한 세포사멸 현상 과정의 가장 대표적인 작용 기전은 세포내로 전달된 각종 DNA 손상 자극에 의해 p53 유전자가 활성화되면서 시작된다. 활성화된 p53 유전자는 Bax나 PUMA, NOXA 같은 단백질 발현을 증가시켜 미토콘드리아 막에 존재하는 씨토크롬-C (cytochrome c) 단백질을 유리시키고, 유리된 씨토크롬-C 단백질은 Apaf-1 단백질과 카스파제-9 전구효소 (pro-Caspase-9)와 결합하여 아포프토좀(apoptosome)이라는 단백 복합체를 이루어 카스파제-9 효소를 활성화시킨다(Li 등, Cell 1997,91,479-489; Srinvasula 등 Mol Cell 1998,1,949-957; Yoshida 등, Cell 1998,94,739-750). 활성화된 카스파제-9은 곧 이어서 비활성 카스파제-3 또는 카스파제-7 전구효소(proenzyme)를 절단시켜 이들 효소를 활성화시킨다(Hakem 등, Cell 1998,94,339-352). 활성화된 카스파제-3 또는 카스파제-7은 세포의 생리적 활성에 중요한 각종 기질 단백질을 분해시켜 결과적으로 세포의 죽음이 유도된다. Caspase is one of the cysteine proteases that induce apoptosis, and is usually present in the form of pro-caspase, an inactive proenzyme. Pro-caspase proteins consist of pro-domain, small-subunit, and large-subunit structures. The pro-domain and large-subunit and the small-subunit and large-subunit are cleaved Small-subunits and large-subunits are recombined and converted to active enzymes. The most representative mechanism of action of apoptosis caused by DNA damage stimulation begins with the activation of p53 gene by various DNA damage stimuli delivered into cells. Activated p53 gene increases the expression of proteins such as Bax, PUMA, and NOXA to release the cytochrome c protein present in the mitochondrial membrane, and the free cytochrome-C protein is Apaf-1 protein and caspase- 9 Pro-Caspase-9 binds to a protein complex called apoptosome to activate the caspase-9 enzyme (Li et al., Cell 1997,91,479-489; Srinvasula et al. Mol Cell 1998,1,949 -957; Yoshida et al., Cell 1998,94,739-750). Activated caspase-9 shortly activates these enzymes by cleaving inactive caspase-3 or caspase-7 proenzymes (Hakem et al., Cell 1998,94,339-352). Activated caspase-3 or caspase-7 degrades various matrix proteins that are important for the physiological activity of the cell, resulting in cell death.
카스파제-3과 카스파제-7은 다른 종류의 카스파제와 유사하게 비활성형의 전구체(proenzyme)로 존재하다가 세포사멸 신호 자극에 의해 활성화되는 카스파제-8 또는 카스파제-9에 의해 잘려져서 활성화 된다. 활성화된 카스파제-3과 카스파제-7은 세포 생리 현상 유지에 중요한 다양한 기질 단백질을 직접 분해시키는 효소이므로, 카스파제-3과 카스파제-7의 활성을 파악하면 세포사멸 진행 여부를 용이하게 판단할 수 있으며, 카스파제-3과 카스파제-7의 활성화 여부는 전구체형이 활성형의 단편으로 잘려지는 정도를 분석함으로써 판단할 수 있다. Caspase-3 and Caspase-7, like other caspases, are cut off and activated by caspase-8 or caspase-9, which are present as inactive proenzymes and are activated by apoptosis signal stimulation. do. Activated caspase-3 and caspase-7 are enzymes that directly degrade various matrix proteins that are important for maintaining cell physiology. Therefore, if the activity of caspase-3 and caspase-7 is determined, it is easy to determine whether apoptosis is progressing. The activation of caspase-3 and caspase-7 can be determined by analyzing the extent to which the precursor form is cut into fragments of the active form.
칼콘(chalcone) 화합물은 식물의 이차대사물 생성과정에서 발견되거나 이차대사물로 알려진 폴리페놀 계열 화합물의 일종으로 치환기에 따라서 다양한 구조를 가진 유도체들의 생성이 가능하므로 많은 종류의 칼콘 화합물들이 발견되어 왔다. 이들 중 일부는 항암효과가 있다고 알려져 있다(Yadav VR, Prasad S, Sung B, Aggarwal BB. Int Immunopharmacol. 2011 Mar;11(3):295-309). 칼콘 화합물은 C6-C3-C6의 골격을 가진 화합물로서 플라보노이드 계열 화합물이 갖는 탄소 골격과 동일한 구성을 가진다. 단지 플라보노이드 계열 화합물은 C6-C3-C6를 구성하는 탄소 골격이 모두 환(ring)으로 구성되어 있는 반면 칼콘 화합물은 양쪽의 두 C6골격은 환으로 구성되고 가운데 C3 골격은 에논(enone)으로 구성되었다는 점에서 차이를 보인다. 칼콘 화합물을 구성하는 양쪽의 두 환은 방향족 환(aromatic ring)으로 이루어져 있기 때문에 다양한 치환기가 위치할 수 있는 구조를 갖는다. 그 결과 치환기의 종류와 위치에 따라서 다양한 종류의 유도체들이 존재하게 된다. 치환기가 히드록시(hydroxy)일 때는 친수성의 성질을 증가시키고, 메톡시(methoxy)일 때는 소수성의 성질을 증가시키며 또 다른 방향족 환이 치환기로 더해지게 되면 소수성을 더욱 증가시키게 된다. 친수성의 증가는 생체의 대부분을 구성하는 물과의 친화력을 용이하게 하는 반면 세포를 구성하는 세포막을 투과하기 어렵게 하는 단점을 갖는다. 따라서 칼콘 화합물에 메톡시기와 벤조기를 치환기로 덧붙이게 되면 세포막의 투과성을 높여주는 장점을 가질 것이므로 본 발명자들은 칼콘 화합물에 치환기로서 메톡시기와 벤조기를 붙이고 생체를 구성하는 물과의 친화력을 약간 가지도록 하기 위해 히드록시기도 치환기로 가진 벤조히도록시메톡시칼콘 화합물을 선택하여 항암효과를 보이는지 여부에 대한 실험을 수행하였다.Chalcone (chalcone) compound is a kind of polyphenol-based compound that is found during the production of secondary metabolites of plants or known as secondary metabolites, and many kinds of chalcone compounds have been found since they can generate derivatives having various structures according to substituents. . Some of these are known to have anticancer effects (Yadav VR, Prasad S, Sung B, Aggarwal BB. Int Immunopharmacol. 2011 Mar; 11 (3): 295-309). The chalcone compound is a compound having a skeleton of C6-C3-C6 and has the same structure as the carbon skeleton of the flavonoid compound. Only the flavonoid compounds consist of rings that contain all of the C6-C3-C6 carbon backbones, while the chalcone compounds consist of two C6 backbones on both sides of the ring, and the C3 backbone consists of enones. There is a difference in this respect. Both rings constituting the chalcone compound are composed of aromatic rings, and thus have a structure in which various substituents can be located. As a result, various kinds of derivatives exist depending on the type and position of the substituent. When the substituent is hydroxy (hydroxy) increases the hydrophilic properties, when methoxy (methoxy) it increases the hydrophobic properties, and when another aromatic ring is added as a substituent it is further increased hydrophobicity. Increasing hydrophilicity has the disadvantage of facilitating affinity with water, which constitutes the majority of the living body, while making it difficult to penetrate the cell membranes that make up the cell. Therefore, adding a methoxy group and a benzo group as a substituent to the chalcone compound will have the advantage of improving the permeability of the cell membrane. Therefore, the present inventors attach a methoxy group and a benzo group as a substituent to the chalcone compound to have a little affinity with water constituting the living body. In order to test the anticancer effect was selected by selecting a benzohydroxy methoxychalcone compound having a hydroxyl group as a substituent.
이 화합물에 대한 암세포 콜로니 형성능 평가를 수행한 결과 HCT116 사람 대장암 세포와 Capan-1 사람 췌장암 세포의 콜로니 형성능을 현저하게 감소시킨다는 사실을 확인하였다. 추가적으로 본 발명의 벤조히도록시메톡시칼콘 화합물에 의한 항암 작용 기전을 분석한 결과, 벤조히도록시메톡시칼콘 화합물은 카스파제-3과 카스파제-7을 활성화시켜 암세포의 세포사멸을 유도하는 항암효과를 확인하였다. 또한 Capna-1 췌장암 세포를 이식한 마우스 동물실험을 통하여 벤조히도록시메톡시칼콘 화합물을 생체에 투여하였을 때 생성된 종양이 효과적으로 억제된다는 결과를 확인함으로써, 본 발명을 완성하였다.Evaluation of cancer cell colony forming ability against this compound confirmed that the colony forming ability of HCT116 human colon cancer cells and Capan-1 human pancreatic cancer cells was significantly reduced. In addition, as a result of analyzing the anticancer action mechanism by the benzohydroxymethoxychalcone compound of the present invention, the benzohydroxymethoxychalcone compound activates caspase-3 and caspase-7 to induce apoptosis of cancer cells. It was confirmed. In addition, the present invention was completed by confirming that the tumors produced when the benzohydroxymethoxychalcone compound was administered to the living body were effectively inhibited through mouse animal experiments in which Capna-1 pancreatic cancer cells were transplanted.
칼콘 또는 이의 유도체를 약학조성물로 이용한 종래기술로는 MMP 활성을 억제하여 혈관신생 억제효과가 있는 등록특허 제10-0567125호(칼콘 또는 이의 유도체를 함유하는 매트릭스메탈로프로테아제 활성 억제용 약학 조성물), 글리코시다아제에 의해 유발되는 질환을 예방 및 치료하는 효과가 있는 등록특허 제10-0751899호(신규한 칼콘 유도체, 이의 약학적으로 허용가능한 염, 이의 제조방법 및 이들을 유효성분으로 함유하는 글리코시다아제에 의해 유발되는 질환의 예방 및 치료용 조성물) 등이 있다.
Conventional techniques using a chalcone or a derivative thereof as a pharmaceutical composition have been disclosed in Korean Patent No. 10-0567125 (A matrix composition for inhibiting matrix metalloprotease activity containing a chalcone or a derivative thereof) by inhibiting MMP activity. Patent No. 10-0751899 (new chalcone derivatives, pharmaceutically acceptable salts thereof, preparation method thereof and glycosidase containing them as an active ingredient) which has the effect of preventing and treating diseases caused by glycosidase. Composition for the prevention and treatment of diseases caused by).
본 발명의 목적은, 세포사멸 조절 단백질인 카스파제(caspase) 활성에 의해 암세포의 성장억제 및 사멸작용을 갖는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물 및 이를 포함하는 암 질환의 예방 및 치료용 약학 조성물을 제공함에 있다.An object of the present invention, benzohydroxymethoxychalcone compound having a growth inhibitory and killing effect of cancer cells by caspase activity, which is a cell death regulating protein and a pharmaceutical for the prevention and treatment of cancer diseases comprising the same In providing a composition.
또한, 본 발명의 다른 목적은 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물을 포함하는 암 질환의 예방 및 개선용 건강기능식품을 제공함에 있다.
In addition, another object of the present invention to provide a health functional food for the prevention and improvement of cancer diseases comprising a benzohydroxymethoxychalcone compound.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물 또는 이의 약학적으로 허용되는 염을 제공한다.In order to achieve the above object, the present invention provides a benzohydroxymethoxychalcone compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
또한, 본 발명은 상기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물의 제조방법에 있어서, (1) 2-히드록시-4-메톡시-아세토페논(2-hydroxy-4-methoxy-1-acetophenone)과 1-나프타알데하이드(4-naphthaaldehyde)를 에탄올에 용해시키는 단계;와 (2) 상기 (1)단계에 의한 혼합용액에 50% KOH 수용액을 첨가하는 단계;와 (3) 상기 (2)단계에 의해 제조된 혼합용액을 상온에서 교반한 후 4℃로 냉각시키는 단계;와 (4) 상기 (3)단계에 의해 냉각된 혼합용액에 6N HCl용액을 첨가하여 중화시킨 후 클로로폼으로 2번 추출하는 단계;와 (5) 상기 (4)단계에 의해 추출된 각각의 유기 용매를 합친 후 황산마그네슘을 가하여 물을 제거하는 단계; 및 (6) 상기 (5)단계에서 물이 제거되고 남은 혼합물을 감압여과 및 건조한 다음 관 크로마토그라피로 분리정제하는 단계;를 포함하는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물의 제조방법을 제공한다.
In addition, the present invention is a method for producing a benzohydroxymethoxychalcone compound represented by the formula (1), (1) 2-hydroxy-4-methoxy-acetophenone (2-hydroxy-4-methoxy -1-acetophenone) and 1-naphthaaldehyde (4-naphthaaldehyde) in ethanol; and (2) adding a 50% KOH aqueous solution to the mixed solution according to step (1); and (3) the Stirring the mixed solution prepared by step (2) at room temperature and then cooling to 4 ° C .; and (4) neutralizing by adding 6N HCl solution to the mixed solution cooled by step (3) and neutralizing it. (2) combining the respective organic solvents extracted by step (4) and then adding magnesium sulfate to remove water; It provides a method for producing a benzohydroxymethoxychalcone compound comprising a; and (6) the water is removed in step (5) and the remaining mixture is filtered under reduced pressure and dried and separated by column chromatography. .
또한, 본 발명은 대장암, 위암, 전립선암, 유방암, 신장암, 간암, 뇌종양, 폐암, 자궁암, 결장암, 방광암, 췌장암, 혈액암으로 구성된 그룹에서 선택되는 암세포에 대하여 세포사멸 조절 단백질인 카스파제(caspase) 활성에 의해 암세포의 성장억제 및 사멸작용을 갖는 상기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물과 약학적으로 허용되는 염을 포함하여 이루어지는 암 질환의 예방 및 치료용 약학조성물을 제공한다.In addition, the present invention is a caspase is apoptosis control protein for cancer cells selected from the group consisting of colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, blood cancer Pharmaceuticals for the prevention and treatment of cancer diseases comprising a benzohydroxymethoxychalcone compound represented by the formula (1) and a pharmaceutically acceptable salt having growth inhibition and death of cancer cells by the caspase activity To provide a composition.
상기 약학조성물은 약학적으로 허용 가능한 1종 이상의 담체, 희석제 또는 부형제를 포함하는 것을 특징으로 한다.The pharmaceutical composition is characterized in that it comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
상기 약학조성물은 제2의 항암제 또는 항암 보조제를 포함하는 것을 특징으로 한다.The pharmaceutical composition is characterized in that it comprises a second anticancer agent or anticancer adjuvant.
상기 제2의 항암제는 인터페론(interferon), 인터루킨-2(interleukin-2), 파클리탁셀(paclitaxel), 빈크리스틴(vincristine), 빈블라스틴(vinblastin), 독소루비신(doxorrubicin), 에토포시드(etoposide), 이리노테칸 히드로클로라이드(irinotecan hydrochloride), 시스플라틴(cisplatin), 암사크린(amsacrine), 사이토신 아라비노시드(cytosine arabinoside), 플루오로우라실(fluoro uracil) 및 탁솔(taxol)로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 한다.
The second anticancer agent is interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorrubicin, etoposide, etoposide, Irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluorouracil and taxol It is done.
또한, 본 발명은 상기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물 및 식품학적으로 허용 가능한 식품 보조 첨가제를 포함하는 암 질환의 예방 및 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing and improving cancer diseases, including a benzohydroxymethoxychalcone compound represented by the formula (1) and a food supplement acceptable food supplement.
상기 건강기능식품은 정제, 캡슐제, 환제 또는 액제 형태인 것을 특징으로 한다.
The health functional food is characterized in that the tablet, capsule, pills or liquid form.
상기와 같은 본 발명에 따르면, 세포사멸 조절 단백질인 카스파제(caspase) 활성에 의해 암세포의 성장억제 및 사멸작용을 갖는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물 및 이를 포함하는 암 질환의 예방 및 치료용 약학 조성물과 암 질환의 예방 및 개선용 건강기능식품을 제공함으로써, 암 질환을 예방 및 치료하는데 유용하게 사용될 수 있을 뿐만 아니라, 암 질환의 예방 및 개선을 위한 건강기능식품에 유용하게 사용될 수 있는 효과가 있다.
According to the present invention as described above, benzohydroxymethoxychalcone compound having a growth inhibitory and killing effect of cancer cells by the caspase (caspase) activity which is apoptosis control protein and the prevention and treatment of cancer diseases including the same By providing a pharmaceutical composition and a health functional food for preventing and improving cancer diseases, not only can be usefully used for preventing and treating cancer diseases, but also can be useful for health functional foods for preventing and improving cancer diseases. It works.
도 1 은 벤조히드록시메톡시칼콘 화합물의 수소핵자기공명분광스펙트럼(400MHz 브루커 핵자기공명분광기기 사용).
도 2 는 벤조히드록시메톡시칼콘 화합물의 탄소핵자기공명분광스펙트럼(100MHz 브루커 핵자기공명분광기기 사용).
도 3 은 Capan-1 사람 췌장암 세포와 HCT116 사람 대장암 세포에서 벤조히드록시메톡시칼콘 화합물에 의한 암세포 콜로니 형성 억제 효과를 나타낸 것.
도 4 는 유세포 분리 측정기(fluorescent activating cell sorting, FACS)를 이용하여 벤조히드록시메톡시칼콘 화합물의 세포주기 진행 억제 및 세포사멸 효과를 분석한 것.
도 5 는 벤조히드록시메톡시칼콘 화합물에 의한 카스파제(caspase) 활성 효과를 면역블롯법으로 분석한 것.
도 6 은 벤조히드록시메톡시칼콘 화합물에 의해 카스파제-7(caspase-7)과 핵내 DNA가 잘려지는 현상을 형광현미경으로 관찰한 것.
도 7 은 벤조히드록시메톡시칼콘 화합물의 종양 억제 효과를 나타낸 것.1 is a hydrogen nuclear magnetic resonance spectra of the benzohydroxymethoxychalcone compound (using a 400 MHz Bruker nuclear magnetic resonance spectrometer).
2 is a carbon nuclear magnetic resonance spectroscopy spectrum of a benzohydroxymethoxychalcone compound (using a 100 MHz Bruker nuclear magnetic resonance spectrometer).
Figure 3 shows the inhibitory effect of cancer cell colony formation by the benzohydroxymethoxychalcone compound in Capan-1 human pancreatic cancer cells and HCT116 human colon cancer cells.
Figure 4 analyzes the cell cycle progression inhibition and apoptosis effect of benzohydroxymethoxychalcone compound using a fluorescent activating cell sorting (FACS).
Figure 5 is an analysis of the caspase (caspase) activity effect by the benzohydroxymethoxychalcone compound by immunoblot method.
Figure 6 is observed by the fluorescence microscope the phenomenon that the caspase-7 (caspase-7) and the nuclear DNA is cut by the benzohydroxymethoxychalcone compound.
Figure 7 shows the tumor suppression effect of the benzohydroxymethoxychalcone compound.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 하기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물 또는 이의 약학적으로 허용되는 염을 제공한다The present invention provides a benzohydroxymethoxychalcone compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof
[화학식 1][Formula 1]
본 발명은 상기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물의 제조방법에 있어서, (1) 2-히드록시-4-메톡시-아세토페논(2-hydroxy-4-methoxy-1-acetophenone)과 1-나프타알데하이드(4-naphthaaldehyde)를 에탄올에 용해시키는 단계;와 (2) 상기 (1)단계에 의한 혼합용액에 50% KOH 수용액을 첨가하는 단계;와 (3) 상기 (2)단계에 의해 제조된 혼합용액을 상온에서 교반한 후 4℃로 냉각시키는 단계;와 (4) 상기 (3)단계에 의해 냉각된 혼합용액에 6N HCl용액을 첨가하여 중화시킨 후 클로로폼으로 2번 추출하는 단계;와 (5) 상기 (4)단계에 의해 추출된 각각의 유기 용매를 합친 후 황산마그네슘을 가하여 물을 제거하는 단계; 및 (6) 상기 (5)단계에서 물이 제거되고 남은 혼합물을 감압여과 및 건조한 다음 관 크로마토그라피로 분리정제하는 단계;를 포함하는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물의 제조방법을 제공한다.
The present invention is a method for producing a benzohydroxymethoxychalcone compound represented by the formula (1), (1) 2-hydroxy-4-methoxy-acetophenone (2-hydroxy-4-methoxy-1 -acetophenone) and 1-naphthaaldehyde (4-naphthaaldehyde) dissolving in ethanol; and (2) adding a 50% KOH aqueous solution to the mixed solution of step (1); and (3) the (2) Stirring the mixed solution prepared by the step at room temperature and then cooling to 4 ° C .; and (4) adding 6N HCl solution to the mixed solution cooled by the step (3) and neutralizing the mixture. Extracting once; and (5) combining the respective organic solvents extracted by step (4) and then adding magnesium sulfate to remove water; It provides a method for producing a benzohydroxymethoxychalcone compound comprising a; and (6) the water is removed in step (5) and the remaining mixture is filtered under reduced pressure and dried and separated by column chromatography. .
본 발명은 대장암, 위암, 전립선암, 유방암, 신장암, 간암, 뇌종양, 폐암, 자궁암, 결장암, 방광암, 췌장암, 혈액암으로 구성된 그룹에서 선택되는 암세포에 대하여 세포사멸 조절 단백질인 카스파제(caspase) 활성에 의해 암세포의 성장억제 및 사멸작용을 갖는 상기 화학식 1로 표시되는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물과 약학적으로 허용되는 염을 포함하여 이루어지는 암 질환의 예방 및 치료용 약학조성물을 제공한다.The present invention is a caspase (caspase) which is an apoptosis regulating protein for cancer cells selected from the group consisting of colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, and blood cancer. A pharmaceutical composition for the prevention and treatment of cancer diseases comprising a benzohydroxymethoxychalcone compound represented by
상기 약학조성물은 약학적으로 허용 가능한 1종 이상의 담체, 희석제 또는 부형제를 포함할 수 있다. 상기 담체, 희석제 또는 부형제로는 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있으나 이에 한정되지는 않는다.The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers, diluents or excipients. The carrier, diluent or excipient may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
상기 약학조성물은 제2의 항암제 또는 항암 보조제를 포함할 수 있다. 상기 제2의 항암제 또는 항암 보조제로는 인터페론(interferon), 인터루킨-2(interleukin-2), 파클리탁셀(paclitaxel), 빈크리스틴(vincristine), 빈블라스틴(vinblastin), 독소루비신(doxorrubicin), 에토포시드(etoposide), 이리노테칸 히드로클로라이드(irinotecan hydrochloride), 시스플라틴(cisplatin), 암사크린(amsacrine), 사이토신 아라비노시드(cytosine arabinoside), 플루오로우라실(fluoro uracil) 및 탁솔(taxol)을 들 수 있으나 이에 한정되지는 않는다.The pharmaceutical composition may include a second anticancer agent or an anticancer adjuvant. The second anticancer agent or anticancer agent includes interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorrubicin, etoposide (etoposide), irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluoro uracil and taxol It is not limited.
본 발명의 벤조히드록시메톡시칼콘 화합물을 함유하는 약학조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상세하게는 제제화할 경우에는 보통 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제, 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제는 상기 벤조히드록시메톡시칼콘 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 액제, 유제, 시럽제 등이 해당되는데, 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, 비수성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.Pharmaceutical compositions containing the benzohydroxymethoxychalcone compounds of the present invention may be oral formulations, external preparations, suppositories, or sterile injections of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to conventional methods, respectively. It can be formulated and used in the form of a solution. Specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like which are commonly used. Solid preparations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like with the benzohydroxymethoxychalcone compound. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups, 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. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 벤조히드록시메톡시칼콘 화합물의 바람직한 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 벤조히드록시메톡시칼콘 화합물 또는 이의 약학적으로 허용 가능한 염은 0.0001 내지 100㎎/㎏으로, 바람직하게는 0.001 내지 100㎎/㎏의 양을 일일 1회 내지 수회로 나누어 투여할 수 있다. 전체 약학조성물에서 본 발명의 벤조히드록시메톡시칼콘 화합물 또는 이의 약학적으로 허용 가능한 염은 0.0001 내지 10중량%, 바람직하게는 0.001 내지 1중량%로 포함되어야 한다.Preferred dosages of the benzohydroxymethoxychalcone compounds of the present invention vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the benzohydroxymethoxychalcone compound of the present invention or a pharmaceutically acceptable salt thereof is 0.0001 to 100 mg / kg, preferably in an amount of 0.001 to 100 mg / kg once a day It can be administered in divided doses. The benzohydroxymethoxychalcone compound or pharmaceutically acceptable salt thereof of the present invention in the entire pharmaceutical composition should be included in 0.0001 to 10% by weight, preferably 0.001 to 1% by weight.
본 발명의 약학조성물은 위, 마우스, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있으며, 투여의 방식은 경구, 직장, 정맥, 근육, 피하 등 다양한 방법으로 주사에 의해 투여될 수 있다.
The pharmaceutical composition of the present invention can be administered to mammals such as stomach, mouse, livestock, human, etc. by various routes, and the mode of administration can be administered by injection in various ways such as oral, rectal, intravenous, intramuscular, subcutaneous. .
또한, 본 발명은 상기 화학식 1로 표시되는 벤조히드록시메톡시칼콘 화합물 및 식품화학적으로 허용 가능한 식품 보조 첨가제를 포함하는 암 질환의 예방 및 개선용 건강기능식품을 제공한다. 예를 들면, 건강기능식품으로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료, 차, 드링크제, 알콜 음료 및 비타민 복합제 등에 벤조히드록시메톡시칼콘 화합물 또는 이의 약학적으로 허용 가능한 그의 염을 첨가하여 제조할 수 있다.
The present invention also provides a health functional food for preventing and improving cancer diseases, including the benzohydroxymethoxychalcone compound represented by
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1. 벤조히드록시메톡시칼콘 화합물(DK49)의 합성Example 1 Synthesis of Benzohydroxymethoxychalcone Compound (DK49)
본 발명에서는 화학식 1로 표시되는 벤조히드록시메톡시칼콘을 아래 반응식 1에 나타낸 방법을 사용하여 다음과 같이 합성하였다.In the present invention, benzohydroxymethoxychalcone represented by
[반응식 1][Reaction Scheme 1]
상기 칼콘 화합물을 합성은 2-히드록시-4-메톡시-아세토페논(2-hydroxy-4methoxy-1-acetophenone, 830 mg, 5 mmol)과 1-나프타알데하이드(4-naphthaldehyde, 936 mg, 6 mmol)를 30 ㎖의 에탄올에 녹인 후에 4℃에서 50% KOH 수용액 8 ㎖을 약 5분간 천천히 가하였다. 위 혼합용액을 상온에서 22시간 교반한 후에 온도를 약 4℃로 낮추었다. 위의 냉각된 용액에 6 N HCl용액 20 ㎖ 를 가하여 중화시킨 후, 이 수용액을 클로로폼 70 ㎖로 두 번 추출하였다. 추출된 각각의 유기 용매를 합친 후 황산마그네슘을 가하여 물을 제거하고, 남은 혼합물을 감압여과 하였다. 다음으로, 회전증발기를 이용하여 여액의 용매를 제거한 후 남은 혼합물을 관 크로마토그라피로 분리정제 함으로써 칼콘 화합물 1 (DK49)을 1185 mg (78 %)의 수율로 얻었다. The chalcone compound was synthesized with 2-hydroxy-4-methoxy-acetophenone (2-hydroxy-4methoxy-1-acetophenone, 830 mg, 5 mmol) and 1-naphthaldehyde (4-naphthaldehyde, 936 mg, 6 mmol). ) Was dissolved in 30 ml of ethanol, and 8 ml of 50% KOH aqueous solution was slowly added at 4 ° C. for about 5 minutes. After stirring the mixed solution at room temperature for 22 hours, the temperature was lowered to about 4 ° C. After neutralizing by adding 20 ml of 6 N HCl solution to the cooled solution, the aqueous solution was extracted twice with 70 ml of chloroform. The combined organic solvents were combined, magnesium sulfate was added to remove water, and the remaining mixture was filtered under reduced pressure. Next, the solvent of the filtrate was removed using a rotary evaporator, and the remaining mixture was separated and purified through column chromatography to obtain Chalcone Compound 1 (DK49) in a yield of 1185 mg (78%).
최종산물의 확인을 위해 핵자기공명분광 실험을 수행하였다. 사용한 기기는 브루커사 400MHz 기기였다. 수소핵자기공명분광스펙트럼과 탄소핵자기공명스펙트럼은 각각 도 1. 및 도 2.에 나타낸 바와 같고 화학적 이동도는 아래와 같다.Nuclear magnetic resonance spectroscopy was performed to confirm the final product. The device used was a
1H-NMR(400MHz) δ : 3.88(s), 6.52(m), 6.52(m), 7.55(m), 7.57(m), 7.61(m), 7.67(d, 15.2), 7.88(m), 7.91(m), 7.95(m), 7.92(m), 8.29(d, 8.5), 8.75(d, 15.2) 1 H-NMR (400 MHz) δ: 3.88 (s), 6.52 (m), 6.52 (m), 7.55 (m), 7.57 (m), 7.61 (m), 7.67 (d, 15.2), 7.88 (m) , 7.91 (m), 7.95 (m), 7.92 (m), 8.29 (d, 8.5), 8.75 (d, 15.2)
13C-NMR(100MHz) δ : 55.8, 101.3, 108.0, 114.3, 123.2, 123.7, 125.4, 125.6, 126.6, 127.2, 129.0, 131.1, 131.5, 132.0, 132.5, 133.9, 141.5, 166.5, 167.0, 191.1
13 C-NMR (100 MHz) δ: 55.8, 101.3, 108.0, 114.3, 123.2, 123.7, 125.4, 125.6, 126.6, 127.2, 129.0, 131.1, 131.5, 132.0, 132.5, 133.9, 141.5, 166.5, 167.0, 191.1
실시예 2. 벤조히드록시메톡시칼콘 화합물(DK49)의 콜로니 형성능 평가를 통한 암세포주 성장 억제 확인Example 2. Confirmation of growth inhibition of cancer cell line through evaluation of colony forming ability of benzohydroxymethoxychalcone compound (DK49)
HCT116 대장암 세포와 Capan-1 췌장암 세포를 ATTC(American Type Culture Collection)로부터 구입하여 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies), Antibiotic-Antimycotic solution (Invitrogen Life Technologies) 포함한 DMEM (Invitrogen Life Technologies) 배양액을 2일에 한 번씩 100-mm 세포배양접시에 1 x 106의 접종 밀도(seed density)로 계대하면서 37℃, 5% CO2 배양기에서 배양하였다. 벤조히드록시메톡시칼콘 화합물(DK49)에 의한 세포증식 억제 효과는 암세포의 콜로니 형성능 평가 (Colonony forming assay)를 통하여 세포 성장이 억제되는지의 여부로 측정하였다. HCT116 대장암 세포와 Capan-1 췌장암 세포를 24-well 배양접시에 well 당 6000개 세포로 분주한 후 0, 5, 10, 20 μM 농도의 벤조히드록시메톡시칼콘 화합물(DK49)을 처리하고, 7일 후 6% 글루타르알데하이드 (glutaraldehyde)와 0.5% 크리스탈바이올렛 (crystal violet) 용액을 1:1로 섞어준 혼합액을 세포에 첨가한 후 15분 동안 반응시켜 남아있는 세포를 염색하였다. 그 결과 도 3.에 나타난 바와 같이 벤조히드록시메톡시칼콘 화합물(DK49)을 10 μM 농도 이상으로 처리했을 때 암세포의 콜로니 형성능이 급격히 감소되는 것이 관찰되었다. 이러한 사실로 부터 벤조히드록시메톡시칼콘 화합물(DK49)은 대장암과 췌장암 세포의 증식을 억제시키는 효과가 있다는 사실을 확인하였다.
HCT116 colorectal cancer cells and Capan-1 pancreatic cancer cells were purchased from the American Type Culture Collection (ATTC) and DMEM (Invitrogen Life Technologies) including 10% FBS (Fetal Bovine Serum, Invitrogen Life Technologies), Antibiotic-Antimycotic solution (Invitrogen Life Technologies) Cultures were incubated in a 5% CO 2 incubator at 37 ° C., passaged once every two days at a seed density of 1 × 10 6 in a 100-mm cell culture dish. The effect of inhibiting cell proliferation by the benzohydroxymethoxychalcone compound (DK49) was measured by whether or not cell growth was inhibited through colony forming assay of cancer cells. HCT116 colorectal cancer cells and Capan-1 pancreatic cancer cells were dispensed at 6000 cells per well in a 24-well culture dish and treated with benzohydroxymethoxychalcone compounds (DK49) at concentrations of 0, 5, 10, and 20 μM, After 7 days, a mixture of 6% glutaraldehyde and 0.5% crystal violet solution in a 1: 1 ratio was added to the cells, and the remaining cells were stained by reacting for 15 minutes. As a result, as shown in FIG. 3, it was observed that colony forming ability of cancer cells was rapidly decreased when the benzohydroxymethoxychalcone compound (DK49) was treated at 10 μM or more. From these facts, it was confirmed that benzohydroxymethoxychalcone compound (DK49) has an effect of inhibiting the proliferation of colorectal cancer and pancreatic cancer cells.
실시예 3. 벤조히드록시메톡시칼콘 화합물(DK49)의 세포주기 억제 및 세포사멸 효과Example 3. Cell Cycle Inhibition and Apoptosis Effect of Benzohydroxymethoxychalcone Compound (DK49)
Capan-1 사람 췌장암 세포에 벤조히드록시메톡시칼콘 화합물(DK49)을 처리하고 1일 간격으로 1% 트립신-EDTA 용액을 첨가해 배양 용기에 부착되어 있던 세포를 떼어내어 세포를 수확한 후, 70% 에탄올을 첨가해 세포를 고정시켰다. PI (Propidium Iodide)를 첨가해 세포의 DNA를 염색한 다음 유세포 분리측정기(Fluorescent Activating Cell Sorting; FACS, BD Science, 미국)를 이용하여 세포의 DNA 함량을 측정하였다.Treated with benzohydroxymethoxychalcone compound (DK49) to Capan-1 human pancreatic cancer cells and added 1% trypsin-EDTA solution at daily intervals to detach the cells attached to the culture vessel and harvest the cells. Cells were fixed by adding% ethanol. PI (Propidium Iodide) was added to stain the DNA of the cells, and the DNA content of the cells was measured using a Fluorescent Activating Cell Sorting (FACS, BD Science, USA).
도 4.에 나타낸 바와 같이, 정상적으로 성장하고 있는 Capan-1 췌장암세포에서 G2/M 주기를 가지는 세포는 약 25.3%였지만 벤조히드록시메톡시칼콘 화합물(DK49)를 처리한 세포군에서는 1일 후 69.3%로 증가하였다. 이후 G2/M 주기 세포는 점차 감소하면서 sub-G0/G1 세포양이 증가되는 것이 관찰되었다. 세포사멸(apoptosis)이 유도되면 G1 주기 세포의 2N DNA 함량보다 작은 양의 DNA를 함유하는 세포(sub-G0/G1)가 생기게 되므로, 본 발명의 DK49 화합물은 HCT116 대장암 세포에서 G2/M 세포주기 진행을 차단하면서 세포사멸을 유도시킨다는 사실을 확인할 수 있었다.
As shown in FIG. 4, about 25.3% of the normally growing Capan-1 pancreatic cancer cells had a G2 / M cycle, but 69.3% after 1 day in the cell group treated with the benzohydroxymethoxychalcone compound (DK49). Increased to. Since the G2 / M cycle cells gradually decreased, it was observed that the amount of sub-G0 / G1 cells increased. Induction of apoptosis results in cells (sub-G0 / G1) containing a smaller amount of DNA than the 2N DNA content of G1 cycle cells, so that the DK49 compounds of the present invention are G2 / M cells in HCT116 colorectal cancer cells. Blocking the cycle progression was found to induce apoptosis.
실시예 4. 벤조히드록시메톡시칼콘 화합물(DK49)에 의한 세포사멸 조절 단백질 카스파제(Caspase) 활성 효과 분석Example 4 Analysis of Effect of Apoptosis-Casting Caspase Activity by Benzohydroxymethoxychalcone Compound (DK49)
벤조히드록시메톡시칼콘 화합물(DK49)에 의한 세포 사멸 기전을 분석하기 위하여, 벤조히드록시메톡시칼콘 화합물(DK49)을 처리한 Capan-1 세포에서 활성형태의 Caspase-9과 Caspase-3, Caspase-7 단백질의 절단형 변화를 면역블롯법으로 조사하였다. Capan-1 췌장암 세포를 60 ㎜ 배양접시에서 1.5 X 106개 되도록 배양하고, 20 μM 농도가 되도록 벤조히드록시메톡시칼콘 화합물(DK49)을 처리한 후 0, 6, 12, 24, 48시간 경과 후 세포를 수확하여, 세포용해액(cell lysis buffer)으로 세포를 용해시킨 후, 고속원심분리하여 세포 용해액만을 수확하였다. 동량의 단백질이 포함하도록 제조된 시료를 SDS-폴리아크릴아마이드 겔(SDS-polyacrylamide gel) 전기영동을 실시하여 세포에 존재하는 단백질들을 분리하였다. 전기영동으로 분리된 단백질을 폴리스틸렌 막 (polystyrene membrane)으로 옮긴 후 절단형의 Caspase-9, Caspase-7. Caspase-3 단백질과 poly(ADP-ribose)polymerase(PARP)와 결합하는 일차 항체(Cell Signaling Technology 회사에서 구입)와 대조군으로서 단백질 발현이 변화되지 않는 GAPDH(glyceraldehyde 3-phosphate dehydrogenase)를 인지하는 일차항체 (Santa Cruz technology 회사에서 구입)를 각각 5시간 반응 시킨 후, 일차항체를 인자하는 이차항체(Cell Signaling Technology 회사에서 구입)를 1시간동안 반응시켰다. 화학형광감지 시스템 (chemiluminescence) (Amersham Pharmacia Biotechnology에서 구입)을 이용하여 Caspase 단백질 활성형을 분석하였다. In order to analyze the cell death mechanism by benzohydroxymethoxychalcone compound (DK49), the active forms of Caspase-9, Caspase-3, Caspase in Capan-1 cells treated with benzohydroxymethoxychalcone compound (DK49) Cleavage change of -7 protein was examined by immunoblot method. Capan-1 pancreatic cancer cells were cultured in a 60 mm culture dish to 1.5 X 10 6 cells, and treated with benzohydroxymethoxychalcone compound (DK49) to a concentration of 20 μM, followed by 0, 6, 12, 24, 48 hours. The cells were harvested, lysed with cell lysis buffer, and then centrifuged at high speed to harvest only the cell lysate. Samples prepared to contain the same amount of protein were subjected to SDS-polyacrylamide gel electrophoresis to separate proteins present in the cells. The proteins separated by electrophoresis were transferred to a polystyrene membrane, and then cut into Caspase-9 and Caspase-7. Primary antibody that binds to Caspase-3 protein and poly (ADP-ribose) polymerase (PARP) (purchased from Cell Signaling Technology) and primary antibody that recognizes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with unchanged protein expression as a control After 5 hours of reaction (purchased from Santa Cruz technology company), a secondary antibody (purchased from Cell Signaling Technology company) that reacts with primary antibody was reacted for 1 hour. The Caspase protein active form was analyzed using a chemiluminescence system (purchased from Amersham Pharmacia Biotechnology).
도 5.에 나타낸 바와 같이 벤조히드록시메톡시칼콘 화합물(DK49)을 HCT116 암세포에 처리하면, 대조 단백질인 GAPDH의 양은 변하지 않았으나 세포사멸을 유도하는 단백분해 효소인 Caspase-9과 Caspase-3, Caspase-7 등의 잘려진 활성형이 증가되었으며, Caspase-3과 Caspase-7의 기질 단백질인 poly(ADP-ribose)polymerase(PARP) 단백질의 절단이 증가되었다. 그러나, 세포질 대사 효소인 GAPDH의 양은 변화되지 않았다.
As shown in FIG. 5, when the benzohydroxymethoxychalcone compound (DK49) was treated to HCT116 cancer cells, the amount of GAPDH, a control protein, was not changed but Caspase-9 and Caspase-3 and Caspase, which are protease enzymes that induce cell death. The truncated active forms such as -7 were increased, and cleavage of poly (ADP-ribose) polymerase (PARP) protein, which is the substrate protein of Caspase-3 and Caspase-7, was increased. However, the amount of cellular metabolic enzyme GAPDH did not change.
실시예 5. 벤조히드록시메톡시칼콘 화합물(DK49)에 의한 Caspase-7 활성 효과와 핵내 DNA 단편(nuclear DNA fragmentation) 현상 분석Example 5 Analysis of Caspase-7 Activity and Nuclear DNA Fragmentation Phenomenon by Benzohydroxymethoxychalcone Compound (DK49)
Caspase 활성과 세포사멸과의 상관관계를 좀 더 알아보기 위하여, 형광 현미경을 이용하여 세포내에 절단형 카스파제-7 단백질 존재와 세포사멸 유도 여부를 조사하였다. Capan-1 췌장암세포를 커버 글라스 (cover glasss)에서 배양한 후 벤조히드록시메톡시칼콘 화합물(DK49)을 24시간 동안 처리한 후 절단형의 Caspase-7 단백질과 결합하는 일차 항체를 90분 동안 반응하고 적색 형광체 Alexa-Fluor 555가 결합된 이차항체(Invitrogen 회사에서 구입)와 30분 동안 반응시켰다. 커버글라스를 PBS 완충용액으로 10분간 세척하고 파랑색으로 DNA를 선택적으로 염색하는 시약 Hoechst 33258 시약(Invitrogen 회사에서 구입)을 첨가한 후 FV-1000 공촛점 형광 현미경 (Olympus 회사에서 구입)을 이용하여 관찰하였다. In order to examine the correlation between caspase activity and apoptosis, the presence of cleaved caspase-7 protein and the induction of apoptosis were investigated by fluorescence microscopy. After culturing Capan-1 pancreatic cancer cells in cover glass, the benzohydroxymethoxychalcone compound (DK49) was treated for 24 hours, and then the primary antibody binding to the cleaved Caspase-7 protein was reacted for 90 minutes. And reacted with a secondary antibody (purchased from Invitrogen) with a red phosphor Alexa-Fluor 555 for 30 minutes. Wash the cover glass with PBS buffer for 10 minutes and add reagent Hoechst 33258 reagent (purchased from Invitrogen) to selectively stain DNA in blue, and then use FV-1000 confocal fluorescence microscope (purchased from Olympus). Observed.
도 6.에 나타낸 바와 같이 벤조히드록시메톡시칼콘 화합물(DK49) 처리에 의해 활성형 Caspase-7(적색으로 염색)을 발현시키는 세포가 증가되었으며, 활성형 Caspase-7이 나타나는 세포에서만 세포사멸의 특징적 현상으로 나타나는 핵내 DNA 단편(nuclear fragmentation; 화살표로 표시)이 관찰되었다. As shown in FIG. 6, the cells expressing the active Caspase-7 (stained in red) were increased by the treatment with the benzohydroxymethoxychalcone compound (DK49), and the cell death was observed only in the cells showing the active Caspase-7. Intranuclear DNA fragments (indicated by arrows) were observed, which were characteristic features.
이러한 결과는, 벤조히드록시메톡시칼콘 화합물(DK49)을 Capan-1 췌장암세포에 처리하면 Caspase-7 등과 같은 Caspase 효소 활성이 증가되어 암세포의 세포사멸을 유도시킨다는 사실을 의미하는 것이다.
These results indicate that treatment of benzohydroxymethoxychalcone compound (DK49) to Capan-1 pancreatic cancer cells increases Caspase enzyme activity, such as Caspase-7, and induces apoptosis of cancer cells.
실시예 6. 벤조히드록시메톡시칼콘 화합물(DK49)에 의한 동물의 종양 억제 효과Example 6. Tumor Suppression Effect of Animals by Benzohydroxymethoxychalcone Compound (DK49)
본 발명의 벤조히드록시메톡시칼콘 화합물(DK49)에 의한 항암 효과를 생체에서 검증하기 위하여 Capan-1 췌장암 세포를 이식하여 암을 유발시킨 Nude 마우스를 이용하여 조사하였다. 5×106 개의 Capan-1 세포를 BALB/c-Foxn1 nu mouse 피하에 주사하고 14일 동안 방치하였다. 육안으로 종양이 관찰되고, 종양 크기가 비슷한 10 마리 마우스를 선택하여, 각 5마리씩 생리식염수만 주사하거나 (NT) 20 mg/kg의 벤조히드록시메톡시칼콘 화합물(DK49)을 복강 내로 주사하였다. 종양의 부피는 버니어캘리퍼스를 이용하여 장축(larger diameter) x 단축(smaller diameter) x 0.5로 계산하였으며, 3일 간격으로 측정하였다. In order to verify the anticancer effect of the benzohydroxymethoxychalcone compound (DK49) of the present invention in vivo, it was investigated using Nude mice transplanted with Capan-1 pancreatic cancer cells to cause cancer. 5 × 10 6 Capan-1 cells were injected subcutaneously in BALB / c- Foxil nu nu mice and left for 14 days. Ten mice were visually observed and 10 tumors of similar tumor size were selected and only 5 physiological saline was injected (5) each or intraperitoneally injected with 20 mg / kg of benzohydroxymethoxychalcone compound (DK49). Tumor volume was calculated using a vernier caliper as a large diameter (smaller diameter) x small diameter (0.5), measured every three days.
도 7A.에 나타난 바와 같이, 27일째부터 종양 크기가 대조군(NT)보다 벤조히드록시메톡시칼콘 화합물(DK49) 투여군에서 현저히 감소되는 것이 관찰되었다. As shown in FIG. 7A, it was observed from
도 7B.는 42일째 부검하기 전의 종양 크기이며, 도 7C.는 부검하여 종양을 적출 한 것이다. 대조군(NT)보다 벤조히드록시메톡시칼콘 화합물(DK49) 투여군에서 육안적 종양 크기가 현저히 감소되어 있음을 확인하였다.
Fig. 7B. Shows tumor size before autopsy at 42 days, and Fig. 7C. Shows tumors extracted at necropsy. It was confirmed that gross tumor size was significantly reduced in the benzohydroxymethoxychalcone compound (DK49) administration group than the control group (NT).
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.
Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
[화학식 1]
A benzohydroxymethoxychalcone compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.
[Formula 1]
(1) 2-히드록시-4-메톡시-아세토페논(2-hydroxy-4-methoxy-1-acetophenone)과 1-나프타알데하이드(4-naphthaaldehyde)를 에탄올에 용해시키는 단계;
(2) 상기 (1)단계에 의한 혼합용액에 50% KOH 수용액을 첨가하는 단계;
(3) 상기 (2)단계에 의해 제조된 혼합용액을 상온에서 교반한 후 4℃로 냉각시키는 단계;
(4) 상기 (3)단계에 의해 냉각된 혼합용액에 6N HCl용액을 첨가하여 중화시킨 후 클로로폼으로 2번 추출하는 단계;
(5) 상기 (4)단계에 의해 추출된 각각의 유기 용매를 합친 후 황산마그네슘을 가하여 물을 제거하는 단계;
(6) 상기 (5)단계에서 물이 제거되고 남은 혼합물을 감압여과 및 건조한 다음 관 크로마토그라피로 분리정제하는 단계;를 포함하는 벤조히드록시메톡시칼콘(benzohydroxymethoxychalcone) 화합물의 제조방법.
In the method for producing a benzohydroxymethoxychalcone compound represented by Formula 1 of claim 1,
(1) dissolving 2-hydroxy-4-methoxy-1-acetophenone and 1-naphthaaldehyde in ethanol;
(2) adding a 50% KOH aqueous solution to the mixed solution of step (1);
(3) stirring the mixed solution prepared by step (2) at room temperature and then cooling to 4 ° C;
(4) neutralizing by adding 6N HCl solution to the mixed solution cooled by step (3) and extracting twice with chloroform;
(5) combining the respective organic solvents extracted by step (4) and then adding magnesium sulfate to remove water;
(6) a method of preparing a benzohydroxymethoxychalcone compound comprising the step of (5) the water is removed and the remaining mixture is filtered under reduced pressure and dried and separated by column chromatography.
The activity of caspase, a protein that regulates apoptosis, is regulated against cancer cells selected from the group consisting of colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, and blood cancer. A pharmaceutical composition for preventing and treating cancer diseases, comprising a benzohydroxymethoxychalcone compound represented by Formula 1 of claim 1 having a growth inhibitory and killing effect of cancer cells and a pharmaceutically acceptable salt.
상기 약학조성물은 약학적으로 허용 가능한 1종 이상의 담체, 희석제 또는 부형제를 포함하는 것을 특징으로 하는 암 질환의 예방 및 치료용 약학조성물.
The method of claim 3, wherein
The pharmaceutical composition is a pharmaceutical composition for the prevention and treatment of cancer diseases, characterized in that it comprises one or more pharmaceutically acceptable carriers, diluents or excipients.
상기 약학조성물은 제2의 항암제 또는 항암 보조제를 포함하는 것을 특징으로 하는 암 질환의 예방 및 치료용 약학조성물.
The method of claim 3, wherein
The pharmaceutical composition is a pharmaceutical composition for preventing and treating cancer diseases, characterized in that it comprises a second anticancer agent or an anticancer adjuvant.
상기 제2의 항암제는 인터페론(interferon), 인터루킨-2(interleukin-2), 파클리탁셀(paclitaxel), 빈크리스틴(vincristine), 빈블라스틴(vinblastin), 독소루비신(doxorrubicin), 에토포시드(etoposide), 이리노테칸 히드로클로라이드(irinotecan hydrochloride), 시스플라틴(cisplatin), 암사크린(amsacrine), 사이토신 아라비노시드(cytosine arabinoside), 플루오로우라실(fluoro uracil) 및 탁솔(taxol)로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 하는 암 질환의 예방 및 치료용 약학조성물.
The method of claim 5, wherein
The second anticancer agent is interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorrubicin, etoposide, etoposide, Irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluorouracil and taxol A pharmaceutical composition for preventing and treating cancer diseases.
A dietary supplement for the prevention and improvement of cancer diseases comprising a benzohydroxymethoxychalcone compound represented by the formula (1) of claim 1 and a food supplement acceptable food supplement.
상기 건강기능식품은 정제, 캡슐제, 환제 또는 액제 형태인 것을 특징으로 하는 암질환 예방 및 개선용 건강기능식품.
The method of claim 7, wherein
The health functional food is a health functional food for cancer disease prevention and improvement, characterized in that the tablet, capsule, pills or liquid form.
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| PCT/KR2012/004183 WO2013054998A1 (en) | 2011-10-13 | 2012-05-25 | Novel chalcone derivative and anticancer composition comprising same as active ingredient |
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| KR101587233B1 (en) * | 2014-08-08 | 2016-01-20 | 제주대학교 산학협력단 | Skin topical composition for treating wound, recovering wound or preventing forming scar |
| CN110452162A (en) * | 2019-09-04 | 2019-11-15 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | A kind of application of chalcone derivative as Fli-1 gene target regulator |
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| FR2896245B1 (en) * | 2006-01-17 | 2010-09-17 | Univ Claude Bernard Lyon | NEW CHALCONE DERIVATIVES WITH ANTIMITOTIC ACTIVITY |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101587233B1 (en) * | 2014-08-08 | 2016-01-20 | 제주대학교 산학협력단 | Skin topical composition for treating wound, recovering wound or preventing forming scar |
| CN110452162A (en) * | 2019-09-04 | 2019-11-15 | 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) | A kind of application of chalcone derivative as Fli-1 gene target regulator |
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