KR20150025603A - A polyphenol with anticancer effect by cell cycle arrest - Google Patents
A polyphenol with anticancer effect by cell cycle arrest Download PDFInfo
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- KR20150025603A KR20150025603A KR20130103360A KR20130103360A KR20150025603A KR 20150025603 A KR20150025603 A KR 20150025603A KR 20130103360 A KR20130103360 A KR 20130103360A KR 20130103360 A KR20130103360 A KR 20130103360A KR 20150025603 A KR20150025603 A KR 20150025603A
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- South Korea
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- cell cycle
- mpbc
- polyphenol
- present
- cells
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Abstract
Description
본 발명은 세포주기 조절을 통한 항암 효과를 보이는 폴리페놀에 관한 것이다.The present invention relates to polyphenols exhibiting an anticancer effect through cell cycle regulation.
일반적으로 대장암은 화학요법제에 의하여 예방될 수 있고 식물 유래된 폴리페놀들이 그러한 것으로 테스트되고 있다. 암 세포 주기가 정상 세포 주기보다 더 빠르기 때문에, 세포 주기 정지는 정상 세포보다 암세포에 더 영향을 미친다. 세포 주기는 G0/G1, S, G2, 및 M 단계에서 고장날 수 있으며 일부 폴리페놀들은 G1 단계에서 세포주기를 정지시키고(Du, G.J.; Zhang, Z.; Wen, X.D.; Yu, C.; Calway, T.; Yuan, C.S.; Wang, C.Z. Nutrients 2012, 4, 1679-1691;Halder, B.; Das Gupta, S.; Gomes, A. FEBS J. 2012, 279, 2876-2891;Thangapazham, R.L.; Singh, A.K.; Sharma, A.; Warren, J.; Gaddipati, J.P.; Maheshwari, R.K. Cancer Lett. 2007, 245, 232-241), 반면 다른 것들은 G2/M 단계에서 세포 주기 증식을 저해한다(Visanji, J.M.; Thompson, D.G.; Padfield, P.J. Cancer Lett . 2006, 237, 130-136). In general, colon cancer can be prevented by chemotherapeutic agents and plant-derived polyphenols have been tested as such. Because the cancer cell cycle is faster than the normal cell cycle, cell cycle arrest affects cancer cells more than normal cells. The cell cycle may fail at the G0 / G1, S, G2, and M stages and some polyphenols may arrest the cell cycle at the G1 stage (Du, GJ; Zhang, Z .; Wen, XD; Yu, , T. Yuan, CS Wang, CZ Nutrients 2012, 4 , 1679-1691, Halder, B .; Das Gupta, S. Gomes, FEBS J. 2012, 279 , 2876-2891; Thangapazham, RL; 2007, 245 , 232-241), while others inhibit cell cycle proliferation at the G2 / M stage (Visanji, R. et al . , Cancer Lett. JM; Thompson, DG; Padfield, PJ Cancer Lett . 2006, 237 , 130-136).
세포 주기 진행은 CDKs(cyclin-dependent kinases)에 의하여 조절된다: CDK2/4는 G1 단계 진행을 조절하고, CDK2는 S 단계 진행을 조절하고, CDK1은 G2/M 단계 진행을 조절한다. CDK 활성화는 cyclins이 필요하고, 이것은 CDK 기질과 결합 및 특정 세포내 위치에 CDK를 타겟팅하는 것에 관여한다. G1 세포 주기 진행은 D-타입 cyclins (cyclin D)에 이하여 조절된다. p21, CIP1, 또는 WAF1로도 알려진 CDKN1A는 CDK-Cyclin 복합체의 활성을 직접적으로 저해하고 세포 주기 진행의 내생적인 조절자로 기능한다.[Sherr, C.J.; Roberts, J.M.Genes Dev . 1999, 13, 1501-1512] Cell cycle progression is regulated by CDKs (cyclin-dependent kinases): CDK2 / 4 regulates G1 stage progression, CDK2 regulates S stage progression, and CDK1 regulates G2 / M stage progression. CDK activation requires cyclins, which are involved in binding CDK substrates and targeting CDKs to specific intracellular locations. G1 cell cycle progression is regulated by D-type cyclins (cyclin D). CDKN1A, also known as p21, CIP1, or WAF1, directly inhibits the activity of the CDK-cyclin complex and functions as an endogenous regulator of cell cycle progression [Sherr, CJ; Roberts, JM Genes Dev . 1999, 13, 1501-1512]
[선행 특허 문헌][Prior Patent Literature]
대한민국 특허공개번호 제10-2013-0044837Korean Patent Publication No. 10-2013-0044837
본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 세포주기 조절을 통한 항암 효과를 보이는 화합물을 제공하는 것이다.The present invention has been made in view of the above needs, and it is an object of the present invention to provide a compound having an anticancer effect through cell cycle regulation.
상기의 목적을 달성하기 위하여 본 발명은 하기 화학식 1의 화합물을 제공한다.In order to achieve the above object, the present invention provides a compound represented by the following general formula (1).
[화학식 1][Chemical Formula 1]
상기 화학식 1에서 R1, R2, 및 R3는 H 또는 OCH3임.Wherein R 1 , R 2 , and R 3 are H or OCH 3 .
또한 본 발명은 상기 본 발명의 화합물을 유효성분으로 포함하는 항암용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for anti-cancer comprising the compound of the present invention as an active ingredient.
또한 본 발명은 상기 본 발명의 화합물을 유효성분으로 포함하는 G1 단계에서 세포 주기를 정지시키는 조성물을 제공한다.The present invention also provides a composition for stopping the cell cycle in the G1 step comprising the compound of the present invention as an active ingredient.
또한 본 발명은 1-하이드록시-2-아세토나프톤(acetonaphthone)과 2,3-디메녹시벤즈알데히드 또는 1-하이드록시-2-아세포나프톤과 3-메톡시벤즈알데히드를 알코올에 용해하고, 그 혼합물에 염기를 첨가하는 단계를 포함하는 하기 화학식 1의 화합물의 제조방법을 제공한다.The present invention also relates to a process for the preparation of 1-hydroxy-2-acetonaphthone, 2,3-dimethoxybenzaldehyde or 1-hydroxy-2-benzonaphthone and 3-methoxybenzaldehyde in an alcohol, And a step of adding a base to the mixture.
[화학식 1][Chemical Formula 1]
상기 화학식 1에서 R1, R2, 및 R3는 H 또는 OCH3임Wherein R 1 , R 2 , and R 3 are H or OCH 3
본 발명의 조성물은 대장암, 위암, 전립선암, 유방암, 신장암, 간암, 뇌종양, 폐암, 자궁암, 결장암, 방광암, 췌장암, 혈액암으로 구성된 그룹에서 선택되는 암세포에 대하여 효과를 가지는 암 질환의 예방 및 치료용 약학조성물을 제공한다.The composition of the present invention is useful for the prevention of cancer diseases having effects on cancer cells selected from the group consisting of colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain cancer, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, And a pharmaceutical composition for therapeutic use.
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 anti-cancer agent or anti-cancer adjuvant. Examples of the second anticancer agent or anticancer agent include interferon, interleukin-2, paclitaxel, vincristine, vinblastin, doxorubicin, but are not limited to, etoposide, irinotecan hydrochloride, cisplatin, amsacrine, cytosine arabinoside, fluoro uracil, and taxol. But is not limited to.
본 발명의 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. The pharmaceutical compositions containing the compounds of the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories or sterile injection solutions, Can be used. In detail, when formulating the composition, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. which are usually used.
경구투여를 위한 고형제제는 상기 본 발명의 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.The solid preparation for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like in the compound of the present invention. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile 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. Examples of the suppository base include withexol, macrogol,
본 발명의 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 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 화합물 또는 이의 약학적으로 허용 가능한 염은 0.0001 내지 10중량%, 바람직하게는 0.001 내지 1중량%로 포함되어야 한다.The preferred dosage of the compound of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention or a pharmaceutically acceptable salt thereof may be administered in an amount of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, once to several times per day . In a total pharmaceutical composition, the compound of the present invention or a pharmaceutically acceptable salt thereof should be contained in an amount of 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, and the like in various routes. The administration route can be administered by various methods such as oral, rectal, intravenous, .
이하 본 발명을 설명한다Hereinafter, the present invention will be described
본 발명에서 본 발명자들은 여덟 종의 다른 스카폴드를 포함하는 27 종의 폴리페놀을 제조하여 정량적 구조-활성 관계 연구에서 G1 단계에서 세포 주기를 정지시키는 구조적 조건에 대한 이해를 얻었다. 본 발명자들은 G1 세포 주기 정지에 관여하는 바이오포어(biophore)를 결정하기 위하여 세포 주기 프로파일을 사용하였고 본 발명에서 동정된 바이오포어들은 G1 세포 주기 정지를 야기하는 폴리페놀을 고안하는데 도움이 될 것이다.In the present invention, the present inventors obtained 27 kinds of polyphenols including eight kinds of different scaffolds and obtained the understanding of the structural conditions for stopping the cell cycle at the G1 stage in the quantitative structure-activity relationship study. We used a cell cycle profile to determine the biophore involved in G1 cell cycle arrest and the biopores identified in the present invention will help to devise a polyphenol that causes G1 cell cycle arrest.
본 발명의 화합물들을 표 1에 리스트하였다. 그들은 세 페닐이미노페놀(phenyliminophenol)(1-3), 두 페닐아세트아마이드(phenylacetamide) (4 및 5), 네 페닐벤조크로메논(phenylbenzochromenone)(6-9), 세 벤조프라바논(benzoflavanone)(10-12), 일곱 크로메닐페닐프로페논(chromenylphenylpropenone)(13-19), 세 비스크로메닐프로페논(bischromenylpropenone)(20-22), 네 크로멘(chromene)(23-26), 및 하나의 페닐크로메닐프로페논(phenylchromenylpropenone)(27)을 포함한다. The compounds of the present invention are listed in Table 1. They include phenyliminophenol (1-3), phenylacetamide (4 and 5), phenylbenzochromenone (6-9), benzoflavanone (6-9) 10-12), seven chromenylphenylpropenone (13-19), bischromenylpropenone (20-22), chromene (23-26), and one Phenylchromenylpropenone (27).
세포 주기 프로파일을 유동 세포분석법을 사용하여 분석하였다. 폴리페놀 1-27의 구조들 중 관계를 정량화하고 G1 단계에서 세포 주기 정지에 대한 그들의 효과를 정량화하기 위하여, 3차원 (3D) QSAR 연구는 비교 분자 장 분석(comparative molecular field analysis;CoMFA) 및 비교 분자 유사성 지수 분석(comparative molecular similarity indices analysis;CoMSIA)을 사용하여 수행하였다. QSAR 계산을 위하여, 생물학적 활성을 다음과 같이 결정하였다: 도 1은 유동 세포분석법에 의하여 얻어진 폴리페놀 9의 DNA 히스토그램을 나타낸다. 부위 M1-M4는 각각 sub-G1, G1, S, 및 G2/M 단계를 나타낸다. 화합물이 G1 단계에서 세포 주기를 정지시킬 때, G1 단계에서 세포의 퍼센트는 G2/M 단계에 비하여 증가되어야 한다. 초기에는 히스토그램에서 G1 단계에서 세포의 퍼센트는 52.47%이고; 24 시간 후, 그것은 51.96%이고; 48 시간 후, 그것은 79.03%가 되었다. 유사하게, 히스토그램에서 G2/M 단계에서 초기 세포 퍼센트는 32.11%이고; 24 시간 후, 그것은 35.78%이고; 48 시간 후, 그것은 12.62%가 된다. 이것은 폴리페놀 9 처리가 G1 단계에서 세포 주기를 정지시켰다는 것을 시사한다. 따라서 G2/M 단계에 대한 G1 단계에서 세포 퍼센트의 비율은 G1 단계에서 세포 주기 진행에 대한 ㅌ테테스트 화합물의 저해 효과에 대한 정보를 제공한다. 본 발명의 27 종 폴리페놀에 대한 비율을 이 형태로 얻었다(표1). 이 값들의 로그 스케일이 원 데이터보다 더 좋은 분포를 가지므로, 로그 값들을 QSAR 계산을 위한 생물학적 데이터로 사용하였다(표 1). 폴리페놀 9 또는 2-(3-methoxyphenyl)-2H-benzo[h]chromen-4(3H)-one (MPBC)는 G1 단계 세포 주기 진행을 가장 잘 저해하여 주형으로 사용하였다.Cell cycle profiles were analyzed using flow cytometry. To quantify the relationships among the structures of polyphenol 1-27 and to quantify their effect on cell cycle arrest at the G1 stage, three-dimensional (3D) QSAR studies were performed using comparative molecular field analysis (CoMFA) And comparative molecular similarity indices analysis (CoMSIA). For QSAR calculation, the biological activity was determined as follows: Figure 1 shows a DNA histogram of
CoMFA로부터 생성된 모델 중, 최고 교차 인증(cross-validated) 상관 계수(q2 = 0.754)를 가지는 모델을 추가 분석을 위하여 선택하였다. 부분 최소 자승(PLS) 분석을 27 폴리페놀의 생물학적 활성과 결과 필드 매트릭스 사이의 선형 관계를 확립하기 위하여 사용하였다. 교차 인증된 분석은 LOO(leave-one-out) 방법을 사용하여 수행하였다. 최종 비 교차 인증(r2 = 0.999) 분석은 LOO 방법으로부터 얻어진 구성요소의 최적 수(n = 6)를 사용하여 수행하였다. PLS 분석에서, 그 추정치의 표준 에러는 0.008이었고 그 F-값은 2068.154이었다. CoMFA 모델을 평가하기 위하여, 트레이닝 세트에서 화합물들의 활성을 예측하고 그 시험 데이터와 비교하였다(표 2). 트레이닝 세트에 대한 실험값과 예측값 사이의 잔차는 0.03%에서 0.67% 범위이었다. QSAR 모델을 인증하기 위하여, 다섯 화합물들(5, 10, 16, 22, 및 23)을 테스트 세트로 임의적으로 선택하였다. 그들의 잔차는 4.61%에서 14.09% 범위이었다. 따라서 결과 CoMFA 모델은 신뢰할만하다. 실험 데이터는 예측값에 EOG서 작도하였다(도 7). CoMFA는 입체 및 정전기 효과에 대한 정보만을 제공하지만, CoMSIA는 소수성, H-결합 공여체 및 수용체 효과에 대한 정보도 제공한다. 몇 CoMSIA 모델 중에서, 최고 교차 인증 값(q2 = 0.673)을 가지는 모델을 선택하였다. 상관 계수(r2)는 0.993이었다. 여섯 구성요소가 PLS 통계 파라미터에 있고, 추정치의 표준 에러는 0.020이고, 그 F-값은 337.846이었다. CoMFA와 같이, CoMSIA 모델을 평가하기 위하여, 트레이닝 세트 및 테스트 세트에 대한 활성을 예측하고 실험 데이터와 비교하였다(표 2). 실험 데이터를 예측 값에 대해서 작도하였다(도 8). 트레이닝 세트에서, 실험값과 예측값 사이의 잔차(residual)들은 0에서 1.74% 범위이었고; 테스트 세트에서, 그 범위는 4.35-13.10%이었다. 따라서, 그 CoMSIA 모델은 신뢰성이 있다.Among the models generated from CoMFA, a model with the highest cross-validated correlation coefficient (q 2 = 0.754) was selected for further analysis. Partial least squares (PLS) analysis was used to establish a linear relationship between the biological activity of the 27 polyphenols and the resulting field matrix. Cross-certified analyzes were performed using the leave-one-out (LOO) method. Final non cross-validation (r 2 = 0.999) analysis was performed using the optimal number of components (n = 6) obtained from the LOO method. In the PLS analysis, the standard error of the estimate was 0.008 and the F-value was 2068.154. To evaluate the CoMFA model, the activity of the compounds in the training set was predicted and compared with the test data (Table 2). The residuals between the experimental and predicted values for the training set ranged from 0.03% to 0.67%. To validate the QSAR model, five compounds (5, 10, 16, 22, and 23) were randomly selected as test sets. Their residuals ranged from 4.61% to 14.09%. The resulting CoMFA model is therefore reliable. Experimental data was constructed by EOG on predicted values (Fig. 7). CoMFA provides information only on stereoscopic and electrostatic effects, but CoMSIA also provides information on hydrophobic, H-bond donor and receptor effects. Of the several CoMSIA models, a model with the highest cross-certified value (q 2 = 0.673) was selected. The correlation coefficient (r 2 ) was 0.993. The six components are in the PLS statistical parameter, the standard error of the estimate is 0.020, and the F-value is 337.846. To assess the CoMSIA model, such as CoMFA, activity on the training set and test set was predicted and compared with the experimental data (Table 2). Experimental data were plotted against predicted values (Figure 8). In the training set, the residuals between the experimental and predicted values ranged from 0 to 1.74%; In the test set, the range was 4.35-13.10%. Therefore, the CoMSIA model is reliable.
27 종 폴리페놀의 구조와 세포 주기 진행의 G1 단계의 그들의 저해 사이 관계를 육안화하기 위하여, CoMFA 등고선 지도(contour map)를 프로그램 SYBYL 7.3를 사용하여 만들었다. 입체 및 정전기 장 디스크립터는 각각 44.7% 및 55.3%이었다. 입체 장에 대해서, 입체 벌크를 선호하는 부위는 19%이었고 벌크를 선호하지 않는 부위는 81%이었다. MPBC는 최고 저해 효과를 가졌고 등고선 지도에 들어있다(도 2). 유사하게 정전기 장에 대한 등고선 지도를 만들었고, 양전기 및 음전기 그룹을 선호하는 부위는 각각 14% 및 86%이었다. 크로멘 그룹의 C-2에서 벌키한 치환체는 폴리페놀 6-12가 크로멘의 C-2에서 벌키한 치환체가 없는 다른 폴리페놀보다 더 큰 활성을 나타내므로 활성에 포지티브하게 기여한다. 폴리페놀 6-9의 활성이 10-12의 것보다 더 활성이 우수하므로 7,8-naphtho 그룹은 5,6-naphtho 그룹보다 더 좋은 활성을 나타낸다. 13-22의 프로페논에서 크로메닐 그룹은 활성을 감소시켰다. 크로멘 부위의 C-4에서 음전기 그룹은 활성을 증가시켰다.To visualize the relationship between the structure of the 27 polyphenols and their inhibition at the G1 stage of cell cycle progression, a CoMFA contour map was generated using the program SYBYL 7.3. The stereoscopic and electrostatic field descriptors were 44.7% and 55.3%, respectively. For the steric field, 19% of the sites favored solid bulk and 81% sites where bulk was not preferred. MPBC has the highest inhibitory effect and is in the contour map (Fig. 2). Likewise, a contour map was created for the electrostatic field, with 14% and 86% of sites favoring the positive and negative groups respectively. The bulky substituent at C-2 in the chromene group positively contributes to the activity since polyphenol 6-12 has greater activity than other polyphenols without substituents bulky at the C-2 of the chromene. Since the activity of polyphenol 6-9 is more active than that of 10-12, the 7,8-naphtho group shows better activity than the 5,6-naphtho group. In propenone of 13-22, the chromenyl group reduced activity. At C-4 in the chromene region, the negative charge group increased activity.
CoMFA와 동일한 방법을 사용하여, CoMSIA 등고선 지도를 만들었다. 입체 및 정전기 장에 대한 정보 이외에, CoMSIA는 소수성, H-결합 공여체 및 수용체 장에 대한 정보를 제공하고, 그 CoMSIA 모델은 단지 입체, 정전기 및 H-결합 수용체 장 디스크립터만을 선택하였고 각각 30.8%, 51.0%, 및 18.2%이었다. CoMSIA 등고선 지도는 더 벌크, 덜 벌크 음전기 그룹, 양전기 그룹 및 H-결합 수용체가 선호되고, H-결합 수용체는 선호되지 않았다(도 3). 입체 장과 관련하여, 입체적 벌크를 선호하는 부위는 12%이고 벌크를 선호하지 않는 부위는 88%이었다. 정전기 장에 관하여, 양전기 그룹을 선호하는 부위가 90%이고, 양전기 그룹을 선호하는 부위는 10%이었다. H-결합 수용체 장에 대해서, H-결합 수용체를 선호하는 부위는 79%이고 비선호 부위는 21%이었다. CoMSIA 및 CoMFA 입체 및 정전기 장 등고선 지도는 CoMSIA 및 CoMFA 모두에서 정전기장 디스크립터보다 입체 장 디스크립터가 덜 기여하므로 유사한 경향을 나타내었다; 따라서 H-결합 수용체 장에 대한 등고선 지도를 분석하였다. 크로멘 부위의 C-4에서 수소결합 수용체는 활성에 기여하는 반면, 크로멘 부위의 C-3 위치에서 치환체의 수소결합 수용체는 그러하지 않았다.Using the same method as the CoMFA, a CoMSIA contour map was created. In addition to information on steric and electrostatic field, CoMSIA provided information on hydrophobic, H-bond donor and acceptor field, and the CoMSIA model selected only three-dimensional, electrostatic and H-bond acceptor intestinal descriptors, and 30.8%, 51.0 %, And 18.2%, respectively. CoMSIA contour maps favored bulk, less bulk negative groups, positively charged groups and H-coupled receptors, and H-coupled receptors were not preferred (FIG. 3). Regarding the steric field, 12% of sites favored the three-dimensional bulk and 88% sites where bulk was not preferred. Regarding the electrostatic fields, 90% preferred the positively charged group and 10% preferred the positively charged group. For H-binding receptor sites, the preferred site for H-binding receptors was 79% and the non-preferred site was 21%. The CoMSIA and CoMFA stereoscopic and electrostatic field contour maps showed a similar trend because of the less contribution of steric descriptors to electrostatic field descriptors in both CoMSIA and CoMFA; Therefore, contour maps for H-binding receptor sites were analyzed. At the C-4 of the chromene moiety, the hydrogen bond acceptor contributes to the activity, while the substituent hydrogen bond acceptor does not at the C-3 position of the chromene moiety.
CoMFA 및 CoMSIA 등고선 지도의 분석에 기초하여, G1 단계에서 현저하게 세포 주기 진행을 저해하는 바이오포어를 도 4에 나타낸 것과 같이 묘사할 수 있다. 굵은 선들은 벤질 부위가 폴리페놀을 배열하는데 사용된다는 것을 나타내고 점선은 테스트한 폴리페놀들 중 일부에 치환체가 포함된다는 것을 시사한다.Based on the analysis of CoMFA and CoMSIA contour maps, a biopore that significantly inhibits cell cycle progression in the G1 step can be depicted as shown in FIG. The thick lines indicate that the benzyl moiety is used to align the polyphenols, and the dotted line indicates that some of the polyphenols tested contain substituents.
세포 주기 진행의 저해는 종종 항증식 기작과 관련된다. 폴리페놀 9 (2-(3-methoxyphenyl)-2H-benzo[h]chromen-4(3H)-one, MPBC)의 항증식 활성을 평가하기 위하여, 지수적으로 성장하는 HCT116 세포를 여러 농도의 MPBC로 24 또는 48 시간 노출하고, 세포 생존률을 Cell Counting Kit-8 Assay 키트를 사용하여 측정하였다. 세포 생종률의 감소가 MPBC를 처리한 세포에서 용량 및 시간 의존적으로 관찰되었다(도 5A, 왼쪽 그래프). 세포 증식률을 DNA 합성 동안 BrdU(bromo-deoxyUridine) 삽입에 기반한 Cell Proliferation Assay Kit를 사용하여 측정하였다. MPBC로 처리는 미처리 대조군 세포와 비교하여 용량 및 시간 의존적으로 세포 증식 속도를 감소시켰다(도 5A, 우측 그래프). 집락형성 생존(clonogenic survival) 에세이는 단일 종양 세포가 생존 콜로니로 성장하는 활성에 기반하고 항암제의 효과를 예측하기 위한 신뢰 테스트로 간주된다. 종양 세포 성장을 저해하는 MPBC의 장기간 효과를 평가하기 위하여 집락형성 생존 에세이를 수행하였다. 7일간 MPBC의 처리는 개별 세포가 생존 콜로니로 증식하는 능력의 용량 의존적인 상실을 야기한다(도 5B). CDK 저해제 p21은 G1 세포 주기 진행을 억제하는데 관여하는 가장 강력한 세포 주기 조절자로 간주된다. p21 발현은 종양 억제자 p53-의존적 및 p53-비의존적 형태로 조절된다. MPBC-유도된 G1 세포 주기 정지의 분자 기작을 조사하기 위하여, p53 및 p21의 발현 레벨을 조사하였다. 웨스턴 블럿 분석은 p53 단백질의 양은 MPBC 처리 6시간 이내에 증가하고, 약 24 시간에 피크에 도달한 후 그 후에 48시간까지 감소하는 것을 나타내었다(도 5C). p21 단백질의 양은 6시간 후에 증가되기 시작하고 약 24 시간에 최대를 가지고 48시간까지 상당하게 감소하나 여전히 기준선에 비하여서는 높게 유지하였다. 반면, cyclins D1 및 B1의 레벨 모두는 24시간 이내에 감소하였고 이것은 MPBC-유도된 p53 및 p21 단백질의 축적은 G1 세포 주기 정지를 유도하고, 그것에 의하여 G1 및 G2/M 조절 cyclins의 발현을 변경한다는 것을 시사한다.Inhibition of cell cycle progression is often associated with antiproliferative mechanisms. To evaluate the antiproliferative activity of polyphenol 9 (2- (3-methoxyphenyl) -2 H -benzo [ h ] chromen-4 (3 H ) -one, MPBC), exponentially growing HCT116 cells Of MPBC for 24 or 48 hours, and cell viability was measured using a Cell Counting Kit-8 Assay kit. A decrease in cell lysis rate was observed in MPBC-treated cells in a dose- and time-dependent manner (Fig. 5A, left graph). Cell proliferation was measured using Cell Proliferation Assay Kit based on BrdU (bromo-deoxyuridine) insertion during DNA synthesis. Treatment with MPBC reduced cell proliferation rate in a dose- and time-dependent manner compared to untreated control cells (Fig. 5A, right graph). The clonogenic survival assay is based on the activity of single tumor cells to grow into viable colonies and is considered a confidence test to predict the effect of anticancer agents. To assess the long-term effects of MPBC inhibiting tumor cell growth, a colony formation survival assay was performed. Treatment with 7 days of MPBC causes a dose-dependent loss of the ability of individual cells to proliferate into viable colonies (Fig. 5B). The CDK inhibitor p21 is considered to be the most potent cell cycle regulator involved in inhibiting G1 cell cycle progression. p21 expression is regulated in tumor suppressor p53-dependent and p53-independent forms. To investigate the molecular mechanism of MPBC-induced G1 cell cycle arrest, expression levels of p53 and p21 were investigated. Western blot analysis indicated that the amount of p53 protein increased within 6 hours of MPBC treatment, reached a peak at about 24 hours and then decreased by 48 hours (Figure 5C). The amount of p21 protein began to increase after 6 hours and was maximal at about 24 hours and significantly reduced to 48 hours but still higher than baseline. On the other hand, both levels of cyclins D1 and B1 decreased within 24 hours, indicating that the accumulation of MPBC-induced p53 and p21 proteins induced G1 cell cycle arrest, thereby altering the expression of G1 and G2 / M regulated cyclins It suggests.
MPBC-유도된 p21 발현이 p53-의존적인 전사 활성화를 통하여 매개되는지를 확립하기 위하여, p21 프로모터 리포터 에세이를 수행하였다. HCT116 세포를 p21-Luc(-2400/+1), 2.4 kb의 5' 프랭킹 서열을 가지는 p21 프로모터-리포터 구축물 또는 p21-Luc(-952/+70), p53 컨센서스 결합 부위가 결손된 결손 구축물로 트랜스팩션하였다. 그 다음, luciferase 활성을 측정하였다. MPBC로 처리는 -2400/+1 구축물의 촉진을 야기하였으나, -952/+70 구축물은 그러하지 않았다(도 5D). 이들 데이터는 MPBC가 p21 발현을 p53-의존적인 p21 유전자의 전사 활성화를 통하여 증가시킨다는 것을 시사한다.To establish whether MPBC-induced p21 expression is mediated through p53-dependent transcriptional activation, the p21 promoter reporter assay was performed. HCT116 cells were transfected with p21-Luc (-2400 / + 1), a p21 promoter-reporter construct or p21-Luc (-952 / + 70) with a 2.4 kb flanking sequence, a p53 consensus binding site- . Then, luciferase activity was measured. Treatment with MPBC resulted in the acceleration of the -2400 / + 1 construct, while the -952 / + 70 construct did not (Fig. 5D). These data suggest that MPBC increases p21 expression through transcriptional activation of the p53-dependent p21 gene.
p53의 MPBC-유도된 p21 발현에서의 역할을 제공하기 위하여 본 발명자들은 p53-null HCT116 세포를 사용하였다. 도 6A에 나타낸 것과 같이, both p53 및 p21 발현 모두는 야생형 HCT116 세포(p53+/+)의 MPBC 노출에 수반하여 시간 의존적으로 증가하였느나, p53-null HCT116 세포(p53-/-)는 그러하지 않았다. 또한, MPBC-유도된 G1 세포 주기 정지는 p53-null HCT116 세포에서는 손상되었다(도 6B). 이 결과들은 MPBC-유도된 G1 세포 주기 정지가 HCT116 세포에서 p53의 전사적 활성화를 수반하는 p21의 업레귤에이션을 통하여 매개되는 것을 시사한다.In order to provide a role in MPBC-induced p21 expression of p53, we used p53-null HCT116 cells. As shown in Figure 6A, both p53 and p21 expression increased in a time-dependent manner with MPBC exposure of wild-type HCT116 cells (p53 + / + ), whereas p53-null HCT116 cells (p53 - / - ) did not . In addition, MPBC-induced G1 cell cycle arrest was impaired in p53-null HCT116 cells (Fig. 6B). These results suggest that MPBC-induced G1 cell cycle arrest is mediated through upregulation of p21 with transcriptional activation of p53 in HCT116 cells.
표 1은 27 종 폴리페놀의 구조와 명칭 그리고 그들의 HCT116 인간 대장암 세포에서 G1 단계의 세포 주기 진행의 저해의 로그 스케일을 나타낸 표. 별표(*)는 정량적인 구조-활성 관계를 계산하는데 사용된 테스트 세트를 나타냄.Table 1 shows the structure and name of the 27 polyphenols and the log scale of the inhibition of cell cycle progression in the G1 phase in their HCT116 human colon cancer cells. An asterisk (*) indicates the set of tests used to calculate the quantitative structure-activity relationship.
표 2는 트레이닝 세트 및 테스트 세트에 대한 잔차 값들을 가지는 예측된 값 및 실험값. 별표(*)는 테스트 세트를 의미.
Table 2 shows predicted values and experimental values with residual values for the training set and the test set. An asterisk (*) denotes a test set.
본 발명을 통하여 알 수 있는 바와 같이, 2-(3-methoxyphenyl)-2H-benzo[h]chromen-4(3H)-one (폴리페놀 9, MPBC)은 세포 주기 저해제 p21의 전사 인자 p53-매개된 업레귤레이션을 유도하는 것을 알 수 있다. p53 단백질의 사일런싱은 MPBC-유도된 G1 세포 주기 정지를 폐지하였다. 이들 데이터에 기반하여, 본 발명자들은 MPBC가 G1 단계에서 세포 주기 진행의 p53-매개된 저해를 통하여 항종양 활성을 나타낸다는 것을 시사하였다.본 발명에서 얻어진 바이오포어는 G1 세포 주기 정지를 촉발하는 폴리페놀을 고안하는데 도움을 줄 것이다.As can be seen from the present invention, 2- (3-methoxyphenyl) -2 H -benzo [h] chromen-4 (3 H) -one (
도 1은 유동 세포분석법을 사용하여 얻어진 폴리페놀 9 (MPBC)의 DNA 히스토그램. M1-M4 부위는 각각 sub-G1, G1, S, 및 G2/M 단계를 나타냄.
도 2는 프로그램 SYBYL 7.3을 사용하여 만들어진 CoMFA 등고선 지도. 입체 및 정전기 장 디스트립터는 각각 44.7% 및 55.3%이었다. 입체장과 관련하여, 입체 벌크를 선호하는 부위는 19%이었고 벌크를 비선호하는 부위는 81%이었다. 유사하게, 정전기장의 등고선 지도를 만들었고, 양전기 및 음전기 그룹을 선호하는 부위는 각각 14% 및 86%이었다. MPBC는 최고 저해 효과를 가지고 등고선 지도에 들어있다.
도 3은 프로그램 SYBYL 7.3을 사용하여 만들어진 CoMSIA 등고선 지도. 입체 및 정전기 및 수소 결합 수용체 디스크립터는 각각 30.8%, 51.0%, 및 18.2%이었다. 입체장과 관련하여, 입체 벌크를 선호하는 부위는 12%이고 벌크를 비선호하는 부위는 88%이었으며; 정전기장에서, 양전기 그룹을 선호하는 부위는 90%이고 음전기 그룹을 선호하는 부위는 10%이었다. 수소 결합 수용체 장에 대해서, H-결합 수용체 선호 부위는 79%이고 비선호 부위는 21%이었다.
도 4는 HCT116 인간 대장암 세포에서 G1 단계에서 세포주기 진행의 더 큰 저해를 야기하는 구조 조건을 나타냄.
도 5는 MPBC의 처리는 용량 및 시간 의존적으로 세포 생존(좌) 및 세포 증식(우)을 현저하게 감소하는 것을 보여줌(A). 7일간 MPBC 처리는 개별 세포가 생존 콜로니로 증식하는 능력을 용량 의존적으로 상실하게 하는 것을 나타냄(B).웨스턴 블럿 분석은 MPBC 처리 6시간 이내에 p53 단백질의 양을 증가시키고 약 24 시간 부근에서 피크에 도달한 후 48시간 까지 감소하는 것을 나타냄(C). MPBC 처리는 -2400/+1 구축물의 촉진을 야기하였으나 -952/+70 구축물은 그러하지 않은 것을 나타냄(D).
도 6은 (A) p53 및 p21 모두의 발현은 야생형 HCT116 세포 (p53+/+)의 MPBC 노출을 수반하여 시간 의존적으로 증가하였으나, p53-null HCT116 세포(p53-/-)에서는 그러하지 않는다는 것을 나타냄. (B) MPBC-유도된 G1 세포 주기 정지는 p53-null HCT116 세포에서 손상된 것을 나타냄.
도 7은 CoMFA 모델로부터 얻은 예측된 값 대 실험 데이터 값의 그림.
도 8은 CoMSIA 모델로부터 얻은 예측된 값 대 실험 데이터 값의 그림.
도 9는 폴리페놀 6 - 27의 3차 구조를 얻는데 사용된 스카폴드, 그 구조는 X-선 결정학적 구조에 기반하여 구축됨.
도 10은 폴리페놀 9에 대한 트레이닝 세트 및 테스트 세트의 배열
도 11은 계통학적 클러스터 분석
도 12는 폴리페놀 6 및 9의 제조과정.Figure 1 is a DNA histogram of polyphenol 9 (MPBC) obtained using flow cytometry. The M1-M4 region represents sub-G1, G1, S, and G2 / M, respectively.
Figure 2 is a CoMFA contour map created using the program SYBYL 7.3. The steric and electrostatic field destructors were 44.7% and 55.3%, respectively. Regarding the steric field, 19% of the sites favored solid bulk and 81% of sites where bulk was not preferred. Similarly, a contour map of the electrostatic field was created, with 14% and 86% of sites favoring the positive and negative groups, respectively. MPBC has the highest inhibitory effect and is on the contour map.
Figure 3 is a CoMSIA contour map created using the program SYBYL 7.3. The stereoscopic and electrostatic and hydrogen bond acceptor descriptors were 30.8%, 51.0%, and 18.2%, respectively. Regarding the steric field, the region favoring the solid bulk was 12% and the region where the bulk was not preferred was 88%; In the electrostatic field, 90% preferred the positively charged group and 10% preferred the negatively charged group. For the hydrogen bond acceptor site, the preferred site for the H-binding receptor was 79% and the non-preferred site was 21%.
Figure 4 shows the structural conditions leading to greater inhibition of cell cycle progression at the G1 stage in HCT116 human colorectal cancer cells.
Figure 5 shows that treatment of MPBC (A) significantly reduces cell viability (left) and cell proliferation (right) in a dose- and time-dependent manner. (B) 7 days of MPBC treatment results in a dose dependent loss of the ability of individual cells to proliferate into viable colonies (B). Western blot analysis increased the amount of p53 protein within 6 hours of MPBC treatment and peaked at around 24 hours (C) indicating that it has decreased to 48 hours after reaching. MPBC treatment resulted in the acceleration of the -2400 / + 1 construct, while the -952 / + 70 construct did not.
Figure 6 shows that (A) expression of both p53 and p21 increased in a time-dependent manner with MPBC exposure of wild-type HCT116 cells (p53 + / + ), but not in p53-null HCT116 cells (p53 - / - ) . (B) MPBC-induced G1 cell cycle arrest indicates damage to p53-null HCT116 cells.
Figure 7 is a plot of predicted versus experimental data values from the CoMFA model.
Figure 8 is a plot of predicted versus experimental data values from the CoMSIA model.
9 is a scaffold used to obtain the tertiary structure of polyphenol 6-27, the structure of which is constructed based on the X-ray crystallographic structure.
Figure 10 shows a training set for
Figure 11 shows the results of a systematic cluster analysis
12 shows the production process of
이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.
6과 9를 제외한 본 발명의 모든 폴리페놀은 보고된 방법을 사용하여 합성하였다.[Chattopadhyay, B.; Basu, S.; Chakraborty, P.; Choudhuri, C.K.; Mukherjee, A.K. J. Mol. Struct. 2009, 932, 90-96;Nguyen, T.B.; Wang, Q.; Gueritte, F. Synth. Commun. 2012, 42, 2648-2663;Rueping, M.; Lin, M.Y. Chem. -Eur. J. 2010, 16, 4169-4172;Rad, M.N.S.; Khalafi-Nezhad, A.; Behrouz, S.; Amini, Z.; Behrouz, M. Phosphorus Sulfur Silicon Relat. Elem. 2010, 185, 1658-1671;Williams, R.; Roose, P.; De Saegher, J.J. PCT Int. Appl. 2010, 156 pp;Rao, M.A.; Nayak, A.; Rout, M.K. J. Inst. Chem. (India) 1972, 44, 151-154;Fang, X.T.; Hu, L.H.; Zhang, J. Hubei Huagong 2002, 19, 24-25;Williams, A.C.; Camp, N. Sci. Synth. 2003, 14, 347;Lu, Y.; Liu, J.; Song, H.; Cai, M. Studies on flavonoids XX. Chinese Sci. Bull. 1993, 38, 1607-1611;Yoon, H.; Ahn, S.; Hwang, D.; Jo, G.; Kim, D.W.; Kim, S.H.; Koh, D.; Lim, Y. Magn. Reson. Chem. 2012, 50, 759-764;Zhang, J.M.; Lou, C.L.; Hu, Z.P.; Yan, M. ARKIVOC 2009, 362-375;Kaye, P.T.; Nocanda, X.W. J. Chem. Soc., Perkin Trans. 1 2002, 1318-1323;Nair, V.; Sinu, C.R.; Rejithamol, R.; Seetha Lakshmi, K.C.; Suresh, E. Org. Biomol. Chem. 2011, 9, 5511-5514;Murray, I.A.; Flaveny, C.A.; Chiaro, C.R.; Sharma, A.K.; Tanos, R.S.; Schroeder, J.C.; Amin, S.G.; Bisson, W.H.; Kolluri, S.K.; Perdew, G.H. Mol. Pharmacol. 2011, 79, 508-519] 신규한 폴리페놀 6 및 9에 대한 합성 과정, 및 그것을 동정하기 위하여 사용된 NMR 및 질량 분광법(MS) 데이터를 나타낸다. 녹는점(m.p.)을 Fisher-Johns melting 장치 (Fisher Scientific, Lafayette, CO) 상에서 결정하고 보정하지 않았다. 모든 NMR 실험들은 Avance 400 분광계 시스템(9.4 T; Bruker, Karlsruhe, Germany) 상에서 298 K에서 수행하였다. 그 합성된 화합물들을 중수소 치환된 dimethyl sulfoxide (DMSO-d6)에서 용해하였다. 상세한 실험 방법은 보고된 방법들을 참고하였다.[Yoon, H.; Ahn, S.; Hwang, D.; Jo, G.; Kim, D.W.; Kim, S.H.; Koh, D.; Lim, Y. Magn. Reson. Chem. 2012, 50, 759-764] 모든 질량 스펙트럼은 대한민국 대구 소재 한국 기초 과학지원 연구원의 도움으로 고해상 전자 충격 이온화 질량 분광계(HREIMS; JMS700, JEOL, Tokyo, Japan) 상에서 수집하였다. 컬럼 크로마토그래피 정제는 Silica gel 60 (70-230 mesh, Merck, Whitehouse Station, NJ) 상에서 수행하였다.[Yong, Y.; Ahn, S.; Hwang, D.; Yoon, H.; Jo, G.; Kim, Y.H.; Kim, S.H.; Koh, D.; Lim, Y. Magn. Reson. Chem. 2013, 51, 364-370] All polyphenols of the present invention, except 6 and 9, were synthesized using the reported method. [Chattopadhyay, B .; Basu, S .; Chakraborty, P .; Choudhuri, CK; Mukherjee, AK J. Mol. Struct. 2009, 932 , 90-96; Nguyen, TB; Wang, Q .; Gueritte, F. Synth. Commun. 2012, 42 , 2648-2663; Rueping, M .; Lin, MY Chem. -Eur. J. 2010, 16 , 4169-4172; Rad, MNS; Khalafi-Nezhad, A .; Behrouz, S.; Amini, Z .; Behrouz, M. Phosphorus Sulfur Silicon Relat. Elem. 2010, 185 , 1658-1671; Williams, R .; Roose, P .; De Saegher, JJ PCT Int. Appl. 2010 , 156 pp; Rao, MA; Nayak, A .; Rout, MK J. Inst. Chem. (India) 1972, 44 , 151-154; Fang, XT; Hu, LH; Zhang,
2-(2,3-dimethoxyphenyl)-22- (2,3-dimethoxyphenyl) -2 HH -benzo[-benzo [ hh ]chromen-4(3] chromen-4 (3 HH )-one (6)의 합성 ) Synthesis of -one (6)
1-Hydroxy-2-acetonaphthone (558 mg, 3 mmol) 및 2,3-dimethoxybenzaldehyde (498 mg, 3 mmol)을 40 ml의 에탄올에 용해하고 온도를 얼음조에서 3-4℃로 맞추었다. 냉장된 반응 혼합물에 2 ml의 50% KOH 수용액을 첨가하고, 그 반응 혼합물을 상온에서 24시간 동안 교반하였다. 그 반응 혼합물을 냉장된 물(60 ml)에 넣고 6 N HCl 용액으로 산성화하여 고체화하였다. 그 생성된 침전물을 여과하고 찬 에탄올로 세척하여 다음 반응에 사용될 순수한 찰콘 중간체를 만들었다. 10ml의 DMF(dimethyl formamide) 내 chalcone (668 mg, 2 mmol)의 용액에 촉매량의 6 N HCl를 첨가하고 그 혼합물을 20시간 동안 환류하였다. 냉각 후, 그 생성된 용액을 냉각된 물(200 ml)에 넣어 조(crude) 고체를 생성하였다. 그 혼합물을 CH2Cl2 (20 ml x3)로 추출하였다. 그 혼합된 유기층을 무수 MgSO4 로 건조하고 그 용매를 진공에서 증류하였다. 프래쉬 컬럼 크로마토그래피(ethyl acetate:n-hexane = 1:20) 후에, 순수한 플라바논 6를 53% 수율로 얻었다; m.p. 112-114℃. 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, 1H, H-8a, J = 8.3 Hz), 7.95 (d, 1H, H-7a, J = 8.1 Hz), 7.80 (d, 1H, H-5, J = 8.7 Hz), 7.70 (ddd, 1H, H-7b, J = 1.1, 7.0, 8.1 Hz), 7.58 (m, 1H, H-8b), 7.56 (d, 1H, H-6, J = 8.7 Hz), 7.30 (dd, 1H, H-6', J = 1.5, 8.0 Hz), 7.21 (dd, 1H, H-5', J = 8.0, 8.0 Hz), 7.15 (dd, 1H, H-4', J = 1.5, 8.0 Hz), 6.05 (dd, 1H, H-2, J = 2.9, 13.7 Hz), 3.86 (s, 3H, 3'-OCH3), 3.81 (s, 3H, 2'-OCH3), 3.36 (dd, 1H, H-3, J = 13.7, 16.8 Hz), 2.83 (dd, 1H, H-3, J = 2.9, 16.8 Hz); 13C NMR (100 MHz, DMSO-d6) δ 191.2 (C-4), 159.2 (C-9), 152.4 (C-3'), 146.1 (C-2'), 136.9 (C-7), 131.9 (C-1'), 129.8 (C-7b), 128.0 (C-7a), 126.7 (C-8b), 124.4 (C-5'), 124.2 (C-8), 123.0 (C-8a), 121.3 (C-5), 120.9 (C-6), 118.7 (C-6'), 115.0 (C-10), 113.4 (C-4'), 75.4 (C-2), 60.7 (2'-OCH3), 55.8 (3'-OCH3); HREIMS (m/z): Calcd. for C21H18O4 (M+): 334.1205; found 334.1201. 1-Hydroxy-2-acetonaphthone (558 mg, 3 mmol) and 2,3-dimethoxybenzaldehyde (498 mg, 3 mmol) were dissolved in 40 ml of ethanol and the temperature was adjusted to 3-4 ° C in an ice bath. To the chilled reaction mixture was added 2 ml of 50% aqueous KOH solution and the reaction mixture was stirred at ambient temperature for 24 hours. The reaction mixture was placed in chilled water (60 ml) and acidified with 6 N HCl solution to solidify. The resulting precipitate was filtered and washed with cold ethanol to give a pure chalcone intermediate to be used in the next reaction. A catalytic amount of 6 N HCl was added to a solution of chalcone (668 mg, 2 mmol) in 10 ml DMF (dimethyl formamide) and the mixture was refluxed for 20 hours. After cooling, the resulting solution was taken up in cold water (200 ml) to give a crude solid. The mixture was extracted with CH 2 Cl 2 (20 ml x 3). The combined organic layers were dried over anhydrous MgSO 4 and the solvent was distilled off in vacuo. After flash column chromatography (ethyl acetate: n- hexane = 1: 20), pure flavanone 6 was obtained in 53% yield; mp 112-114 [deg.] C. 1 H NMR (400 MHz, DMSO -d 6) δ 8.20 (d, 1H, H-8a, J = 8.3 Hz), 7.95 (d, 1H, H-7a, J = 8.1 Hz), 7.80 (d, 1H , H-5, J = 8.7 Hz), 7.70 (ddd, 1H, H-7b, J = 1.1, 7.0, 8.1 Hz), 7.58 (m, 1H, H-8b), 7.56 (d, 1H, H- 6, J = 8.7 Hz), 7.30 (dd, 1H, H-6 ', J = 1.5, 8.0 Hz), 7.21 (dd, 1H, H-5', J = 8.0, 8.0 Hz), 7.15 (dd, 1H, H-4 ', J = 1.5, 8.0 Hz), 6.05 (dd, 1H, H-2, J = 2.9, 13.7 Hz), 3.86 (s, 3H, 3'-OCH 3), 3.81 (s, 3H, 2'-OCH 3), 3.36 (dd, 1H, H-3, J = 13.7, 16.8 Hz), 2.83 (dd, 1H, H-3, J = 2.9, 16.8 Hz); 13 C NMR (100 MHz, DMSO-d 6 )? 191.2 (C-4), 159.2 (C-9), 152.4 C-8a), 124.9 (C-1 '), 129.8 (C-7b), 128.0 , 121.3 (C-5), 120.9 (C-6), 118.7 (C-6 '), 115.0 OCH 3), 55.8 (3'- OCH 3); HREIMS ( m / z ): Calcd . for C 21 H 18 O 4 ( M +): 334.1205; found 334.1201.
2-(3-methoxyphenyl)-22- (3-methoxyphenyl) -2 HH -benzo[-benzo [ hh ]chromen-4(3] chromen-4 (3 HH )-one (9)의 합성) Synthesis of -one (9)
폴리페놀 9의 합성에도 동일한 과정을 사용하였으나, 시작은 1-hydroxy-2-acetonaphthone 및 3-methoxybenzaldehyde로부터 출발하였다. 그 화합물을 47% 수율로 얻었다; m.p. 126-128℃. 1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, 1H, H-8a, J = 7.9 Hz), 7.95 (d, 1H, H-7a, J = 8.1 Hz), 7.78 (d, 1H, H-5, J = 8.7 Hz), 7.71 (ddd, 1H, H-7b, J = 0.8, 6.9, 8.1 Hz), 7.61 (ddd, 1H, H-8b, J = 0.8, 6.9, 7.9 Hz), 7.55 (d, 1H, H-6, J = 8.7 Hz), 7.39 (dd, 1H, H-5', J = 8.1, 8.1 Hz), 7.21 (m, 1H, H-2), 7.21 (m, 1H, H-6'), 6.98 (dd, 1H, H-4', J = 2.4, 8.1 Hz), 5.88 (dd, 1H, H-2, J = 3.1, 13.0), 3.80 (s, 3H, 3'-OCH3), 3.34 (dd, 1H, H-3, J = 13.0, 16.7 Hz), 2.98 (dd, 1H, H-3, J = 3.1, 16.7 Hz); 13C NMR (100 MHz, DMSO-d6) δ 191.0 (C-4), 159.4 (C-3'), 158.9 (C-9), 140.4 (C-1'), 136.9 (C-7), 129.8 (C-7b), 129.8 (C-5'), 128.0 (C-7a), 126.7 (C-8b), 124.2 (C-8), 123.0 (C-8a), 121.2 (C-5), 120.9 (C-6), 118.5 (C-6'), 115.2 (C-10), 113.8 (C-4'), 112.2 (C-2'), 79.5 (C-2), 55.1 (3'-OCH3), 42.8 (C-3); HREIMS (m/z): Calcd. for C20H16O3 (M+): 304.1099; found 304.1097.The same procedure was used for the synthesis of
세포 주기 프로파일을 유동 세포분석법을 사용하여 분석하였다. HCT116 세포를 비히클(dimethyl sulfoxide, DMSO) 또는 20 mM 폴리페놀 화합물로 24시간 또는 48시간 동안 처리하고, 70% 에탄올로 고정하고, phosphate-buffered saline로 2회 세척한 후, 50 mg/ml propidium iodide로 염색하였다.[29] 세포 DNA를 NucleoCounter NC-3000 (ChemoMetec, Allerod, Denmark)을 사용하여 측정하였다. 세포 주기의 네 특징적인 단계는 세포 DNA 양에 기반하였다: G1 (갭 1; 2배체), S (DNA 합성; 2배체 및 4배체 사이), G2 (갭 2; 4배체), 및 M (mitosis; 4배체).Cell cycle profiles were analyzed using flow cytometry. HCT116 cells were treated with dimethyl sulfoxide (DMSO) or 20 mM polyphenol for 24 hours or 48 hours, fixed with 70% ethanol, washed twice with phosphate-buffered saline, and resuspended in 50 mg / ml propidium iodide . [29] Cell DNA was measured using a NucleoCounter NC-3000 (ChemoMetec, Allerod, Denmark). Four distinct stages of the cell cycle were based on the amount of cellular DNA: G1 (
폴리페놀 1-27의 구조 및 G1 단계에서 세포 주기 정지에 대한 그들의 효과 사이의 관계를 정량화하기 위하여, 3차원(3D) QSAR 연구를 비교 분자 장 분석(CoMFA) 및 비교분자 유사성 지수 분석(CoMSIA)를 사용하여 수행하였다. 모든 계산을 분자 모델링 패키지 SYBYL 7.3 (Tripose, St. Louis, MO)를 가지는 Intel Core 2 Quad Q6600 (2.4 GHz) Linux PC 상에서 수행하였다. 합성된 폴리페놀 10-19의 3차 구조를 2',3,5-trimethoxychalcone의 X-선 결정 구조에 기반하여 구축하였다(도 9A).[Lim, Y.; Koh, D. Acta Crystallogr. E 2013, E69, o514] 그 구축된 폴리페놀 10-19을 SYBYL 7.3에 의하여 제공된 분자 역학 알고리즘을 사용하여 에너지 최소화를 하였다. 입체 형태 검색을 SYBYL의 그리드 검색 방법을 사용하여 수행하고, 그 선택된 결합을 0°에서 360°까지 15°씩 증가하면서 회전시키고 그 형태를 Tripos force 장 및 Gasteiger-Hukel 차지를 사용하여 최소화하였다. 최소화를 전체 에너지의 수렴(0.05 kcal/mol·Å) 상에서 정지시켰다. 유사하게, 폴리페놀 6-9 및 20-27의 3차 구조를 2'-hydroxy-3',4'-naphtho-3,5-dimethoxychalcone 및 8-methoxy-2H-chromene-3-carbaldehyde의 X 선 결정 구조에 기반하여 구축하였다(도 9B 및 C).폴리페놀 1-5의 3차원 구조를 분자 모델링을 사용하여 결정하였다. QSAR 계산을 위하여, 데이터 세트의 화합물들이 균질하지 아니하므로, 벤질 부분에 포함된 중원자(heavy atom)들을 일관된 중첩이 관찰될 때 배열을 위하여 사용하였다. 배열 과정은 프로그램 SYBYL에서 DATABASE Alignment 모듈을 사용하여 수행하였다. 배열된 구조들을 도 10에 나타내었다.(3D) QSAR studies were performed using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) to quantify the relationship between the structure of polyphenol 1-27 and their effect on cell cycle arrest at G1 stage. . All calculations were performed on an
표 1에서 27 종 폴리페놀들을 22 종 화합물의 트레이닝 세트 및 다섯 화합물(5, 10, 16, 22, 및 23)들의 테스트 세트로 임의적으로 나누었다. 트레이닝 세트를 QSAR 모델을 만들기 위하여 사용하고, 테스트 세트를 그 모델을 인증하기 위하여 사용하였다. 테스트 세트를 계통학적 클러스터링을 사용하여 분석하였다.[Pirhadi, S.; Ghasemi, J.B. Eur. J. Med. Chem. 2010, 45, 4897-4903] 테스트 세트를 위하여 선택된 화합물들은 개별적 구조적 그룹에 속하였다 (도 11). 따라서, 테스트 세트를 실험에서 QSAR 모델을 신뢰할 수 있는지를 인증하기 위하여 사용될 수 있다. QSAR 계산은 CoMFA 및 CoMSIA를 사용하여 수행되고 그 실험적 과정은 보고된 방법을 따랐다.[Shin, S.Y.; Woo, Y.; Hyun, J.; Yong, Y.; Koh, D.; Lee, Y.H.; Lim, Y. Bioorg. Med. Chem. Lett. 2011, 21, 6036-6041] In Table 1, the 27 polyphenols were randomly divided into a training set of 22 compounds and a test set of five compounds (5, 10, 16, 22, and 23). The training set was used to create the QSAR model, and the test set was used to certify the model. Test sets were analyzed using phylogenetic clustering [Pirhadi, S .; Ghasemi, JB Eur. J. Med. Chem. 2010, 45 , 4897-4903] The compounds selected for the test set belonged to a separate structural group (Fig. 11). Therefore, the test set can be used to verify that the QSAR model is reliable in the experiment. QSAR calculations were performed using CoMFA and CoMSIA, and the experimental procedure followed the reported method [Shin, SY; Woo, Y .; Hyun, J .; Yong, Y .; Koh, D .; Lee, YH; Lim, Y. Bioorg. Med. Chem. Lett. 2011, 21 , 6036-6041]
세포 성장 저해 에세이를 위하여, HCT116 인간 대장암 세포를 American Type Culture Collection (Manassas, VA)으로부터 구입하고 10% 우태아 혈청(FBS; HyClone, Logan, UT)이 보충된 Dulbecco's modified Eagle's 배지(DMEM; Life Technologies, Seoul, Korea)에서 유지하였다. 그 HCT116 세포(2 x 103 세포/샘플)를 96-웰 플레이트에 시딩하고 MPBC로 증가되는 농도(0, 5, 10, 및 20 μM)로 24시간 또는 48시간 동안 처리하였다. 세포 생존에 대한 MPBC 저해효과를 제조업자의 지시에 따라서 기질로 수용성 tetrazolium 염 WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, 나트륨 염)을 가지고 Cell Counting Kit-8(Dojindo Molecular Technologies, Gaithersburg, MD)을 사용하여 결정하였다.[34] 세포 증식에 대한 MPBC의 저해 효과를 제조업자의 지시에 따라 Cell Proliferation Assay Kit (Cell Signaling Technology)를 사용하여 측정하였다. 집락형성 생존 에세이를 위하여, HCT116 세포를 계수하고 10% FBS이 보충된 DMEM에서 24 웰 조직 배양 플레이트 (BD Falcon Becton Dickson Immunocytometry System, Franklin Lakes, NJ; 5 x103 세포/웰) 상에 플레이팅하였다. 부착 후, 세포를 7일간 MPBC로 처리하였다. 그 다음, 세포들을 6% glutaraldehyde로 고정하고 0.1% crystal violet으로 염색하였다. 웨스턴 블럿 분석을 위하여, 세포를 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 ㎍/ml leupeptin, 및 1 mM phenylmethylsulfonyl fluoride (PMSF)를 포함하는 버퍼에서 파쇄하였다. 웨스턴 블럿팅을 Shin, S.Y.; Choi, C.; Lee, H.G.; Lim, Y.; Lee, Y.H. Carcinogenesis 2012, 33, 2467-2476에 기재된 것과 같이 수행하였다. 신호들을 증강 화학발광 검출 시스템(Amersham Pharmacia Biotech, Piscataway, NJ)을 사용하여 현상하였다.HCT116 human colon cancer cells were purchased from the American Type Culture Collection (Manassas, Va.) And cultured in Dulbecco's modified Eagle's medium (DMEM; Life) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) Technologies, Seoul, Korea). The HCT116 cells (2 x 10 3 cells / sample) were seeded in 96-well plates and treated with MPBC at increasing concentrations (0, 5, 10, and 20 μM) for 24 or 48 hours. The MPBC inhibitory effect on cell viability was determined by the manufacturer's instructions, using the water soluble tetrazolium salt WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- ) -2 H -tetrazolium, sodium salt) with Cell Counting Kit-8 (Dojindo Molecular Technologies, Gaithersburg, Md.). [34] The inhibitory effect of MPBC on cell proliferation was measured using a Cell Proliferation Assay Kit (Cell Signaling Technology) according to the manufacturer's instructions. For colony formation survival assay, HCT116 cells were counted and plated on 24-well tissue culture plates (BD Falcon Becton Dickson Immunocytometry System, Franklin Lakes, NJ; 5 x 10 3 cells / well) in DMEM supplemented with 10% FBS . After attachment, cells were treated with MPBC for 7 days. The cells were then fixed with 6% glutaraldehyde and stained with 0.1% crystal violet. For Western blot analysis, cells were resuspended in 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 ug / ml leupeptin, And 1 mM phenylmethylsulfonyl fluoride (PMSF). Western blotting Shin, SY; Choi, C .; Lee, HG; Lim, Y .; Lee, YH Carcinogenesis 2012, 33 , 2467-2476. Signals were developed using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).
프로모터 리포터 에세이를 위하여, HCT116 세포를 cells were seeded onto 12-웰 플레이트 상에 시딩하고 LipofectAMINE 2000 (Invitrogen Life Technologies, San Diego, CA)을 사용하여 0.2 ㎍의 두 p21 프로모터 구축물, p21-Luc(-2400/+1) 또는 p21-Luc(-952/+70) 중 하나로 트랜스팩션하였다. 트랜스팩션 효율을 모니터하기 위하여, Renilla luciferase를 코딩하는 pRL-null 플라즈미드(50 ng)는 모든 트랜스팩션에서 포함되었다. 트랜스팩션 24 시간 후, 그 세포들을 20 uM MPBC로 처리하였다. 8 시간 후, 반딧불이 및 Renilla 루시퍼레이즈 활성을 제조업자의 지시에 따라 Dual-Glo Luciferase Assay System을 사용하여 단일 샘플로부터 연속적으로 측정하였다. 발광은 광도계(Centro LB960; Berthold Tech, Bad Wildbad, Germany)로 측정하였다.
For the promoter reporter assay, HCT116 cells were seeded onto 12-well plates and seeded with 0.2 μg of two p21 promoter constructs, p21-Luc (-2400 (Invitrogen Life Technologies, San Diego, Calif.) Using LipofectAMINE 2000 / + 1) or p21-Luc (-952 / + 70). To monitor transfection efficiency, pRL-null plasmid (50 ng) encoding Renilla luciferase was included in all transfections. After 24 hours of transfection, the cells were treated with 20 uM MPBC. After 8 hours, the firefly and Renilla luciferase activities were continuously measured from a single sample using the Dual-Glo Luciferase Assay System according to the manufacturer's instructions. The luminescence was measured with a photometer (Centro LB960; Berthold Tech, Bad Wildbad, Germany).
Claims (5)
[화학식 1]
상기 화학식 1에서 R1, R2, 및 R3는 H 또는 OCH3임.A compound of the formula
[Chemical Formula 1]
Wherein R 1 , R 2 , and R 3 are H or OCH 3 .
[화학식 1]
상기 화학식 1에서 R1, R2, 및 R3는 H 또는 OCH3임
Acetonaphthone, 2,3-dimenoxybenzaldehyde or 1-hydroxy-2-benzonaphthone and 3-methoxybenzaldehyde were dissolved in alcohol, and a base was added to the mixture. ≪ / RTI > wherein R < 1 >
[Chemical Formula 1]
Wherein R 1 , R 2 , and R 3 are H or OCH 3
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