KR102427539B1 - Two-photon fluorescent probe for detecting COX-2 and its use - Google Patents
Two-photon fluorescent probe for detecting COX-2 and its use Download PDFInfo
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- KR102427539B1 KR102427539B1 KR1020210015892A KR20210015892A KR102427539B1 KR 102427539 B1 KR102427539 B1 KR 102427539B1 KR 1020210015892 A KR1020210015892 A KR 1020210015892A KR 20210015892 A KR20210015892 A KR 20210015892A KR 102427539 B1 KR102427539 B1 KR 102427539B1
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Abstract
Description
본 발명은 하기 화학식 1로 표시되는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 검출용 신규 화합물과 이의 용도에 관한 것이다. 상세하게, 본 발명에 따른 형광 프로브 화합물은 사이클로옥시게나아제-2에 대해서 우수한 선택성으로 반응하며, 우수한 광물리적 특성과 낮은 세포독성을 가지고 있어 암 세포를 효과적으로 이미징하여 암 진단에 널리 응용될 수 있다.The present invention relates to a novel compound for detecting cyclooxygenase-2 represented by the following formula (1) and use thereof. Specifically, the fluorescent probe compound according to the present invention reacts with excellent selectivity to cyclooxygenase-2, and has excellent photophysical properties and low cytotoxicity, so it can be widely applied to cancer diagnosis by effectively imaging cancer cells. .
[화학식 1][Formula 1]
암은 전세계적으로 생명을 위협하는 주요 질병으로서, 세계보건기구 (WHO) 에 의하면 매년 7백만명의 암에 의해 사망하며 건강 관리 시스템에 대한 엄청난 사회적, 경제적 압력에 큰 비중을 차지하는 것으로 보고되고 있다. 하지만 화학 요법의 신중한 모니터링과 병행하여 수술 및 화학 요법을 받는 경우 암 진행 초기 단계에 진단받은 암 환자의 30%는 사망을 피할 수 있는 것으로 추정되고 있다. 예를 들어 국소 전이 또는 전이 전대장암 환자의 5년 생존률은 90%를 넘지만, 원격 전이의 경우 10% 미만으로 급격히 감소한다.Cancer is a major life-threatening disease worldwide, and according to the World Health Organization (WHO), it is reported that 7 million people die each year from cancer, contributing to the enormous social and economic pressures on the health care system. However, it is estimated that 30% of cancer patients diagnosed at an early stage of cancer progression can avoid death if surgery and chemotherapy are used in combination with careful monitoring of chemotherapy. For example, the 5-year survival rate for patients with local or metastatic pre-colon cancer exceeds 90%, but for distant metastases it drops sharply to less than 10%.
특히 대장암 (Colorectal cancer, CRC)은 세계에서 가장 흔한 유형의 암 중 하나이다. 대장암의 조기 진단은 생존율을 90 %까지 향상시킬 수 있지만 암 진단 및 치료의 발전에도 불구하고 대장암의 조기 발견의 어려움은 여전히 환자에게 심각한 문제를 초래한다. 일반적으로 대장암은 대장 내시경 및 헤마톡실린 및 에오신 (H & E) 염색과 같은 조직학적 방법으로 진단되지만, 이러한 방법은 다단계 절차를 수반하며 관찰자 간 변동성으로 인한 오진 위험도 주요 관심사이다. In particular, colorectal cancer (CRC) is one of the most common types of cancer in the world. Early diagnosis of colorectal cancer can improve the survival rate up to 90%, but despite advances in cancer diagnosis and treatment, the difficulty of early detection of colorectal cancer still causes serious problems for patients. In general, colorectal cancer is diagnosed by colonoscopy and histological methods such as hematoxylin and eosin (H&E) staining, but these methods involve a multi-step procedure and the risk of misdiagnosis due to inter-observer variability is also a major concern.
따라서 대장암의 빠르고 정확한 분석을 위한 대체 방법의 개발에 대한 강한 요구되고 있다. 이러한 문제를 해결하기 위해, 질병 바이오마커를 선택적으로 식별할 수 있는 질병 특이적 프로브를 발견하기 위한 다양한 이미징 기술이 개발되었다. 이 중 형광 이미징 방법은 높은 선택성과 감도, 비교적 낮은 계측 비용 및 조작 편의성으로 인해 많은 주목을 받았다. 하지만, 비특이적인 생체 분포 및 비 표적 기관으로부터의 형광 프로브의 비효율적인 제거는 투여 후 높은 신호 대 잡음비를 얻는데에 장애가 된다. 이와 관련하여, 대상 부위에 선택적으로 유지될 수 있는 유도적 형광 조영제의 개발은 대상이 아닌 부위로부터 프로브를 제거하기에 충분한 시간을 제공함으로써 신호 대 잡음 비율을 향상시킬 수 있는바 이 문제를 해결하는 가장 좋은 방법이 될 수 있다. 프로스타글란딘-엔도퍼옥사이드 신타아제 (PTGS) 효소로도 알려진 사이클로옥시게나아제 (Cyclooxygenase, COX)는 두 개의 동종효소인 COX-1과 COX-2의 계열로 분류되며, 그 중 COX-2는 염증 반응에서의 프로스타글란딘 생합성과 중추신경계에서 중요한 역할을 한다 임상 데이터에 의하면 COX-2는 초기 전암 단계에서 전이단계까지 암의 모든 단계에서 과발현 되는 것으로 알려져 있다. 특히 대장암, 위암, 췌장암에서 높은 수준의 COX-2 과발현이 일어나는 것으로 알려져 있다. 따라서 COX-2는 고효율 차세대 종양 표적 유닛의 유력한 후보로 고려되고 있다. COX-2의 검출 및 이미징은 조기 징후 (및 증상) 인지는 물론, 암 치료를 위한 해결책을 안내해줄 수 있다. 이처럼 COX-2는 암의 모든 단계, 즉 전암 상태에서 전이 및 확산에 이르기까지 과발현 되기 때문에 암의 조기 진단을 위한 매력적인 바이오마커이다.Therefore, there is a strong demand for the development of alternative methods for rapid and accurate analysis of colorectal cancer. To solve this problem, various imaging techniques have been developed to discover disease-specific probes that can selectively identify disease biomarkers. Among them, the fluorescence imaging method has received much attention due to its high selectivity and sensitivity, relatively low measurement cost, and ease of operation. However, non-specific biodistribution and inefficient removal of fluorescent probes from non-target organs are obstacles to obtaining a high signal-to-noise ratio after administration. In this regard, the development of an inducible fluorescent contrast agent that can be selectively retained in the target site can improve the signal-to-noise ratio by providing sufficient time to remove the probe from the non-target site. This could be the best way. Cyclooxygenase (COX), also known as prostaglandin-endoperoxide synthase (PTGS) enzyme, is classified into a family of two isoenzymes, COX-1 and COX-2, of which COX-2 is an inflammatory reaction It plays an important role in prostaglandin biosynthesis in the central nervous system. According to clinical data, COX-2 is known to be overexpressed at all stages of cancer, from the early precancerous stage to the metastatic stage. In particular, it is known that a high level of COX-2 overexpression occurs in colon cancer, stomach cancer, and pancreatic cancer. Therefore, COX-2 is being considered as a strong candidate for a high-efficiency next-generation tumor targeting unit. Detection and imaging of COX-2 can guide early sign (and symptoms) recognition as well as solutions for cancer treatment. As described above, COX-2 is an attractive biomarker for early diagnosis of cancer because it is overexpressed in all stages of cancer, from the precancerous state to metastasis and spread.
한편, 이광자 현미경 (Two-photon microscopy, TPM) 이미징 기술은 두 개의 저에너지 광자를 여기 소스로 사용하며 생물학적 샘플의 손상과자가 형광을 최소화하는 동시에 심부 조직 영역 내에서 오랜 시간 동안 고품질 이미지를 얻는 이점이 있다.On the other hand, two-photon microscopy (TPM) imaging technology uses two low-energy photons as an excitation source and has the advantage of obtaining high-quality images for a long time within the deep tissue region while minimizing damage and autofluorescence of biological samples. have.
이러한 배경 하에서, 본 발명자들은 암 세포에 고선택성을 갖는 암 세포 검출용 형광 프로브 화합물을 개발하고자 하였다. Under this background, the present inventors sought to develop a fluorescent probe compound for detecting cancer cells having high selectivity for cancer cells.
본 발명자들은 세포 내 효소 수준을 반영하고 대장 암 환자에서 암 조직과 정상 조직을 구별할 수 있는 사이클로옥시게나아제-2 (Cyclooxygenase-2, COX-2)에 대한 이광자 프로브를 개발하고자 하였다. 본 발명자가 발굴한 SCX는 페난트렌 공여체 (phenanthrene donor)와 벤조티아졸 수용체 (benzothiazole acceptor) 및 COX-2 표적화 단위로 인도메타신(indomethacin)을 보유한 새로운 이광자 형광단 (PBT)을 기반으로 한다. 체외 및 세포 이미징 연구를 통해 SCX가 암 세포와 정상 세포에서 COX-2 발현 수준을 구별할 수 있음을 확인할 수 있었다. 또한 COX-2에 대해 선택성을 갖는 이광자 탐침을 사용하여 인간 생검 샘플에서 처음으로 정상 조직과 암 조직을 명확하게 구분할 수 있었다. 따라서, 본 발명에 따른 형광 프로브 화합물은 사이클로옥시게나아제-2에 대해서 우수한 선택성으로 반응하며, 우수한 광물리적 특성과 낮은 세포독성을 가지고 있어 암 세포를 효과적으로 이미징할 수 있어 암 진단에 유용할 것으로 판단하였다.The present inventors attempted to develop a two-photon probe for cyclooxygenase-2 (COX-2) that reflects the level of intracellular enzymes and can distinguish cancerous and normal tissues in colorectal cancer patients. The SCX discovered by the present inventors is based on a novel two-photon fluorophore (PBT) with a phenanthrene donor and benzothiazole acceptor and indomethacin as the COX-2 targeting unit. In vitro and cellular imaging studies confirmed that SCX was able to discriminate the level of COX-2 expression in cancer cells and normal cells. In addition, using a two-photon probe that is selective for COX-2, it was possible for the first time to clearly differentiate between normal and cancerous tissues in human biopsy samples. Therefore, the fluorescent probe compound according to the present invention responds with excellent selectivity to cyclooxygenase-2, and has excellent photophysical properties and low cytotoxicity, so that it can effectively image cancer cells. did.
따라서, 본 발명은 화학식 로 표시되는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 검출용 이광자 형광 프로브와 이의 용도를 제공하는데 그 목적이 있다. Accordingly, an object of the present invention is to provide a two-photon fluorescent probe for detecting cyclooxygenase-2 represented by the formula and uses thereof.
상기와 같은 목적을 해결하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 제공한다.In order to solve the above object, the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
또한 본 발명은 상기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 검출용 이광자 형광 프로브를 제공한다. In addition, the present invention provides a two-photon fluorescent probe for detecting cyclooxygenase-2, comprising the compound represented by Formula I or a pharmaceutically acceptable salt thereof.
또한, 본 발명은 상기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 사이클로옥시게나아제-2 검출용 키트를 제공한다.In addition, the present invention provides a kit for detecting cyclooxygenase-2 comprising the compound represented by Formula I or a pharmaceutically acceptable salt thereof.
또한, 본 발명은 상기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 이광자 형광 프로브를 세포 내 주입하여 사이클로옥시게나아제-2와 반응시키는 단계; 및 상기 반응시킨 반응물의 형광신호를 이광자 현미경(TPM)으로 이미지를 관측하는 단계를 포함하는 사이클로옥시게나아제-2 활성의 정량적 검출 방법을 제공한다. In addition, the present invention comprises the steps of injecting a two-photon fluorescent probe containing the compound represented by Formula I or a pharmaceutically acceptable salt thereof into the cell to react with cyclooxygenase-2; And it provides a quantitative detection method of cyclooxygenase-2 activity comprising the step of observing an image of the fluorescence signal of the reacted reactant with a two-photon microscope (TPM).
또한, 본 발명은 (a) 피검체로부터 분리된 시료를 제공하는 단계; (b) 상기 제공된 시료에 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 이광자 형광 프로브를 첨가하는 단계; 및 (c) 상기 이광자 형광 프로브가 첨가된 시료의 형광을 측정하는 단계를 포함하는 생체 내 사이클로옥시게나아제-2 (Cyclooxygenase-2)를 검출하여 암 질환을 예측 또는 진단하기 위하여 정보를 제공하는 방법을 제공한다.In addition, the present invention comprises the steps of (a) providing a sample isolated from a subject; (b) adding a two-photon fluorescent probe comprising the compound represented by
본 발명에 따른 형광 프로브 화합물은 사이클로옥시게나아제-2에 대해서 우수한 선택성으로 반응하며, 우수한 광물리적 특성과 낮은 세포독성을 가지고 있어 암 세포를 효과적으로 이미징할 수 있도록 한다. 또한, 이는 생물학적으로 관계된 과산화수소 (H2O2), t-부틸 하이드로퍼옥사이드 (TBHP), 차아염소산염 (OCl-), 과산화물 (O2 -), 산화질소 (NO), t-부톡시 라디칼 (·OtBu), 히드록실 라디칼 (·OH) 및 퍼옥시니트라이트 (ONOO-)와 같은 간섭물과 함께 반응하여도 암세포 및 조직 내부의 사이클로옥시게나아제-2 효소 활성을 선택적으로 검출할 수 있다.The fluorescent probe compound according to the present invention reacts with excellent selectivity to cyclooxygenase-2, and has excellent photophysical properties and low cytotoxicity, thereby enabling effective imaging of cancer cells. In addition, it is biologically related to hydrogen peroxide (H 2 O 2 ), t-butyl hydroperoxide (TBHP), hypochlorite (OCl - ), peroxide (O 2 - ), nitric oxide (NO), t-butoxy radical ( ·OtBu), hydroxyl radical (·OH) and peroxynitrite (ONOO - ) can selectively detect cyclooxygenase-2 enzyme activity inside cancer cells and tissues even by reacting with interfering substances such as peroxynitrite (ONOO - ).
또한 본 발명에 따른 형광 프로브는 세포에 선택적 표지가 가능하고, 광안정성이 뛰어나다.In addition, the fluorescent probe according to the present invention can selectively label cells and has excellent photostability.
도 1은 SCX의 구조식과 이광자 형광 스펙트럼을 통한 암세포 이미징 결과이다.
도 2는 SCX의 13C-NMR 스펙트럼 (100 MHz) 측정 결과를 나타낸 것이다.
도 3은 SCX HRMS 스펙트럼 측정 결과를 나타낸 것이다.
도 4는 (a) 일광자 형광 스펙트럼 측정 결과 및 (b) PBS 버퍼 (10.0mM, pH 7.4) 내에서 SCX의 농도에 대한 형광 강도 플롯을 나타낸 것이다. 여기 파장은 375nm 이였다.
도 5는 (a) 일광자 형광 스펙트럼 및 (b) 다른 용매 조건에서 SCX (1μM)의 이광자 형광 스펙트럼 측정 결과를 나타낸 것이다. 일광자 형광 스펙트럼의 여기 소스는 1,4-디옥산의 경우 388 nm, DMF의 경우 398 nm, EtOH의 경우 392 nm, EtOH : PBS = 1 : 1 (v / v)의 경우 376 nm, PBS 버퍼의 경우 37 5nm 이였다.
도 6은 (a, c) 일광자 형광 스펙트럼, 및 (b, d) 버퍼 (100 mM Tris-HCl, pH 8.0, 0.05% Triton-x) 내 (a, b) COX-2 (0-5 μg mL-1) 및 (c, d) COX-1 (0-5 μg mL-1)의 농도를 증가시켜 추가 시, 525 nm에서 SCX (1 μM)의 형광 강도 플롯을 나타낸 것이다. 이때 여기 파장은 375nm 이였다.
도 7은 다양한 반응성 산소 (reactive oxygen) 및 질소종 (nitrogen species) (200 μM)에 대한 SCX (1μM)의 형광 강도를 측정한 것이다. 반응성 종을 첨가한 후 0 내지 2 시간 내 측정한 것이다. 데이터는 PBS 버퍼 (10 mM, pH 7.4)에서 37℃에서 수집되었다. 여기 파장은 375nm 이였다.
도 8은 37℃에서 범용 버퍼 용액 내 SCX (1μM)의 pH에 따른 형광 강도를 측정한 결과이다. 여기 파장은 375nm 이였다.
도 9는 SCX 존재 하에서 MTT 분석을 사용하여 HT-29 세포, Huh7 세포, HeLa 세포, CCD-18Co 세포, Chang 세포 및 Raw 264.7 세포의 생존력을 측정한 결과이다. 세포는 0 내지 50 μM의 SCX와 함께 8 시간 동안 배양되었다.
도 10은 (a) 30 분 동안 SCX (2 μM)로 염색한 HeLa 세포의 이광자 형광 이미지이고, (b) 시간의 함수로서 TPM 이미지에서 A-C의 상대적인 이광자 형광 강도를 나타낸 것이고, (c) SCX 표지 (2 μM) HeLa 세포의 형광 스펙트럼을 측정한 결과이다. 400 - 600 nm에서 810 nm 여기 소스까지 이광자 형광 강도를 수집하였다.
도 11은 30 분 동안 SCX (2 μM)로 염색한 (a-c) HT-29 세포 및 (d-f) HeLa 세포의 이광자 형광 이미지 결과이다. (a, e) 대조 이미지이고, (b, f) 10 μM 및 (c, g) 20 μM의 셀레콕시브(celecoxib)로 세포에 30 분 동안 전처리한 후 얻은 이미지이다. (i) HT-29 세포 및 (j) HeLa 세포 이미지의 평균 형광 강도이다. (d, h) COX-2의 웨스턴 블롯 분석 결과이다. COX-2의 밴드 강도는 각각의 α-튜불린 밴드로 정규화 되었다. 여기 파장은 810 nm 이고, 이미지는 400 내지 600 nm에서 얻었다. 스케일 바 = 20 μm.
도 12는 (a, b) Raw 264.7 세포 및 (d, e) Chang 세포에 대해 30 분 동안 SCX (2 μM)로 염색한 후 이광자 형광 이미지를 측정한 결과이다. (a, e) 대조 이미지이고, (b, f) 세포를 LPS (1 μg mL-1)로 12 시간 동안 전처리한 후 얻은 이미지이다. (c, g) LPS 처리 전후의 TPM 이미지의 평균 형광 강도 측정 결과이다. (d, h) COX-2의 웨스턴 블롯 분석 결과이다. COX-2의 밴드 강도는 각각의 α-튜불린 밴드로 정규화 되었다. 여기 파장은 810 nm 이고, 이미지는 400 내지 600 nm에서 얻었다. 스케일 바 = 20 μm.
도 13은 30분 동안 SCX (2μM)로 염색된 (a-c) 암 세포 (HT-29, Huh7, HeLa) 및 (d-f) 정상 세포 (CCD-18Co, Chang, Raw 264.7)의 이광자 형광 이미지를 측정한 결과이다. 이때 (a) HT-29 세포, (b) Huh7 세포, (c) HeLa 세포, (d) CCD-18Co 세포, (e) Chang 세포 및 (f) Raw 264.7 세포이다. (g) 해당 TPM 이미지의 평균 형광 강도를 나타낸 것이다. 여기 파장은 810 nm이고, 이미지는 400 내지 600 nm에서 얻었다. 스케일 바 = 20 μm.
도 14는 인간 결장에서, (a) 암 조직 및 (c) 정상 조직에 대해 90 분 동안 SCX (20 μM)로 염색한 후 이광자 형광 이미지 측정한 결과이다. (b) 약 70 내지 120 μm 깊이에서 z 방향을 따라 측정한 인간 결장암 조직의 단면 이미지이다. (d) 상응하는 인간 결장암 및 정상 이미지의 평균 형광 강도 측정 결과이다. 여기 파장은 810 nm 이고, 이미지는 400 내지 600 nm에서 얻었다. 스케일 바 = 50 μm.1 is a structural formula of SCX and a cancer cell imaging result through a two-photon fluorescence spectrum.
Figure 2 shows the 13 C-NMR spectrum (100 MHz) measurement result of SCX.
Figure 3 shows the SCX HRMS spectrum measurement results.
4 shows (a) a result of one-photon fluorescence spectrum measurement and (b) a fluorescence intensity plot against the concentration of SCX in PBS buffer (10.0 mM, pH 7.4). The excitation wavelength was 375 nm.
5 shows (a) one-photon fluorescence spectrum and (b) two-photon fluorescence spectrum measurement results of SCX (1 μM) in different solvent conditions. The excitation source of the one-photon fluorescence spectrum is 388 nm for 1,4-dioxane, 398 nm for DMF, 392 nm for EtOH, 376 nm for EtOH:PBS = 1:1 (v/v), PBS buffer In the case of , it was 37 5 nm.
6 shows (a, c) day-photon fluorescence spectra, and (b, d) (a, b) COX-2 (0-5 μg) in buffer (100 mM Tris-HCl, pH 8.0, 0.05% Triton-x). mL −1 ) and (c, d) fluorescence intensity plots of SCX (1 μM) at 525 nm upon addition of increasing concentrations of COX-1 (0-5 μg mL −1 ) are shown. At this time, the excitation wavelength was 375 nm.
7 is a measurement of the fluorescence intensity of SCX (1 μM) with respect to various reactive oxygen and nitrogen species (200 μM). Measured within 0 to 2 hours after addition of reactive species. Data were collected at 37° C. in PBS buffer (10 mM, pH 7.4). The excitation wavelength was 375 nm.
8 is a result of measuring the fluorescence intensity according to the pH of SCX (1 μM) in a general-purpose buffer solution at 37 °C. The excitation wavelength was 375 nm.
9 is a result of measuring the viability of HT-29 cells, Huh7 cells, HeLa cells, CCD-18Co cells, Chang cells and Raw 264.7 cells using the MTT assay in the presence of SCX. Cells were incubated with 0-50 μM SCX for 8 h.
10 is (a) two-photon fluorescence images of HeLa cells stained with SCX (2 μM) for 30 min, (b) relative two-photon fluorescence intensity of AC in TPM images as a function of time, (c) SCX label (2 μM) The result of measuring the fluorescence spectrum of HeLa cells. Two-photon fluorescence intensities were collected from 400-600 nm to an 810 nm excitation source.
11 is a two-photon fluorescence image of (ac) HT-29 cells and (df) HeLa cells stained with SCX (2 μM) for 30 minutes. (a, e) is a control image, and (b, f) is an image obtained after pretreatment of cells with 10 μM and (c, g) 20 μM of celecoxib for 30 minutes. Mean fluorescence intensity of (i) HT-29 cells and (j) HeLa cells images. (d, h) Western blot analysis results of COX-2. The band intensity of COX-2 was normalized to each α-tubulin band. The excitation wavelength was 810 nm, and images were obtained from 400 to 600 nm. Scale bar = 20 μm.
12 is a result of measuring two-photon fluorescence images after (a, b) Raw 264.7 cells and (d, e) Chang cells were stained with SCX (2 μM) for 30 minutes. (a, e) is a control image, and (b, f) is an image obtained after pretreatment of cells with LPS (1 μg mL -1 ) for 12 hours. (c, g) Mean fluorescence intensity measurement results of TPM images before and after LPS treatment. (d, h) Western blot analysis results of COX-2. The band intensity of COX-2 was normalized to each α-tubulin band. The excitation wavelength was 810 nm, and images were obtained from 400 to 600 nm. Scale bar = 20 μm.
Figure 13 shows two-photon fluorescence images of (ac) cancer cells (HT-29, Huh7, HeLa) and (df) normal cells (CCD-18Co, Chang, Raw 264.7) stained with SCX (2 μM) for 30 minutes. is the result Here, (a) HT-29 cells, (b) Huh7 cells, (c) HeLa cells, (d) CCD-18Co cells, (e) Chang cells, and (f) Raw 264.7 cells. (g) Shows the average fluorescence intensity of the corresponding TPM image. The excitation wavelength was 810 nm, and images were obtained from 400 to 600 nm. Scale bar = 20 μm.
14 is a two-photon fluorescence image measurement results after staining with SCX (20 μM) for 90 minutes for (a) cancer tissue and (c) normal tissue in human colon. (b) Cross-sectional images of human colon cancer tissue measured along the z-direction at a depth of about 70 to 120 μm. (d) Mean fluorescence intensity measurement results of corresponding human colon cancer and normal images. The excitation wavelength was 810 nm, and images were obtained from 400 to 600 nm. Scale bar = 50 μm.
이하, 일실시예를 통해 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail through an embodiment.
종래의 이광장 프로브는 인체 조직에 적용되지 않았다. 효소 발현 수준에 따라 정상 조직과 암 조직 또는 세포를 구분하기 위해서는 큰 이광자 작용 단면적 값(TPA)을 갖는 민감한 이광자 프로브를 개발해야하는 필요성이 있다. Conventional two-square probes have not been applied to human tissue. There is a need to develop a sensitive two-photon probe with a large two-photon action cross-sectional area (TPA) in order to distinguish normal tissue from cancer tissue or cells according to the level of enzyme expression.
본 발명에서는 살아있는 세포와 인간 결장 조직에서 COX-2를 검출하기 위한 이광자 프로브를 개발하였다 (반응식 1). 본 발명자가 발굴한 이광자 프로브인 SCX는 페난트렌 공여체 (phenanthrene donor)와 벤조티아졸 수용체 (benzothiazole acceptor) 및 COX-2 표적화 단위로 인도메타신 (indomethacin)을 보유한 새로운 이광자 형광단(PBT)을 기반으로 한다. SCX는 스펙트럼 이동에서 환경 민감도와 소수성 환경에서 형광 강도의 극적인 증가뿐만 아니라 160GM 이상의 큰 TPA 값을 보여주었다. SCX는 형광 강도를 높이기 위해 COX-2로 선택적으로 반응하였다. TPM 이미징 연구는 COX-2 선택적 억제제와 세포 내 염증 반응을 사용하여 SCX가 COX-2 발현 수준의 변화를 직접 감지 할 수 있음을 보여주었다. 따라서, 본 발명에 따른 형광 프로브 화합물은 사이클로옥시게나아제-2에 대해서 우수한 선택성으로 반응하며, 우수한 광물리적 특성과 낮은 세포독성을 가지고 있어 암 세포 또는 조직을 효과적으로 이미징할 수 있어 암 진단에 유용할 수 있다고 판단하여 본 발명을 완성하였다.In the present invention, a two-photon probe for detecting COX-2 in living cells and human colon tissues was developed (Scheme 1). SCX, a two-photon probe discovered by the present inventors, is based on a novel two-photon fluorophore (PBT) having a phenanthrene donor, a benzothiazole acceptor, and indomethacin as a COX-2 targeting unit. do it with SCX showed large TPA values over 160 GM, as well as dramatic increases in environmental sensitivity in spectral shift and fluorescence intensity in hydrophobic environments. SCX was selectively reacted with COX-2 to increase fluorescence intensity. TPM imaging studies have shown that SCX can directly detect changes in COX-2 expression levels using a COX-2 selective inhibitor and an intracellular inflammatory response. Therefore, the fluorescent probe compound according to the present invention reacts with excellent selectivity to cyclooxygenase-2, and has excellent photophysical properties and low cytotoxicity, so that it can effectively image cancer cells or tissues, which can be useful for cancer diagnosis. It was determined that the present invention was completed.
이에, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 제공한다.Accordingly, the present invention provides a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
또한, 본 발명은 상기 화학식 Ⅰ로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 검출용 이광자 형광 프로브를 제공한다. In addition, the present invention provides a two-photon fluorescent probe for detecting cyclooxygenase-2, comprising the compound represented by Formula I or a pharmaceutically acceptable salt thereof.
본 발명에서 상기 화학식 Ⅰ로 표시되는 화합물은 N-(6-(2-(1- 4-클로로벤조일)-5-메톡시-2-메틸-1H-인돌-3-일)아세트아미도)헥실)-2-(7-(디메틸아미노)-9,10-디히드로페난트렌-2-일)벤조[d]티아졸-6-카르복사미드)[N-(6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)hexyl)-2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[d]thiazole-6-carboxamide]이며,‘SCX’로 명명할 수 있다 (도 1).In the present invention, the compound represented by Formula I is N- (6-(2-(1-4-chlorobenzoyl)-5-methoxy-2-methyl- 1H -indol-3-yl)acetamido) Hexyl)-2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ d ]thiazole-6-carboxamide)[ N- (6-(2-(1) -(4-chlorobenzoyl)-5-methoxy-2-methyl-1 H -indol-3-yl)acetamido)hexyl)-2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ d ]thiazole-6-carboxamide], and can be named 'SCX' (FIG. 1).
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 검출용 이광자 형광 프로브를 제공한다.In addition, the present invention provides a two-photon fluorescent probe for detecting cyclooxygenase-2, comprising the compound represented by
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 이광자 형광 프로브를 세포 내 주입하여 사이클로옥시게나아제-2와 반응시키는 단계; 및 상기 반응시킨 반응물의 형광신호를 이광자 현미경(TPM)으로 이미지를 관측하는 단계를 포함하는 사이클로옥시게나아제-2 활성의 정량적 검출 방법을 제공한다. In addition, the present invention comprises the steps of injecting a two-photon fluorescent probe containing the compound represented by
또한, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 검출용 키트를 제공한다.In addition, the present invention provides a kit for detecting cyclooxygenase-2, comprising the compound represented by
또한, 본 발명은 (a) 피검체로부터 분리된 시료를 제공하는 단계; (b) 상기 제공된 시료에 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 이광자 형광 프로브를 첨가하는 단계; 및 (c) 상기 이광자 형광 프로브가 첨가된 시료의 형광을 측정하는 단계를 포함하는 생체 내 사이클로옥시게나아제-2 (Cyclooxygenase-2)를 검출하여 암 질환을 예측 또는 진단하기 위하여 정보를 제공하는 방법을 제공한다.In addition, the present invention comprises the steps of (a) providing a sample isolated from a subject; (b) adding a two-photon fluorescent probe comprising the compound represented by
상기 시료는 조직, 세포 및 혈액으로 이루어진 군에서 선택되는 것일 수 있다.The sample may be selected from the group consisting of tissue, cells, and blood.
상기 사이클로옥시게나아제-2 (Cyclooxygenase-2)를 검출하기 위해, 형광을 측정하는 단계에서 여기원으로서 700 내지 850 nm 범위의 파장을 사용할 수 있다.In order to detect the cyclooxygenase-2, a wavelength in the range of 700 to 850 nm may be used as an excitation source in the step of measuring fluorescence.
상기 암 질환은 대장암, 유방암, 간암, 자궁경부암, 폐암 및 뇌암으로 이루어진 군에서 선택될 수 있다.The cancer disease may be selected from the group consisting of colon cancer, breast cancer, liver cancer, cervical cancer, lung cancer and brain cancer.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공하는 것이다. Hereinafter, preferred examples are presented to help the understanding of the present invention, but the following examples are only illustrative of the present invention and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
<실험예><Experimental example>
하기 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.
1. 분광측정(Spectroscopic Measurements)1. Spectroscopic Measurements
흡수 스펙트럼과 형광 스펙트럼을 각각 UV-Vis 분광 광도계 (S-3100)와 형광 분광 광도계(FS-2)로 기록하였다. 형광 양자 수율 및 이광자 작용 단면적은 9,10-디페닐안트란센 (시클로헥산 내 Φ = 0.93) 및 로다민 6G를 기준으로 사용하여 측정하였다. 1H NMR 스펙트럼 및 13C NMR 스펙트럼은 400 MHz NMR 분광계 (OXFORD AS400)를 사용하여 얻었다. 형광 이미지는 스펙트럼 공초점 및 다광자 현미경 (Leica TCS SP8)으로 파장 810 nm 및 출력 전력 2.94 W로 설정된 모드-잠금 티타늄 사파이어 레이저 소스 (mode-locked titanium-sapphire laser source, Mai Tai HP)를 사용하여 얻었다. 이는 초점면 (focal plane)에서 약 6.94 X 108 W cm-2 평균 출력에 해당한다.Absorption and fluorescence spectra were recorded with a UV-Vis spectrophotometer (S-3100) and a fluorescence spectrophotometer (FS-2), respectively. The fluorescence quantum yield and two-photon action cross-sectional area were measured using 9,10-diphenylantrancene (Φ = 0.93 in cyclohexane) and rhodamine 6G as standards. 1 H NMR spectra and 13 C NMR spectra were obtained using a 400 MHz NMR spectrometer (OXFORD AS400). Fluorescence images were taken with a spectral confocal and multiphoton microscope (Leica TCS SP8) using a mode-locked titanium-sapphire laser source (Mai Tai HP) set at a wavelength of 810 nm and an output power of 2.94 W. got it This corresponds to about 6.94 X 10 8 W cm -2 average power in the focal plane.
2. 물 용해도 측정(Water Solubility)2. Water Solubility
소량의 SCX를 DMSO에 용해시켜 10 mM 스톡 용액을 제조하였다. 용액을 1.0 × 10-5 ~ 2.0 × 10-7 M으로 희석하고 마이크로 주사기를 사용하여 2.0 mL의 PBS 버퍼 (10 mM, pH 7.4)가 들어있는 큐벳에 첨가하였다. 모든 경우에 완충액의 DMSO 농도는 0.1 %로 유지되었습니다. 염료 농도에 대한 형광 강도의 플롯은 저농도에서는 선형이었고 고농도에서는 하향 곡률을 나타냈다 (도 4). 선형 영역의 최대 농도를 용해도로 취했습니다. PBS 버퍼에서 SCX의 용해도는 ~ 3.0 μM이다.A 10 mM stock solution was prepared by dissolving a small amount of SCX in DMSO. The solution was diluted to 1.0 × 10 -5 to 2.0 × 10 -7 M and added to a cuvette containing 2.0 mL of PBS buffer (10 mM, pH 7.4) using a micro-syringe. In all cases, the DMSO concentration in the buffer was maintained at 0.1%. The plot of fluorescence intensity versus dye concentration was linear at low concentrations and exhibited downward curvature at high concentrations (Fig. 4). The maximum concentration in the linear region was taken as the solubility. The solubility of SCX in PBS buffer is ~3.0 µM.
3. 선택성 분석(Selectivity Assay)3. Selectivity Assay
SCX의 선택성에 대해서 분석하였다. 상세하게, 200 μM의 반응성 산소 및 질소 종을 PBS 완충액 (10 mM, pH 7.4)에서 1 μM의 SCX에 투여하고 형광 스펙트럼을 시간으로 측정하였다. 온도는 37℃에서 2 시간 동안 유지하였다. t-부틸하이드로퍼옥사이드 (TBHP, 416665), NaOCl (239305), KO2 (278904), NO (D184)는 Sigma-Aldrich에서 구입하였다. 하이드록실 라디칼 (·OH)과 t-부톡시 라디칼 (·OtBu)은 TBHP와 H2O2 반응에서 FeSO4에 의해 생성되었다. Peroxynitrite (ONOO-)는 알려진 기존의 방법에 따라 제조하였다.SCX selectivity was analyzed. Specifically, 200 μM of reactive oxygen and nitrogen species were administered to 1 μM of SCX in PBS buffer (10 mM, pH 7.4) and fluorescence spectra were measured with time. The temperature was maintained at 37° C. for 2 hours. t-Butylhydroperoxide (TBHP, 416665), NaOCl (239305), KO 2 (278904), NO (D184) were purchased from Sigma-Aldrich. The hydroxyl radical (·OH) and the t-butoxy radical (·OtBu) were generated by FeSO 4 in the reaction of TBHP and H 2 O 2 . Peroxynitrite (ONOO - ) was prepared according to a known conventional method.
4. 세포 배양 및 결장 조직(Colon Tissues)의 준비4. Cell Culture and Preparation of Colon Tissues
모든 세포를 유리 바닥 접시 (NEST)에서 배양하고 이미징 하기 전에 2 일 동안 37℃에서 공기 중 5% CO2의 가습 분위기에서 배양하였다. 세포를 SCX (2μM)로 처리하고 30 분 동안 배양한 다음 무혈청 배지를 사용하여 두 번 세척하였다. 각 세포 배양 배지에 10% FBS (WelGene), 페니실린 (100 unit mL-1) 및 스트렙토마이신(100 μg mL-1)을 보충하였다. 배양배지는 HeLa 세포의 경우 DMEM (고 포도당), CCD-18Co 세포 및 Raw 264.7 세포, Huh7 및 Chang 세포의 경우 DMEM (낮은 포도당), HT-29 세포의 경우 RPMI1640을 이용하였다. 조직 영상 실험을 위해, 아주대학교 의료원에서 동의를 받은 3명의 환장에게 대장 내시경 검사를 통해 환자로부터 결장 조직을 채취하였다. 조직 수집은 기관 검토위원회로부터 승인을 받아 진행하였다 (승인 번호 AJIRB-BMR-SMP-17-164). 우리는 환자의 정상 조직과 암 조직을 조사하였다. 결장 조직에 SCX (20 μM)를 90분 동안 처리하였고 이미지를 70 내지 120 μm 범위에서 관찰하였다.All cells were cultured in glass bottom dishes (NEST) and cultured in a humidified atmosphere of 5% CO 2 in air at 37° C. for 2 days prior to imaging. Cells were treated with SCX (2 μM) and incubated for 30 min, then washed twice using serum-free medium. Each cell culture medium was supplemented with 10% FBS (WelGene), penicillin (100 unit mL -1 ) and streptomycin (100 μg mL -1 ). The culture medium was DMEM (high glucose) for HeLa cells, CCD-18Co cells and Raw 264.7 cells, DMEM (low glucose) for Huh7 and Chang cells, and RPMI1640 for HT-29 cells. For tissue imaging experiments, colonic tissues were collected from patients through colonoscopy from three patients who obtained consent from Ajou University Medical Center. Tissue collection was performed with approval from the Institutional Review Board (Approval No. AJIRB-BMR-SMP-17-164). We examined normal and cancerous tissues of patients. Colon tissue was treated with SCX (20 μM) for 90 minutes and images were observed in the range of 70 to 120 μm.
5. 세포 생존력(Cell Viability)5. Cell Viability
세포 독성을 평가하기 위해 MTT 키트 (AbCareBio CL) 분석을 수행하였다. 각 세포를 96-웰 플레이트에서 24 시간 동안 배양한 후 각기 다른 농도의 SCX를 첨가하였다. 8 시간 동안 배양한 후 배양된 배지를 10 % MTT가 포함된 무혈청 배지로 교체하고 추가로 2 시간 동안 배양하였다. MTT 함유 배지를 제거하고 DMSO를 첨가하여 형성된 포르마잔 (formazan) 침전물을 용해시켰다. 600 nm에서 흡광도를 측정하였다.MTT kit (AbCareBio CL) assay was performed to assess cytotoxicity. Each cell was cultured in a 96-well plate for 24 hours, and then SCX at different concentrations was added. After culturing for 8 hours, the cultured medium was replaced with a serum-free medium containing 10% MTT and cultured for an additional 2 hours. The MTT-containing medium was removed and DMSO was added to dissolve the formed formazan precipitate. Absorbance was measured at 600 nm.
6. 웨스턴 블롯 분석(Western Blot Analysis)6. Western Blot Analysis
세포를 PBS로 세척하고, 가열한 나트륨도데실설페이트-폴리아크릴아미드 겔 전기영동 (SDS-PAGE) 샘플 버퍼[62.5 mM Tris (pH 6.8), 1% SDS, 10% 글리세롤 및 5% β-머캅토에탄올]에 용해시켰다. 용해물을 5 분 동안 끓이고 SDS-PAGE로 분리하고 Immobilon 막 (Bio-Rad Laboratories, Hercules, CA, USA)으로 옮겼다. 5% 탈지유를 사용하여 비특이적 결합 부위를 1 시간 동안 차단한 후, 막을 특정 항체와 함께 2 시간 동안 배양하였다. 이어서 막을 Tris-Buffered Saline Tween-20 (TBST)으로 3회 세척하고 horseradish peroxidase-conjugated anti-rabbit 또는 horseradish peroxidase-conjugated anti-mouse antibody와 함께 추가로 1 시간 더 배양하였다. 단백질 밴드의 시각화는 ECL (GE Healthcare, Chicago, IL, USA)을 사용하여 수행하였다. 다음과 같은 1 차 항체가 사용되었다 : Santa Cruz Biotechnology (Santa Cruz, CA, USA)의 α-tubulin (sc-32293); Proteintech의 COX-2 (12375-1-AP), Santa Cruz의 COX-2 (sc-7951), 및 Abcam의 COX-2 (ab15191). 토끼 IgG HRP (G-21234) 및 마우스 IgG HRP (G-21040)를 포함한 2 차 항체는 Molecular Probes로부터 얻었다.Cells were washed with PBS, heated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [62.5 mM Tris (pH 6.8), 1% SDS, 10% glycerol and 5% β-mercapto ethanol]. Lysates were boiled for 5 min, separated by SDS-PAGE and transferred to Immobilon membranes (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the non-specific binding site for 1 hour using 5% skim milk, the membrane was incubated with a specific antibody for 2 hours. Then, the membrane was washed three times with Tris-Buffered Saline Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated anti-rabbit or horseradish peroxidase-conjugated anti-mouse antibody for an additional 1 hour. Visualization of protein bands was performed using ECL (GE Healthcare, Chicago, IL, USA). The following primary antibodies were used: α-tubulin (sc-32293) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); COX-2 from Proteintech (12375-1-AP), COX-2 from Santa Cruz (sc-7951), and COX-2 from Abcam (ab15191). Secondary antibodies including rabbit IgG HRP (G-21234) and mouse IgG HRP (G-21040) were obtained from Molecular Probes.
7. 광안정성(Photostability)7. Photostability
SCX의 광 안정성은 SCX로 표지된 HeLa 세포 (2μM) 의 3 개의 지정된 위치에서 시간에 따른 이광자 형광 강도의 변화를 모니터링 함으로써 확인하였다. 이광자 형광 강도는 xyt 모드를 사용하여 1 시간 동안 2 초 간격으로 기록되었으며 강도는 1 시간 동안 거의 동일하게 유지되어 높은 광 안정성을 나타냄을 확인하였다.The photostability of SCX was confirmed by monitoring the change of two-photon fluorescence intensity with time at three designated locations in SCX-labeled HeLa cells (2 μM). The two-photon fluorescence intensity was recorded at 2-second intervals for 1 hour using the xyt mode, and the intensity remained almost the same for 1 hour, confirming high light stability.
<실시예 1> SCX 합성<Example 1> SCX synthesis
본 발명에 따른 SCX를 헥사 메틸렌 디아민 링커를 사용하여 IMC와 2-(7-(디메틸아미노)-9,10-디히드로 페난트렌-2-일)벤조[d]티아졸-6-카르복실산 형광단 사이의 아미드 커플링 반응을 이용하여 합성하였다. 프로브의 링커 길이가 COX-2 (8.294-9.294 Å)의 큰 소수성 채널에 대한 결합 부위보다 짧기 때문에 결합 포켓에 들어갈 수 없고 COX-2 라벨링에 실패하므로 적절한 길이의 링커를 선택하는 것이 중요한다. 이에 본 발명자들은 신규한 COX-2 프로브를 개발하고자 하였다. SCX 및 각 중간체에 대한 자세한 합성 방법은 아래와 같다 (반응식 1).SCX according to the present invention was combined with IMC using a hexamethylene diamine linker with 2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ d ]thiazole-6-carboxylic acid It was synthesized using an amide coupling reaction between fluorophores. Since the linker length of the probe is shorter than the binding site for the large hydrophobic channel of COX-2 (8.294-9.294 Å), it cannot fit into the binding pocket and fails to label the COX-2, so it is important to select a linker of the appropriate length. Accordingly, the present inventors tried to develop a novel COX-2 probe. Detailed synthesis methods for SCX and each intermediate are as follows (Scheme 1).
먼저 화합물 1, 화합물2 및 화합물3은 종래에 알려진 방법 [Tetrahedron 70 (2014) 7551-7559, Org. Prep. Proced. Int. 35 (2003) 520-524, J. Mater. Chem. B 2 (2014) 502-510]로 제조하였으며, 화합물 4는 Sigma-Aldrich (I7378)에서 구입하였다. PBT, 화합물 5, 화합물 6 및 SCX의 합성은 아래와 같다.First,
[반응식 1][Scheme 1]
1-1. 2-(7-(디메틸아미노)-9,10-디하이드로페난트렌-2-일)벤조[1-1. 2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ dd ]티아졸-6-카르복실산[2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[]thiazole-6-carboxylic acid [2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ dd ]thiazole-6-carboxylic acid] (PBT) 제조]thiazole-6-carboxylic acid] (PBT) production
DMF (30 mL) 내 화합물 1 (241 mg, 0.96 mmol), 화합물 2 (484 mg, 1.44 mmol) 및 p-톨루엔설폰산 (p-TSA, 60 mg, 0.32 mmol) 혼합물을 12 시간 동안 환류시켰다. 반응 혼합물을 물 (200 mL)에 붓고 형성된 고체 생성물을 여과하였다. 이후 컬럼 크로마토 그래피 (DCM/MeOH = 9 : 1)로 정제하여 PBT (242 mg)를 노란색 분말로 얻었다. 반응 혼합물을 물 (200 mL)에 붓고 형성된 고체 생성물을 여과하였다. 이후 컬럼 크로마토그래피 (DCM/MeOH = 9 : 1)로 정제하여 노란색 분말 형태로 PBT (242 mg)를 얻었다.A mixture of compound 1 (241 mg, 0.96 mmol), compound 2 (484 mg, 1.44 mmol) and p-toluenesulfonic acid (p-TSA, 60 mg, 0.32 mmol) in DMF (30 mL) was refluxed for 12 h. The reaction mixture was poured into water (200 mL) and the solid product formed was filtered. Then, it was purified by column chromatography (DCM/MeOH = 9: 1) to obtain PBT (242 mg) as a yellow powder. The reaction mixture was poured into water (200 mL) and the solid product formed was filtered. After purification by column chromatography (DCM/MeOH = 9: 1), PBT (242 mg) was obtained in the form of a yellow powder.
수득율: 63%; 1HNMR (DMSO, 400MHz): δ(ppm) 8.73 (s, 1H), 8.07 (s, 2H), 7.96 (d, J = 7.2 Hz, 2H), 7.83 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 8.8, 2.4 Hz, 1H), 6.64 (s, 1H), 2.97 (s, 6H), 2.88-2.92 (m, 2H), 2.80-2.84 (m, 2H); 13C NMR (DMSO, 100 MHz): δ (ppm) 151.1, 141.2, 139.1, 138.9, 137.0, 134.9, 130.0, 128.2, 127.4, 126.8, 125.9, 124.7, 123.5, 122.7, 121.7, 112.1, 111.6, 29.9, 29.7, 29.2.Yield: 63%; 1 HNMR (DMSO, 400 MHz): δ (ppm) 8.73 (s, 1H), 8.07 (s, 2H), 7.96 (d, J = 7.2 Hz, 2H), 7.83 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 8.8, 2.4 Hz, 1H), 6.64 (s, 1H), 2.97 (s, 6H), 2.88-2.92 (m, 2H), 2.80 -2.84 (m, 2H); 13 C NMR (DMSO, 100 MHz): δ (ppm) 151.1, 141.2, 139.1, 138.9, 137.0, 134.9, 130.0, 128.2, 127.4, 126.8, 125.9, 124.7, 123.5, 122.7, 121.7, 112.1, 111.6, 29.9, 29.7, 29.2.
1-2. 1-2. tt -부틸(6-(2-(1-(4-클로로벤조일)-5-메톡시-2-메틸-1-Butyl (6- (2- (1- (4-chlorobenzoyl) -5-methoxy-2-methyl-1 HH -인돌-3-일)아세트아미도)헥실)카르바메이트[-indol-3-yl)acetamido)hexyl)carbamate [ tt -butyl (6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-butyl (6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 HH -indol-3-yl)acetamido)hexyl)carbamate]의 제조 (화합물 5).-indol-3-yl)acetamido)hexyl)carbamate] (compound 5).
DMF (10 mL) 중 화합물 4 (360 mg, 1.01 mmol) 및 N-(3-디메틸아미노프로필) -N'-에틸카르보디이미드 염산염 (EDC, 390 mg, 2.03 mmol)의 혼합물을 실온에서 30 분 동안 교반하였다. 반응 혼합물에 화합물 3(330 mg, 1.53 mmol) 및 DIPEA (393 mg, 3.04 mmol)를 첨가하고 12 시간 동안 교반하였다. 용매를 증발시키고 반응 혼합물을 DCM에 용해시킨 다음 물로 세척하였다. 유기상을 감압 농축하고 컬럼크로마토그래피(DCM/MeOH = 9 : 1)로 정제하여 백색 분말인 화합물 5 (270 mg)를 얻었다.A mixture of compound 4 (360 mg, 1.01 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 390 mg, 2.03 mmol) in DMF (10 mL) at room temperature for 30 min stirred for a while. Compound 3 (330 mg, 1.53 mmol) and DIPEA (393 mg, 3.04 mmol) were added to the reaction mixture and stirred for 12 hours. The solvent was evaporated and the reaction mixture was dissolved in DCM and washed with water. The organic phase was concentrated under reduced pressure and purified by column chromatography (DCM/MeOH = 9: 1) to obtain Compound 5 (270 mg) as a white powder.
수득율: 48%; 1HNMR (CDCl3, 400 MHz): δ(ppm) 7.60 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 2.4 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 5.84 (brs, 1H), 4.58 (brs, 1H), 3.79 (s, 3H), 3.61 (s, 2H), 3.16 (q, J = 6.4 Hz, 2H), 3.01 (q, J = 6.8 Hz, 2H), 2.36 (s, 3H), 1.41 (s, 9H) 1.32 - 1.38 (m, 4H), 1.15-1.25 (m, 4H); 13C NMR (CDCl3, 100MHz):δ(ppm)169.8, 168.4, 156.3, 139.6, 136.4, 133.7, 131.3, 131.0, 130.5, 129.3, 115.3, 113.2, 112.4, 101.1, 56.0, 40.5, 39.6, 32.6, 30.3, 29.7, 28.8, 26.6, 26.4, 13.7.Yield: 48%; 1 HNMR (CDCl 3 , 400 MHz): δ(ppm) 7.60 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 2.4 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 5.84 (brs, 1H), 4.58 (brs, 1H), 3.79 (s, 3H), 3.61 (s) , 2H), 3.16 (q, J = 6.4 Hz, 2H), 3.01 (q, J = 6.8 Hz, 2H), 2.36 (s, 3H), 1.41 (s, 9H) 1.32 - 1.38 (m, 4H), 1.15-1.25 (m, 4H); 13 C NMR (CDCl 3 , 100 MHz): δ (ppm) 169.8, 168.4, 156.3, 139.6, 136.4, 133.7, 131.3, 131.0, 130.5, 129.3, 115.3, 113.2, 112.4, 101.1, 56.0, 40.5, 39.6, 32.6, 30.3, 29.7, 28.8, 26.6, 26.4, 13.7.
1-3. 1-3. NN -(6-아미노헥실)-2-(1-(4-클로로벤조일)-5-메톡시-2-메틸-1-(6-aminohexyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 HH -인돌-3-일)아세트아미드[-indol-3-yl)acetamide [ NN -(6-aminohexyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-(6-aminohexyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 HH -indol-3-yl)acetamide]의 제조 (화합물 6).-indol-3-yl)acetamide] (Compound 6).
화합물 5 (130 mg, 0.23 mmol)를 DCM (5 mL)에 용해시킨 다음 트리 플루오로 아세트산 (TFA, 266 mg, 2.34 mmol)을 첨가했다. 상온에서 30 분간 교반한 후 반응 혼합물을 감압 농축하고 컬럼 크로마토 그래피 (CHCl3/MeOH = 8 : 2)로 정제하여 백색 분말인 화합물 6 (106 mg)을 얻었다.Compound 5 (130 mg, 0.23 mmol) was dissolved in DCM (5 mL) followed by addition of trifluoroacetic acid (TFA, 266 mg, 2.34 mmol). After stirring at room temperature for 30 minutes, the reaction mixture was concentrated under reduced pressure and purified by column chromatography (CHCl 3 /MeOH = 8: 2) to obtain Compound 6 (106 mg) as a white powder.
수득율: 99%; 1HNMR (CDCl3, 400 MHz): δ(ppm) 7.58 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 6.03 (t, J = 5.6 Hz, 1H), 3.79 (s, 3H), 3.60 (s, 2H), 3.14 (q, J = 6.8 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 2.35 (s, 3H), 1.51 (p, J = 6.8 Hz, 2H), 1.19-1.43 (m, 8H); 13C NMR (CDCl3, 100 MHz):δ(ppm) 168.4, 156.4, 139.7, 137.1, 133.5, 131.3, 131.0, 130.4, 129.3, 115.2, 112.6, 112.4, 100.8, 55.9, 40.2, 39.4, 31.6, 30.1, 28.8, 27.1, 25.6, 13.7.Yield: 99%; 1 HNMR (CDCl 3 , 400 MHz): δ(ppm) 7.58 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 6.03 (t, J = 5.6 Hz, 1H), 3.79 (s, 3H), 3.60 (s, 2H) ), 3.14 (q, J = 6.8 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 2.35 (s, 3H), 1.51 (p, J = 6.8 Hz, 2H), 1.19-1.43 (m) , 8H); 13 C NMR (CDCl 3 , 100 MHz): δ (ppm) 168.4, 156.4, 139.7, 137.1, 133.5, 131.3, 131.0, 130.4, 129.3, 115.2, 112.6, 112.4, 100.8, 55.9, 40.2, 39.4, 31.6, 30.1 , 28.8, 27.1, 25.6, 13.7.
1-4. 1-4. NN -(6-(2-(1- 4-클로로벤조일)-5-메톡시-2-메틸-1-(6-(2-(1-4-Chlorobenzoyl)-5-methoxy-2-methyl-1 HH -인돌-3-일)아세트아미도)헥실)-2-(7-(디메틸아미노)-9,10-디히드로페난트렌-2-일)벤조[-indol-3-yl)acetamido)hexyl)-2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ dd ]티아졸-6-카르복사미드[]thiazole-6-carboxamide [ NN -(6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-(6-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 HH -indol-3-yl)acetamido)hexyl)-2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[-indol-3-yl)acetamido)hexyl)-2-(7-(dimethylamino)-9,10-dihydrophenanthren-2-yl)benzo[ dd ]thiazole-6-carboxamide]의 제조 (SCX).Preparation of ]thiazole-6-carboxamide] (SCX).
DMF (3 mL)에 녹인 PBT (73 mg, 0.18 mmol), N, N'-디시클로헥실카르보디이 미드 (DCC, 74 mg, 0.36 mmol) 및 하이드록시벤조트리아졸 (HOBt, 49 mg, 0.36 mmol)의 혼합물을 2 시간 동안 실온에서 교반하였다. 이 혼합물에 화합물 6 (91 mg, 0.20 mmol)을 첨가하고 밤새 교반하였다. 용매를 증발시키고 반응 혼합물을 CH3CN에 용해시킨 다음 부산물인 요소를 여과하여 제거하였다. 여액을 감압 농축하고 컬럼크로마토그래피 (CHCl3/MeOH = 9 : 1)로 정제하여 황색 분말 형태로 SCX (34 mg)를 얻었다. 도 2는 SCX의 13C-NMR 스펙트럼(100 MHz) 분석 결과이고, 도 3은 SCX HRMS 스펙트럼 분석 결과이다. PBT (73 mg, 0.18 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 74 mg, 0.36 mmol) and hydroxybenzotriazole (HOBt, 49 mg, 0.36 mmol) in DMF (3 mL) ) was stirred at room temperature for 2 h. To this mixture was added compound 6 (91 mg, 0.20 mmol) and stirred overnight. The solvent was evaporated, the reaction mixture was dissolved in CH 3 CN, and the by-product urea was removed by filtration. The filtrate was concentrated under reduced pressure and purified by column chromatography (CHCl 3 /MeOH = 9: 1) to obtain SCX (34 mg) in the form of a yellow powder. 2 is a 13 C-NMR spectrum (100 MHz) analysis result of SCX, and FIG. 3 is an SCX HRMS spectrum analysis result.
수득율: 23%; 1HNMR (CDCl3, 400 MHz): δ(ppm) 8.38 (s,1H), 8.04 (d,J = 8.8, 1H), 7.95 (d, J = 8.4, 2H), 7.83 (dd, J = 8.8, 2.0 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 6.43 (t, J = 6.0 Hz, 1H), 5.75 (t, J = 6.0 Hz, 1H), 3.81 (s, 3H), 3.65 (s, 2H), 3.42 (q, J = 6.0 Hz, 2H), 3.23 (q, J = 6.4 Hz, 2H), 3.03 (s, 6H), 2.95-2.97 (m, 2H), 2.88-2.91 (m, 2H), 2.40 (s, 3H), 1.25-1.47 (m, 8H); 13CNMR (CDCl3, 100 MHz): δ(ppm) 170.8, 169.9, 168.3, 166.9, 156.1, 156.0, 155.9, 139.6, 139.1, 138.3, 136.8, 136.3, 135.2, 133.5, 131.5, 131.2, 130.9, 130.4, 129.2, 127.1, 126.6, 126.5, 125.3, 124.8, 124.6, 123.0, 122.9, 122.6, 121.4, 121.3, 115.1, 113.0, 112.3, 101.0, 56.0, 39.8, 39.3, 32.5, 29.9, 29.8, 29.7, 29.6, 29.3, 26.1, 26.0, 13.6; HRMS(ESI+): m/z calculated for [C49H49O4N5ClS]+: 838.3188, found: 838.3170.Yield: 23%; 1 HNMR (CDCl 3, 400 MHz): δ(ppm) 8.38 (s,1H), 8.04 (d, J = 8.8, 1H), 7.95 (d, J = 8.4, 2H), 7.83 (dd, J = 8.8) , 2.0 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4) Hz, 2H), 6.90 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.69 (s, 1H), 6.60 ( s, 1H), 6.43 (t, J = 6.0 Hz, 1H), 5.75 (t, J = 6.0 Hz, 1H), 3.81 (s, 3H), 3.65 (s, 2H), 3.42 (q, J = 6.0) Hz, 2H), 3.23 (q, J = 6.4 Hz, 2H), 3.03 (s, 6H), 2.95-2.97 (m, 2H), 2.88-2.91 (m, 2H), 2.40 (s, 3H), 1.25 -1.47 (m, 8H); 13 CNMR (CDCl 3 , 100 MHz): δ (ppm) 170.8, 169.9, 168.3, 166.9, 156.1, 156.0, 155.9, 139.6, 139.1, 138.3, 136.8, 136.3, 135.2, 133.5, 131.5, 131.2, 130.9, 130.4, 129.2, 127.1, 126.6, 126.5, 125.3, 124.8, 124.6, 123.0, 122.9, 122.6, 121.4, 121.3, 115.1, 113.0, 112.3, 101.0, 56.0, 39.8, 39.3, 32.5, 29.9, 29.8, 29.7, 29.6, 29.3, 26.1, 26.0, 13.6; HRMS(ESI+): m/z calculated for [C 49 H 49 O 4 N 5 ClS] + : 838.3188, found: 838.3170.
<실시예 2> SCX의 광물리적 특성<Example 2> Photophysical properties of SCX
인산염 완충 식염수 (PBS 완충액, 10mM, pH 7.4)에서 SCX의 용해도는 약 3μM이었다(도 4). SCX (1μM)의 광 물리적 특성은 1,4-디옥산, 디메틸 포름 아미드(DMF), 에탄올 (EtOH), PBS 완충액 및 EtOH와 PBS 완충액의 1 : 1 혼합물 (v/v)과 같은 서로 다른 극성을 갖는 용매 내에서 측정되었다. PBS 완충액의 경우 SCX는 375 nm에서 흡수 피크 및 547 nm에서 방출 피크를 나타냈다. 반면 1,4-디옥산의 경우 SCX는 390 nm에서 흡수 피크를 보였고 PBS 버퍼에서 보다 약 1.8 배 높은 ε 값을 나타냈다. 1,4-디옥산에서 SCX의 방출 피크는 483 nm 이였다.The solubility of SCX in phosphate buffered saline (PBS buffer, 10 mM, pH 7.4) was about 3 μM ( FIG. 4 ). The photophysical properties of SCX (1 μM) showed different polarities such as 1,4-dioxane, dimethyl formamide (DMF), ethanol (EtOH), PBS buffer and 1:1 mixture (v/v) of EtOH and PBS buffer. was measured in a solvent with For PBS buffer, SCX showed an absorption peak at 375 nm and an emission peak at 547 nm. On the other hand, in the case of 1,4-dioxane, SCX showed an absorption peak at 390 nm and showed an ε value about 1.8 times higher than that in PBS buffer. The emission peak of SCX in 1,4-dioxane was 483 nm.
또한, 형광 스펙트럼이 청색편이 되었으며 용매의 극성이 PBS에서 1,4-디옥산으로 감소함에 따라 형광양자수율 (Φ)이 급격히 증가함을 확인할 수 있었다. PBS 버퍼와 1,4-디옥산의 형광양자수율 (Φ)은 각각 0.02와 0.76이었다 (표 1). 유사하게, SCX의 이광자 작용 단면 (TPA, Φδ)은 용매의 극성이 감소함에 따라 증가하고, PBS 버퍼 및 1,4-디옥산에서 내에서, SCX의 TPA는 810 nm에서 각각 1.5 GM 및 167 GM이었다 (도 5b 및 표 1).In addition, it was confirmed that the fluorescence spectrum became blue-shifted and the fluorescence quantum yield (Φ) rapidly increased as the polarity of the solvent decreased from PBS to 1,4-dioxane. The fluorescence quantum yields (Φ) of PBS buffer and 1,4-dioxane were 0.02 and 0.76, respectively (Table 1). Similarly, the two-photon action cross section (TPA, Φδ) of SCX increases with decreasing solvent polarity, and in PBS buffer and 1,4-dioxane, the TPA of SCX is 1.5 GM and 167 GM at 810 nm, respectively. was (Fig. 5b and Table 1).
TPA 값은 PBS 버퍼와 1,4-디옥산 간에 110 배 차이를 보여 주었으며, 이는 SCX의 형광 강도가 소수성 환경에서 일광자 모드와 이광자 모드 모두에 대해 스펙트럼 블루 이동 (spectral blue shifts)이 극적으로 증가한다는 것을 의미한다. 수성 완충액 (100mM Tris-HCl, pH 8.0, 0.05 % Triton-x)에서 SCX의 형광 강도는 COX-2가 첨가됨에 따라 525 nm에서 점차 증가했지만 COX-1에 대한 반응에는 무시할 수 있을 정도의 변화가 있었다 (도 6).TPA values showed a 110-fold difference between PBS buffer and 1,4-dioxane, indicating that the fluorescence intensity of SCX dramatically increased spectral blue shifts for both single-photon and two-photon modes in a hydrophobic environment. means to do The fluorescence intensity of SCX in aqueous buffer (100 mM Tris-HCl, pH 8.0, 0.05% Triton-x) gradually increased at 525 nm with the addition of COX-2, but there was no negligible change in the response to COX-1. There was (Fig. 6).
<실시예 3> SCX의 선택성과 안정성<Example 3> Selectivity and stability of SCX
프로브가 다양한 ROS 및 RNS 에 의해 영향을 받는지 여부를 평가하기 위해, SCX(1μM)를 포함하는 PBS 완충액 내에 과산화수소 (H2O2), t-부틸 하이드로퍼옥사이드 (TBHP), 차아염소산염 (OCl-), 과산화물 (O2 -), 산화질소 (NO), t-부톡시 라디칼 (·OtBu), 히드록실 라디칼 (·OH) 및 퍼옥시니트라이트 (ONOO-)를 첨가하였다 (도 7).To evaluate whether the probe is affected by various ROS and RNS, hydrogen peroxide (H 2 O 2 ), t-butyl hydroperoxide (TBHP), hypochlorite ( OCl- ) in PBS buffer containing SCX (1 μM) ), peroxide (O 2 − ), nitric oxide (NO), t-butoxy radical (·OtBu), hydroxyl radical (·OH) and peroxynitrite (ONOO − ) were added ( FIG. 7 ).
37℃에서 2 시간 후 SCX의 형광 강도는 변화를 나타내지 않았다. 또한 SCX의 형광 강도는 pH 3.5에서 pH 10까지의 넓은 pH 범위에서 거의 일정하였다 (도 8). 이것은 SCX가 ROS, RNS 및 pH에 쉽게 영향을 받지 않음을 의미한다. 아울러, 세포 이미징 전에 MTT 분석을 사용하여 SCX의 세포 생존력을 평가하였다 (도 9). 모든 세포주에서 세포의 97% 이상이 8 시간 동안 50 μM의 농도에서도 살아남았으며, 이로서 SCX가 무시할 수 있을 정도의 세포 독성을 가지고 있음을 확인하였다.The fluorescence intensity of SCX after 2 hours at 37°C showed no change. In addition, the fluorescence intensity of SCX was almost constant in a wide pH range from pH 3.5 to pH 10 (FIG. 8). This means that SCX is not easily affected by ROS, RNS and pH. In addition, cell viability of SCX was evaluated using MTT assay prior to cell imaging ( FIG. 9 ). In all cell lines, more than 97% of the cells survived even at a concentration of 50 μM for 8 hours, confirming that SCX had negligible cytotoxicity.
<실시예 4> SCX를 이용한 TPM 세포 이미징<Example 4> TPM cell imaging using SCX
TPM 라이브 셀 이미지의 경우 HeLa 세포에서 2 μM SCX를 30 분 동안 염색시켰다. SCX는 810 nm 여기 파장에서 세포 내 영역에서 밝은 형광을 보였다. 세포 스펙트럼의 방출 피크는 472 nm 이였다 (도 10). 따라서 SCX는 1,4-디옥산과 유사한 극성을 가진 세포 내 소수성 환경에 위치하였다. 또한 SCX는 2 초 간격으로 1800 개의 이미지를 1 시간 동안 수집하는 동안 형광 강도가 약 80 %로 유지되었기 때문에 높은 광 안정성을 나타내는 것으로 나타났다. For TPM live cell images, HeLa cells were stained with 2 μM SCX for 30 min. SCX showed bright fluorescence in the intracellular region at 810 nm excitation wavelength. The emission peak of the cell spectrum was 472 nm ( FIG. 10 ). Therefore, SCX was located in an intracellular hydrophobic environment with a polarity similar to that of 1,4-dioxane. In addition, SCX was shown to exhibit high photostability because the fluorescence intensity was maintained at about 80% during 1 h of acquisition of 1800 images at 2-second intervals.
다음으로, 셀레콕시브를 이용하여 세포 억제 분석을 수행하였다. 두 암세포주의 대조군에서 SCX의 형광 강도는 HT-29와 HeLa 세포에서 각각 86.0 ± 11.3 및 76.1 ± 9.8 이었다 (도 11). 그러나 COX-2의 선택적 억제제인 셀레콕시브(celecoxib, 20 μM, 30 분)로 전처리한 후 HT-29 및 HeLa 세포의 형광 강도가 각각 19.5 ± 4.3 및 23.7 ± 5.3으로 급격히 감소하였다 (도 11c 및 도 11g). 따라서 암세포 주에서 SCX의 강력한 형광의 기원은 COX-2 때문이다. 또한 웨스턴 블롯팅 분석을 사용하여 COX-2 발현 수준을 조사하였다 (도 11d 및 도 11h). 그러나 COX-2 발현 수준은 셀레콕시브 (celecoxib)의 치료와 관계없이 거의 변하지 않았다. 이러한 결과는 셀레콕시브(celecoxib)가 COX-2 활성을 억제하지만 효소의 발현 수준을 감소시키지 않는다는 이전에 보고된 연구와 유사한 결과였다(Cancer Res. 67 (2007) 9380-9388, J. Exp. Clin. Cancer Res. 27 (2008) 66, Heart Vessels 27 (2012) 307-315). Next, a cell inhibition assay was performed using celecoxib. The fluorescence intensity of SCX in the control group of the two cancer cell lines was 86.0 ± 11.3 and 76.1 ± 9.8 in HT-29 and HeLa cells, respectively ( FIG. 11 ). However, after pretreatment with celecoxib (20 μM, 30 min), a selective inhibitor of COX-2, the fluorescence intensity of HT-29 and HeLa cells was sharply decreased to 19.5 ± 4.3 and 23.7 ± 5.3, respectively (Fig. 11c and 11g). Therefore, the origin of the strong fluorescence of SCX in cancer cell lines is due to COX-2. Western blotting analysis was also used to examine the level of COX-2 expression ( FIGS. 11D and 11H ). However, COX-2 expression levels remained almost unchanged regardless of treatment with celecoxib. These results were similar to previously reported studies that celecoxib inhibits COX-2 activity but does not reduce the expression level of the enzyme (Cancer Res. 67 (2007) 9380-9388, J. Exp. Clin. Cancer Res. 27 (2008) 66, Heart Vessels 27 (2012) 307-315).
COX-2는 염증 세포에서 고도로 발현되는 것으로 알려져 있는데, 그 결과, 염증 세포 이미지를 수집하여 Raw 264.7과 지질다당류(lipopolysaccharides, LPS)에 의해 유도된 Chang 세포에서 세포 내 COX-2 발현 수준을 측정하였다(도 12). LPS (1μg mL-1, 12 시간)로 전처리한 후 SCX로 염색한 후 두 세포주에서 SCX의 향상된 형광 강도가 관찰하였다. 대조군에서 SCX의 형광 강도는 Raw 264.7 및 Chang 세포에서 각각 27.7 ± 4.6 및 24.7 ± 6.6이었다. 더욱이 염증 세포에서 프로브의 형광 강도는 Raw 264.7 및 Chang 세포에서 각각 76.9 ± 14 및 73.4 ± 11로 크게 증가하였다 (도 12c 및 도 12g). 이러한 영상화 결과를 바탕으로 SCX가 암세포와 정상 세포를 구별할 수 있는 가능성을 조사하였다 (도 13). HT-29 세포(결장직장선암), Huh-7 세포(간세포암), HeLa 세포 (상피성 자궁경부암)를 암 세포주로 사용하고, CCD-18Co (정상 결장 세포), Chang (정상 간 세포) 및 Raw 264.7 (대식세포) 세포를 정상 세포주로 사용하였다.COX-2 is known to be highly expressed in inflammatory cells. As a result, inflammatory cell images were collected and the intracellular COX-2 expression level was measured in Chang cells induced by Raw 264.7 and lipopolysaccharides (LPS). (Fig. 12). After pretreatment with LPS (1 μg mL -1 , 12 hours) and staining with SCX, enhanced fluorescence intensity of SCX was observed in both cell lines. The fluorescence intensity of SCX in the control group was 27.7 ± 4.6 and 24.7 ± 6.6 in Raw 264.7 and Chang cells, respectively. Moreover, the fluorescence intensity of the probe in inflammatory cells was significantly increased to 76.9 ± 14 and 73.4 ± 11 in Raw 264.7 and Chang cells, respectively ( FIGS. 12c and 12g ). Based on these imaging results, the possibility of SCX discriminating between cancer cells and normal cells was investigated (FIG. 13). HT-29 cells (colorectal adenocarcinoma), Huh-7 cells (hepatocellular carcinoma), HeLa cells (epithelial cervical cancer) were used as cancer cell lines, and CCD-18Co (normal colon cells), Chang (normal liver cells) and Raw 264.7 (macrophage) cells were used as normal cell lines.
SCX를 각 세포주로 염색했을 때 암과 정상 세포주 사이에 형광 강도의 유의한 차이가 관찰되었다. HT-29 세포, Huh-7 세포 및 HeLa 세포의 SCX의 형광 강도는 각각 86.0 ± 11.3, 73.8 ± 11.3 및 76.1 ± 9.8 이였다. 반면에, CCD-18Co 세포, Chang 세포 및 Raw 264.7 세포의 SCX의 형광 강도는 각각 31.9 ± 5.3, 24.7 ± 6.6, 27.7 ± 4.6 이였다. 따라서 암세포에서 SCX의 형광 강도는 정상 세포보다 약 3 배 강한 것으로 나타났다.Significant differences in fluorescence intensity were observed between cancer and normal cell lines when SCX was stained with each cell line. The fluorescence intensities of SCX of HT-29 cells, Huh-7 cells and HeLa cells were 86.0 ± 11.3, 73.8 ± 11.3 and 76.1 ± 9.8, respectively. On the other hand, the fluorescence intensities of SCX of CCD-18Co cells, Chang cells and Raw 264.7 cells were 31.9 ± 5.3, 24.7 ± 6.6, and 27.7 ± 4.6, respectively. Therefore, it was shown that the fluorescence intensity of SCX in cancer cells was about 3 times stronger than in normal cells.
<실시예 5> SCX를 사용한 TPM 조직 이미징<Example 5> TPM tissue imaging using SCX
그 결과, 인간 CRC와 정상 조직 모두에서 SCX가 명확하게 구별될 수 있는 가능성을 조사하였다 (도 14). 아주대학교 의료원 대장 내시경 검사를 통해 3 명의 환자로부터 결장 정상 및 암 조직을 채취하였다 (승인번호 AJIRB-BMR-SMP-17-164). CRC 조직에서 SCX는 강한 형광을 방출하며 100 μm 이상의 깊이에서 관찰할 수 있었다. 암 조직과 정상 조직의 형광 강도는 각각 85.3 ± 23.3 및 19.6 ± 6.1로 암 조직에서 SCX의 강도가 정상 조직보다 4.4 배 더 밝았다. 이러한 결과는 SCX가 CRC 환자의 세포와 조직 모두에서 암을 정상과 명확하게 구별할 수 있음을 보여준다 (도 13). 결론적으로, 본 발명은 대장암 (CRC) 환자의 암 조직과 정상 조직을 COX-2의 발현 수준으로 구분할 수 있는 이광자 탐침 프로브 SCX가 개발하였다. 본 발명자들은 SCX를 큰 TPA 값을 갖는 새로운 TP 형광단 (PBT)과 COX-2 선택성 억제제인 IMC 사이의 접합을 통해 합성하였다. SCX는 암세포 주와 정상 세포주를 명확하게 구분했으며, 억제제 및 염증 반응으로 인한 COX-2 발현 수준 변화에 직접 반응하였다. 또한 처음으로 CRC 환자의 살아있는 조직에서 100 μm 이상의 깊이에서 정상 조직과 암 조직을 명확하게 구분할 수 있었다. 이러한 결과는 COX-2 발현 수준의 검출에 대한 통찰력을 제공하고 이 프로브가 암 검출 및 연구를 위한 중요한 전략 개발에 사용될 수 있음을 보여준다.As a result, the possibility that SCX can be clearly distinguished from both human CRC and normal tissues was investigated (FIG. 14). Colonic normal and cancer tissues were collected from 3 patients through colonoscopy at Ajou University Medical Center (approval number AJIRB-BMR-SMP-17-164). In CRC tissue, SCX emits strong fluorescence and can be observed at a depth of more than 100 μm. The fluorescence intensity of cancer tissue and normal tissue was 85.3 ± 23.3 and 19.6 ± 6.1, respectively, indicating that the intensity of SCX in cancer tissue was 4.4 times brighter than in normal tissue. These results show that SCX can clearly distinguish cancer from normal in both cells and tissues of CRC patients ( FIG. 13 ). In conclusion, the present invention has developed a two-photon probe, SCX, capable of distinguishing between cancer and normal tissues of colorectal cancer (CRC) patients by the expression level of COX-2. We synthesized SCX through conjugation between a novel TP fluorophore (PBT) with a large TPA value and IMC, a COX-2 selective inhibitor. SCX clearly differentiated cancer cell lines from normal cell lines and directly responded to changes in COX-2 expression levels due to inhibitors and inflammatory responses. In addition, for the first time, it was possible to clearly distinguish normal and cancerous tissues at a depth of more than 100 μm in living tissues of CRC patients. These results provide insight into the detection of COX-2 expression levels and show that this probe can be used to develop important strategies for cancer detection and research.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
[화학식 1]
A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]
A two-photon fluorescent probe for detecting Cyclooxygenase-2, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.
A kit for detecting cyclooxygenase-2, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.
상기 반응시킨 반응물의 형광신호를 이광자 현미경(TPM)으로 이미지를 관측하는 단계를 포함하는 사이클로옥시게나아제-2 (Cyclooxygenase-2) 활성의 정량적 검출 방법.
The step of injecting a two-photon fluorescent probe containing the compound according to claim 1 or a pharmaceutically acceptable salt thereof into a cell to react with cyclooxygenase-2; and
A quantitative detection method of cyclooxygenase-2 activity, comprising the step of observing an image of the fluorescence signal of the reacted reactant with a two-photon microscope (TPM).
(b) 상기 제공된 시료에 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는 이광자 형광 프로브를 첨가하는 단계; 및
(c) 상기 이광자 형광 프로브가 첨가된 시료의 형광을 측정하는 단계를 포함하는 생체 내 사이클로옥시게나아제-2 (Cyclooxygenase-2)를 검출하여 암 질환을 예측 또는 진단하기 위하여 정보를 제공하는 방법:
[화학식 1]
(a) providing a sample isolated from the subject;
(b) adding a two-photon fluorescent probe comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof to the provided sample; and
(c) a method of providing information to predict or diagnose a cancer disease by detecting cyclooxygenase-2 in vivo, comprising measuring the fluorescence of a sample to which the two-photon fluorescent probe is added:
[Formula 1]
The method according to claim 5, wherein the sample is selected from the group consisting of tissues, cells and blood to detect cyclooxygenase-2 in vivo to predict or diagnose cancer diseases. how to provide.
According to claim 5, wherein in the step of measuring the fluorescence, the excitation source predicts or predicts cancer disease by detecting cyclooxygenase-2 in vivo, characterized in that it has a wavelength in the range of 700 to 850 nm. How to provide information to diagnose.
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