KR20210121661A - Photoactive Phosphor Probe and Cancer Cell Detection Method Using the Same - Google Patents

Photoactive Phosphor Probe and Cancer Cell Detection Method Using the Same Download PDF

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KR20210121661A
KR20210121661A KR1020200038772A KR20200038772A KR20210121661A KR 20210121661 A KR20210121661 A KR 20210121661A KR 1020200038772 A KR1020200038772 A KR 1020200038772A KR 20200038772 A KR20200038772 A KR 20200038772A KR 20210121661 A KR20210121661 A KR 20210121661A
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김해조
이원주
서현석
도재혁
권혁만
정선진
김상엽
박석안
조은아
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한국외국어대학교 연구산학협력단
재단법인 아산사회복지재단
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Abstract

The present invention relates to a method for detecting a photoactive phosphor probe, wherein the photoactive phosphor probe contains a compound represented by chemical formula 1 and several biomarkers, and the present invention includes contents about a method for detecting cancer cells by spatiotemporal manipulation in actual cell experiments. According to the present invention, since the photoactive phosphor probe is manufactured to diagnose cancer or enter mitochondria by introducing a biomarker into a photoactive composition selectively releasing a drug due to structural transformation by light in ex vivo or in vivo, diagnosis of cancer cells and drugs such as antibiotics, anticancer drugs, or inhibitors are introduced, so as to provide an effect of being usefully used for spatiotemporal treatment of cancer through drug release only by structural modification by light in a specific wavelength region.

Description

광활성 형광체 프로브 및 이를 이용한 암세포 검출방법 {Photoactive Phosphor Probe and Cancer Cell Detection Method Using the Same}Photoactive Phosphor Probe and Cancer Cell Detection Method Using the Same}

본 발명은 광활성 형광체 프로브에 관한 것으로서, 더욱 상세하게는 생체 외(ex vivo) 및 생체 내(in vivo)에서 특정 영역의 빛(740 nm, 365 nm)을 조사한 경우에만 선택적으로 약물이 배출되는 광활성 형광체 프로브 및 이를 이용한 암세포 검출방법에 관한 것이다.The present invention relates to a photoactive phosphor probe, and more particularly, a photoactive agent that selectively releases a drug only when irradiated with light (740 nm, 365 nm) of a specific region in ex vivo and in vivo It relates to a fluorescent probe and a method for detecting cancer cells using the same.

광활성 형광체는 1광자 흡수 (one-photon absorption, OPA) 또는 2광자 흡수(two-photon absorption, TPA)를 사용하여 분자 또는 물질이 한 개 혹은 두 개의 광자를 동시에 흡수하여 흥분 상태 (excitation state)에 도달하는 원리이다. 2광자 흡수 형광체는 1광자 흡수 형광체의 절반에 해당하는 파장의 에너지를 받아 들뜬상태로 여기 되는 특성을 가진다. 이로 인해 선택적인 영역의 빛을 조사하면 구조 변화 및 결합이 끊기면서 약물이 배출되어 선택적인 치료가 가능하도록 한다. 자외선 (UV) 조사와 달리 근적외선 영역의 빛을 사용하는 TPA는 조직에 더 깊이 침투하고, 손상이 적으며 3차원 공간에서 정밀도의 증가로 원하는 위치에 집중적으로 조사할 수 있는 장점을 가지고 있다. 이러한 이유로 암 세포에 본 조성물을 주입하여 특정한 파장 영역의 빛 (740 nm, 365 nm)을 조사하는 것은 선택적인 악성 종양의 진단 및 치료에 많은 기여를 할 수 있을 것으로 기대되고 있다.Photoactive phosphors use one-photon absorption (OPA) or two-photon absorption (TPA) to allow a molecule or substance to simultaneously absorb one or two photons to enter an excitation state. The principle of reaching The two-photon absorbing phosphor has a characteristic of being excited by receiving energy of a wavelength corresponding to half that of the one-photon absorbing phosphor. For this reason, when light in a selective area is irradiated, structural changes and bonds are broken, and the drug is discharged, enabling selective treatment. Unlike ultraviolet (UV) irradiation, TPA, which uses light in the near-infrared region, has the advantages of penetrating deeper into the tissue, less damaging, and intensively irradiating a desired location with increased precision in three-dimensional space. For this reason, injecting the present composition into cancer cells and irradiating light (740 nm, 365 nm) of a specific wavelength region is expected to contribute a lot to the diagnosis and treatment of selective malignant tumors.

1. A. Shigenaga, J. Yamamoto, Y. Sumikawa, T. Furuta and A. Otaka, Tetrahedron Lett., 2010, 51, 2868. 1. A. Shigenaga, J. Yamamoto, Y. Sumikawa, T. Furuta and A. Otaka, Tetrahedron Lett., 2010, 51, 2868. 2. H. M. Kim and B. R. Cho, Chem. Rev., 2015, 115, 5014.2. H. M. Kim and B. R. Cho, Chem. Rev., 2015, 115, 5014.

본 발명의 목적은, 암의 치료에 이용되는 많은 화학요법의 단점인 비선택적 세포 공격을 극복하고자 함에 있다. 특정한 파장 영역대의 빛에 의해 작동하여 약물이 선택적으로 배출되는 다양한 광활성 유도체를 검출 센서 및 치료제로 활용하여 생체 외 또는 생체 내에서 악성 종양과 같은 질병의 진단 및 선택적인 치료가 가능하게 하는 검출방법을 제공함에 있다.SUMMARY OF THE INVENTION It is an object of the present invention to overcome non-selective cell attack, which is a disadvantage of many chemotherapy methods used for the treatment of cancer. A detection method that enables the diagnosis and selective treatment of diseases such as malignant tumors in vitro or in vivo by using various photoactive derivatives that operate by light in a specific wavelength range and selectively release drugs as detection sensors and therapeutic agents is in providing.

상기 목적을 달성하기 위하여, 본 발명의 일 형태는 하기 [화학식 1]로 표시되는 OPA 또는 TPA 구조체를 활용한 광활성 형광체 프로브를 제공한다.In order to achieve the above object, one embodiment of the present invention provides a photoactive phosphor probe utilizing the OPA or TPA structure represented by the following [Formula 1].

[화학식 1][Formula 1]

Figure pat00001
Figure pat00001

상기 [화학식1]에서, D (Drug)는 카바메이트 또는 카보네이트 또는 벤질 이써 형태로 연결된 다양한 antibiotics 및 inhibitors (억제제) 등을 포함하는 그룹일 수 있으며, 바람직하게는 하기의 [화학식 2] 또는 [화학식 3]과 같은 독소루비신 (Doxorubicin) 또는 [화학식 4]과 같은 파수딜 (Fasudil, Rho-kinase 억제제) 또는 [화학식 5]과 같은 Y-27632 (lac 억제제) 또는 캄토테신 (Camptothecin, CPT) 일 수 있다.In the [Formula 1], D (Drug) may be a group containing various antibiotics and inhibitors (inhibitors), etc. linked in the form of carbamate or carbonate or benzyl ether, preferably the following [Formula 2] or [Formula 2] 3], such as doxorubicin (Doxorubicin) or [Formula 4], such as Fasudil (Fasudil, Rho-kinase inhibitor) or [Formula 5], such as Y-27632 (lac inhibitor) or camptothecin (Camptothecin, CPT) may be .

[화학식 2][Formula 2]

Figure pat00002
Figure pat00002

[화학식 3][Formula 3]

Figure pat00003
Figure pat00003

<화학식 4><Formula 4>

Figure pat00004
Figure pat00004

<화학식 5><Formula 5>

Figure pat00005
Figure pat00005

상기 [화학식1]에서, BM (Biomarker)은 선형 또는 비선형의 C1 내지 C20의 알킬기, 선형 또는 비선형의 C1 내지 C20의 알킬기를 갖는 케톤기, 선형 또는 비선형의 C1 내지 C20의 알코올기, 선형 또는 비선형의 C1 내지 C20의 알킬기를 갖는 에스테르기, C5 내지 C24의 치환 또는 비치환된 아릴기, C6 내지 C24의 아릴알킬기 또는 비선형의 C4 내지 C18의 알킬기를 갖는 아실기 그룹중 선택된 어느하나와 결합된 Biomarker일 수 있으며, 바람직하게는 (CH2)2O(CH2)2OAcGlu, (CH2)2O(CH2)2OGlu, (CH2)2O(CH2)2PPh3, Glucose, Galactose, 트라이페닐포스핀 (TPP, mitochondria marker), 모르폴린 (Morpholine, endosome marker), 엽산 (Folic acid), 바이오틴 (Biotin) 중 선택된 어느 하나 일 수 있다.In the above [Formula 1], BM (Biomarker) is a linear or nonlinear C1 to C20 alkyl group, a ketone group having a linear or nonlinear C1 to C20 alkyl group, a linear or nonlinear C1 to C20 alcohol group, linear or nonlinear Biomarker bonded to any one selected from an ester group having a C1 to C20 alkyl group, a C5 to C24 substituted or unsubstituted aryl group, a C6 to C24 arylalkyl group, or an acyl group having a nonlinear C4 to C18 alkyl group may be, preferably (CH 2 ) 2 O(CH 2 ) 2 OAcGlu, (CH 2 ) 2 O(CH 2 ) 2 OGlu, (CH 2 ) 2 O(CH 2 ) 2 PPh 3 , Glucose, Galactose , triphenylphosphine (TPP, mitochondria marker), morpholine (Morpholine, endosome marker), folic acid (Folic acid), may be any one selected from biotin (Biotin).

본 발명의 다른 한 일형태는 광활성 형광체 프로브 제조방법을 제공한다.Another aspect of the present invention provides a method for manufacturing a photoactive phosphor probe.

상기 광활성 형광체 프로브 제조방법은 (a) 극성 용매 하에서, 다이에틸 아조다이카복실레이트 (Diethyl azodicarboxylate, DEAD)와 트라이페닐포스핀 (Triphenylphosphine, PPh3), 인돌륨 (1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indol-1-ium bromide) 으로 하기 [화학식2]로 표시되는 화합물을 제조하는 단계, (b) 극성 용매 하에서, 4-나이트로페닐 클로로포메이트 (4-Nitrophenyl Chloroformate)와 N,N-다이아이소프로필에틸아민 (N,N-Diisopropylethylamine, DIPEA)에 독소루비신 염산염 (Doxorubicin hydrochloride, Dox-HCl) 혹은 파수딜 (Fasudil, Rho-kinase 억제제) 혹은 Y-27632 (lac 억제제)으로 하기 [화학식3] 내지 [화학식5]로 표시되는 화합물을 제조하는 단계를 포함하여 [화학식1]로 표시되는 화합물을 제조하는 단계를 포함할 수 있다.The method for preparing the photoactive fluorescent probe is (a) in a polar solvent, diethyl azodicarboxylate (DEAD) and triphenylphosphine (PPh 3 ), indolium (1-(2-hydroxyethyl)-2 ,3,3-trimethyl-3H-indol-1-ium bromide) to prepare a compound represented by the following [Formula 2], (b) under a polar solvent, 4-nitrophenyl chloroformate (4-Nitrophenyl Chloroformate) and N,N-Diisopropylethylamine (DIPEA) with doxorubicin hydrochloride (Dox-HCl) or Fasudil (Rho-kinase inhibitor) or Y-27632 (lac inhibitor) ) to the following [Formula 3] to [Formula 5], including the step of preparing a compound represented by [Formula 1] may include a step of preparing a compound represented by [Formula 1].

[화학식 2][Formula 2]

Figure pat00006
Figure pat00006

[화학식 3][Formula 3]

Figure pat00007
Figure pat00007

[화학식 4][Formula 4]

Figure pat00008
Figure pat00008

[화학식 5][Formula 5]

Figure pat00009
Figure pat00009

상기와 같은 본 발명에 따르면, 생체 외(ex vivo) 혹은 생체 내(in vivo)에서 빛에 의해 작동하는 광활성 형광체 프로브를 활용함으로써, 바이오마커를 통한 암세포의 검출, 약물의 방출로 암세포의 치료 등에 적용할 수 있다. 기존의 화학요법의 단점을 극복하여 시공간적(spatiotemporal)으로 조작이 가능하기 때문에 선택적 암 진단 및 치료용 탐침으로서 유용하게 사용될 수 있는 효과를 기대 할 수 있다.According to the present invention as described above, by utilizing a photoactive fluorescent probe operated by light in ex vivo or in vivo, detection of cancer cells through biomarkers, treatment of cancer cells by release of drugs, etc. can be applied. Since it can be manipulated spatiotemporally by overcoming the disadvantages of conventional chemotherapy, it can be expected to be useful as a probe for selective cancer diagnosis and treatment.

도 1은 실시예 1 내지 실시예 4에 빛을 조사하여 주었을 때의 구조 변화에 따른 UV, 형광 데이터 변화를 분석한 결과로, 도 1의 A와 B는 실시예 3 화합물에 740 nm의 빛을 조사해 주었을 때 구조변화에 따른 UV-Vis 스펙트럼과 형광 스펙트럼에서 관찰되는 데이터 변화를 분석한 결과이며, 도 1의 C는 실시예 2 화합물에 740 nm의 빛을 조사해 주었을 때 구조변화에 따른 형광 스펙트럼에서 관찰되는 데이터 변화를 분석한 결과이고, 도 1의 D는 실시예 1 화합물에 740 nm의 빛을 조사해 주었을 때 구조변화에 따른 형광 스펙트럼에서 관찰되는 데이터 변화를 분석한 결과입니다. 또한 도 1의 E는 실시예 4 화합물에 365 nm의 빛을 조사해 주었을 때 구조변화에 따른 UV-Vis 스펙트럼에서 관찰되는 데이터 변화를 분석한 결과이다.
도 2은 실시예 1 화합물의 헬라 세포에서 마이토콘드리아 표시자인 Rhodamine 123 과의 표지 실험 및 빛의 조사에 따른 세포 사진을 공초점 레이저현미경으로 얻은 것이다.
도 3은 실시예 1 화합물의 헬라 세포에서 빛을 조사하여 주었을 때 혹은 조사하지 않은 대조군의 사진을 공초점 레이저현미경으로 얻은 것이다.
도 4은 실시예 1 화합물의 헬라 세포 내의 마이토콘드리아의 활성을 통해 빛을 조사하여 주었을 때 혹은 조사하지 않은 대조군에서 간접적으로 측정한 결과이다.
도 5는 실시예 2 화합물의 SCC7 세포 내의 마이토콘드리아의 활성을 통해 빛을 조사하여 주었을 때 혹은 조사하지 않은 대조군에서 간접적으로 측정한 결과이다.1 is a result of analyzing UV and fluorescence data changes according to structural changes when irradiated with light in Examples 1 to 4, A and B of FIG. 1 are 740 nm light to the compound of Example 3 It is the result of analyzing the data change observed in the UV-Vis spectrum and the fluorescence spectrum according to the structural change when irradiated, and FIG. It is the result of analyzing the observed data change, and Fig. 1D is the result of analyzing the data change observed in the fluorescence spectrum according to the structural change when the Example 1 compound is irradiated with light of 740 nm. In addition, FIG. 1E is a result of analyzing the data change observed in the UV-Vis spectrum according to the structural change when the Example 4 compound was irradiated with 365 nm light.
FIG. 2 is a confocal laser microscope of the cell of Example 1 according to the labeling experiment with Rhodamine 123, a mitochondrial marker, and light irradiation in HeLa cells of the compound of Example 1. FIG.
3 is a picture of a control group that is not irradiated with or when light is irradiated from the HeLa cells of the compound of Example 1 by a confocal laser microscope.
4 is a result of indirect measurement in a control group that is not irradiated or when irradiated with light through mitochondrial activity in HeLa cells of the compound of Example 1;
5 is a result of indirect measurement in a control group that is not irradiated or when irradiated with light through the activity of mitochondria in SCC7 cells of the compound of Example 2;

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 하기 [화학식 1]로 표시되는 OPA 혹은 TPA 의 구조체를 활용한 광활성 형광체 프로브에 관한 내용이다.The present invention relates to a photoactive phosphor probe using a structure of OPA or TPA represented by the following [Formula 1].

[화학식 1][Formula 1]

Figure pat00010
Figure pat00010

상기 [화학식1]에서, D (Drug)는 카바메이트 또는 카보네이트 또는 벤질 이써 형태로 연결된 다양한 antibiotics 및 inhibitors (억제제) 등을 포함하는 그룹일 수 있으며, 바람직하게는 [화학식 2] 또는 [화학식 3]과 같은 독소루비신 (Doxorubicin) 또는 [화학식 4]과 같은 파수딜 (Fasudil, Rho-kinase 억제제) 또는 [화학식 5]과 같은 Y-27632 (lac 억제제) 또는 캄토테신 (Camptothecin, CPT)일 수 있다. In the [Formula 1], D (Drug) may be a group containing various antibiotics and inhibitors (inhibitors), etc. linked in the form of carbamate or carbonate or benzyl ether, preferably [Formula 2] or [Formula 3] Such as doxorubicin (Doxorubicin) or [Formula 4] such as Fasudil (Fasudil, Rho-kinase inhibitor) or [Formula 5] It may be Y-27632 (lac inhibitor) or camptothecin (Camptothecin, CPT).

상기 [화학식1]에서, BM (Biomarker)은 선형 또는 비선형의 C1 내지 C20의 알킬기, 선형 또는 비선형의 C1 내지 C20의 알킬기를 갖는 케톤기, 선형 또는 비선형의 C1 내지 C20의 알코올기, 선형 또는 비선형의 C1 내지 C20의 알킬기를 갖는 에스테르기,C5 내지 C24의 치환 또는 비치환된 아릴기, C6 내지 C24의 아릴알킬기 또는 비선형의 C4 내지 C18의 알킬기를 갖는 아실기 그룹과 결합된 다양한 생체 내 마커를 포함하는 그룹일 수 있으며, 바람직하게는 (CH2)2O(CH2)2OAcGlu 또는 (CH2)2O(CH2)2OGlu 또는 (CH2)2O(CH2)2PPh3 또는 Glucose 또는 Galactose 또는 트라이페닐포스핀 (TPP, mitochondria marker) 또는 모르폴린 (Morpholine, endosome marker) 또는 엽산 (Folic acid) 또는 바이오틴 (Biotin) 일 수 있다.In the above [Formula 1], BM (Biomarker) is a linear or nonlinear C1 to C20 alkyl group, a ketone group having a linear or nonlinear C1 to C20 alkyl group, a linear or nonlinear C1 to C20 alcohol group, linear or nonlinear of C1 to C20 ester group having an alkyl group, C5 to C24 substituted or unsubstituted aryl group, C6 to C24 arylalkyl group, or nonlinear C4 to C18 acyl group having an acyl group combined with various in vivo markers may be a group comprising, preferably (CH 2 ) 2 O(CH 2 ) 2 OAcGlu or (CH 2 ) 2 O(CH 2 ) 2 OGlu or (CH 2 ) 2 O(CH 2 ) 2 PPh 3 or It may be glucose or galactose or triphenylphosphine (TPP, mitochondria marker) or morpholine (Morpholine, endosome marker) or folic acid or biotin.

[화학식 2][Formula 2]

Figure pat00011
Figure pat00011

[화학식 3][Formula 3]

Figure pat00012
Figure pat00012

[화학식 4][Formula 4]

Figure pat00013
Figure pat00013

[화학식 5][Formula 5]

Figure pat00014
Figure pat00014

상기 [화학식 1]은 (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-다이메틸-2,3,9,9a-테트라하이드로옥사졸[3,2-a]인돌-9a-일)바이닐)페녹시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에톡시)-6-(하이드록시메틸)테트라하이드로-2H-파이렌-3,4,5-트라이올, 5-메톡시-2-나이트로-4-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-트라이하이드록시-6-(하이드록시메틸)테트라하이드로-2H-파이렌-2-일)옥시)에톡시)에톡시)벤질, ((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바메이트, (2-(2-(4-(((((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바모일)옥시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에틸)트라이페닐포스포늄 브로마이드, 4,5-다이메톡시-2-나이트로벤질 4-(아이소퀴놀린-5-일설포닐)-1,4-다이아제페인-1-카복실레이트, 및 4,5-다이메톡시-2-나이트로벤질 ((R)-1-((1r,4R)-1-메틸-4-(피리딘-4-일카바모일)싸이클로헥실)에틸)카바메이트로 이루어진 군에서 선택된 어느 하나일 수 있다.The [Formula 1] is (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-dimethyl-2, 3,9,9a-tetrahydrooxazole [3,2-a] indol-9a-yl) vinyl) phenoxy) methyl) -2-methoxy-5-nitrophenoxy) ethoxy) ethoxy) - 6-(Hydroxymethyl)tetrahydro-2H-pyrene-3,4,5-triol, 5-methoxy-2-nitro-4-(2-(2-(((2R,3R,4S) ,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyren-2-yl)oxy)ethoxy)ethoxy)benzyl, ((2S,3S ,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy- 6,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyren-4-yl)carbamate, (2-(2) -(4-(((((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-( 2-Hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyrene- 4-yl)carbamoyl)oxy)methyl)-2-methoxy-5-nitrophenoxy)ethoxy)ethyl)triphenylphosphonium bromide, 4,5-dimethoxy-2-nitrobenzyl 4- (isoquinolin-5-ylsulfonyl)-1,4-diazepain-1-carboxylate, and 4,5-dimethoxy-2-nitrobenzyl ((R)-1-((1r,4R) It may be any one selected from the group consisting of -1-methyl-4-(pyridin-4-ylcarbamoyl)cyclohexyl)ethyl)carbamate.

상기 [화학식1]로 표시되는 화합물은 빛에 의해 노리쉬 타입 2 (Norrish type 2)인 하기 [반응식1]의 과정으로 구조적 변화를 거쳐 형광체(약물)가 방출되어 UV-Vis 혹은 형광 스펙트럼의 비율척도 (ratiometric) 혹은 턴 온 (Turn-on) 변화할 수 있다.The compound represented by [Formula 1] undergoes structural change in the process of [Scheme 1], which is Norrish type 2, by light, and a phosphor (drug) is emitted. UV-Vis or fluorescence spectrum ratiometric or turn-on can be changed.

상기 [화학식2]로 표시되는 화합물은 [반응식 2] 과정으로 구조적 변화를 거쳐 형광체(약물)가 방출되어 UV 혹은 형광이 비율척도 (ratiometric) 혹은 턴 온 (Turn-on) 변화할 수 있다. 도 1의 A에서 740 nm의 빛을 조사했을 때 419 nm의 흡수 파장이 감소하면서 515 nm의 흡수 파장이 증가하는 변화가 관찰된다. 419nm 의 흡수 파장 대비 515 nm의 흡수 파장의 시간에 따른 흡수량이 비율척도적으로 증가한다. 도 1의 B에서 740 nm의 빛을 조사했을 때 560 nm의 방출 파장을 보이며 기존 형광 대비 약 10배 이상의 턴온 (Turn-on) 변화가 관찰된다.The compound represented by [Formula 2] undergoes a structural change in the process of [Scheme 2] to release a phosphor (drug) UV or fluorescence can be changed ratiometrically or turn-on. In FIG. 1A , when light of 740 nm is irradiated, a change in which the absorption wavelength of 515 nm increases while the absorption wavelength of 419 nm decreases is observed. Compared to the absorption wavelength of 419 nm, the absorption amount of the absorption wavelength of 515 nm increases proportionally with time. In FIG. 1B , when light of 740 nm is irradiated, an emission wavelength of 560 nm is exhibited, and a turn-on change of about 10 times or more is observed compared to conventional fluorescence.

상기 [화학식3] 내지 [화학식5]로 표시되는 화합물은 [반응식 3]의 과정을 통해 형광체(약물)가 방출되어 UV 혹은 형광이 비율척도 (ratiometric) 혹은 턴 온 (Turn-on) 변화할 수 있다. 도 1의 C 와 D에서 370 nm 의 빛을 조사했을 때 595 nm의 방출 파장 증가하는 턴온 (Turn-on) 변화가 관찰된다. 도 1의 E에서 365 nm 의 빛을 조사했을 때 355nm 의 흡수 파장 대비 264 nm의 흡수 파장의 시간에 따른 흡수량이 비율척도적으로 증가한다.The compound represented by the [Formula 3] to [Formula 5] is a phosphor (drug) is emitted through the process of [Scheme 3] UV or fluorescence can be changed ratiometrically or turn-on. When irradiated with light of 370 nm in C and D of FIG. 1, a turn-on change in which the emission wavelength of 595 nm increases is observed. In FIG. 1E , when light of 365 nm is irradiated, the absorption amount of the absorption wavelength of 264 nm compared to the absorption wavelength of 355 nm increases proportionally with time.

[반응식1][Scheme 1]

Figure pat00015
Figure pat00015

[반응식2] [Scheme 2]

Figure pat00016
Figure pat00016

[반응식3][Scheme 3]

Figure pat00017
Figure pat00017

본 발명의 화학식 1 내지 5의 화합물은 각각 상기 [반응식1] 내지 [반응식3]을 거쳐 나이트로 작용기가 활성화되고, 연쇄반응으로 벤질 이써 (ether) 혹은 카바메이트 (carbamate) 혹은 카보네이트 (carbonate) 작용기가 끊어져 일어난 구조 변화에 의해 턴 온 되는 형광 변화를 유도하게 된다.In the compounds of Chemical Formulas 1 to 5 of the present invention, a nitro functional group is activated through the [Scheme 1] to [Scheme 3], respectively, and a benzyl ether or carbamate or carbonate functional group through a chain reaction It induces a change in fluorescence that is turned on by the structural change caused by the breakage.

본 발명은 하기와 같은 광활성 형광체 화합물의 제조방법을 제공한다.The present invention provides a method for preparing a photoactive phosphor compound as follows.

(a) 극성 용매 하에서, 다이에틸 아조다이카복실레이트 (Diethyl azodicarboxylate, DEAD)와 트라이페닐포스핀 (Triphenylphosphine, PPh3), 인돌륨 (1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indol-1-ium bromide) 으로 하기 [화학식2]로 표시되는 화합물을 제조하는 단계 및(a) under a polar solvent, diethyl azodicarboxylate (DEAD) and triphenylphosphine (PPh 3 ), indolium (1- (2-hydroxyethyl) -2,3,3-trimethyl- 3H-indol-1-ium bromide) to prepare a compound represented by the following [Formula 2] and

(b) 극성 용매 하에서, 4-나이트로페닐 클로로포메이트 (4-Nitrophenyl Chloroformate)와 N,N-다이아이소프로필에틸아민 (N,N-Diisopropylethylamine, DIPEA)에 독소루비신 염산염 (Doxorubicin hydrochloride, Dox-HCl) 혹은 파수딜 (Fasudil, Rho-kinase 억제제) 혹은 Y-27632 (lac 억제제)으로 하기 [화학식3] 내지 [화학식5]로 표시되는 화합물을 제조하는 단계를 포함하여 [화학식1]로 표시되는 화합물을 제조하는 단계를 포함할 수 있다. (b) Doxorubicin hydrochloride, Dox-HCl in 4-Nitrophenyl Chloroformate and N,N-Diisopropylethylamine (DIPEA) under a polar solvent ) or Fasudil (Fasudil, Rho-kinase inhibitor) or Y-27632 (lac inhibitor) as a compound represented by [Formula 1], including the step of preparing a compound represented by the following [Formula 3] to [Formula 5] It may include the step of manufacturing.

[화학식 2][Formula 2]

Figure pat00018
Figure pat00018

[화학식 3][Formula 3]

Figure pat00019
Figure pat00019

[화학식 4][Formula 4]

Figure pat00020
Figure pat00020

[화학식 5][Formula 5]

Figure pat00021
Figure pat00021

상기 [화학식3] 으로 표시되는 화합물은 항생제로 사용되는 독소로비신 (Doxorubicin) 을 포함하는 구조로 [반응식3]에 의해 독소로비신으로 배출되어 암 세포를 죽이는 약물 (antibiotics) 일 수 있다. 상기 [화학식4] 내지 [화학식 5]로 표시되는 화합물은 억제제로 사용되는 파수딜 (Fasudil, Rho-kinase 억제제) 혹은 Y-27632 (lac 억제제)를 포함하는 구조로 [반응식3]에 의해 억제제가 배출되어 억제제 (inhibitor) 일 수 있다. The compound represented by [Formula 3] has a structure containing doxorubicin used as an antibiotic, and is discharged as doxorubicin by [reaction formula 3] to kill cancer cells. It may be a drug (antibiotics). The compounds represented by the [Formula 4] to [Formula 5] have a structure containing Fasudil (Rho-kinase inhibitor) or Y-27632 (lac inhibitor) used as an inhibitor, and the inhibitor is It may be excreted and may be an inhibitor.

상기 극성용매는 아세토나이트릴 (acetonitrile), 클로로포름, 테트라하이드로퓨란 (Tetrahydrofuran, THF), 디클로로메탄 (Dichloromethane), 다이메틸포름아마이드 (Dimethylformamide, DMF) 아이소프로필 알코올, 메탄올 및 에탄올로 이루어진 군에서 선택된 하나 이상일 수 있다. The polar solvent is one selected from the group consisting of acetonitrile, chloroform, tetrahydrofuran (Tetrahydrofuran, THF), dichloromethane, dimethylformamide (DMF) isopropyl alcohol, methanol and ethanol may be more than

본 발명은 상기한 광활성 형광체 화합물을 이용한 암세포 검출방법을 제공한다.The present invention provides a method for detecting cancer cells using the photoactive fluorescent compound described above.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

실시예 1. (2-(2-(4-(((((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바모일)옥시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에틸)트라이페닐포스포늄 브로마이드의 제조Example 1. (2-(2-(4-(((((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5, 12-Trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy Preparation of )tetrahydro-2H-pyren-4-yl)carbamoyl)oxy)methyl)-2-methoxy-5-nitrophenoxy)ethoxy)ethyl)triphenylphosphonium bromide

Figure pat00022
Figure pat00022

둥근바닥 플라스크에서 중간화합물 (2-(2-(4-(((((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바모일)옥시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에틸 46 mg, 트라이페닐포스핀 39 mg, 소듐아이오다이드 0.8 mg 을 아세토나이트릴 2 mL와 에틸아세테이트 1 mL를 가하여 상온에서 용해 시키고 85 oC 에서 24 시간 반응시켰다. TLC로 출발물질이 사라지는 것을 확인한 후 감압 농축하였다. 얻어진 농축물을 컬럼 크로마토그래피(용리액: 디클로로메탄/메탄올 = 10/1, v/v)로 정제하여 (2-(2-(4-(((((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바모일)옥시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에틸)트라이페닐포스포늄 브로마이드를 제조하였다. Intermediate compound (2-(2-(4-(((((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3, 5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl )oxy)tetrahydro-2H-pyren-4-yl)carbamoyl)oxy)methyl)-2-methoxy-5-nitrophenoxy)ethoxy)ethyl 46 mg, triphenylphosphine 39 mg, sodium 0.8 mg of iodide was added with 2 mL of acetonitrile and 1 mL of ethyl acetate, dissolved at room temperature, and reacted for 24 hours at 85 ° C. After confirming that the starting material disappeared by TLC, the mixture was concentrated under reduced pressure. Purification by graph (eluent: dichloromethane/methanol = 10/1, v/v) (2-(2-(4-((((2S,3S,4S,6R)-3-hydroxy-2- methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3, 4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyren-4-yl)carbamoyl)oxy)methyl)-2-methoxy-5-nitrophenoxy) Toxy) ethyl) triphenylphosphonium bromide was prepared.

수율: 67 %Yield: 67%

1H NMR ((CD3)2SO, 400MHz): δ 14.05 (s, 1H), 13.27 (s, 1H), 7.91 (d, J = 4.3 Hz, 3H), 7.85 - 7.72 (m, 12H), 7.68-7.61 (m, 9H), 7.47 (s, 1H), 7.24 (d, J = 8.1 Hz, 1H), 7.19 (s, 1H), 5.46 (s, 1H), 5.31 (d, J = 9.9 Hz, 2H), 5.26 (s, 1H), 4.96 (t, J = 4.1 Hz1H), 4.87 (t, J = 5.9 Hz, 1H), 4.80 (d, J = 6.1 Hz, 1H), 4.57 (d, J = 5.9 Hz, 2H), 4.01 - 3.79 (m, 13H), 3.78 - 3.66 (m, 3H), 3.47 (d, J = 29.5 Hz, 5H), 2.97 (d, J = 5.0 Hz, 2H), 2.25 - 2.07 (m, 2H), 1.89 (td, J = 9.6, J = 2.5 Hz, 1H), 1.52 (d, J = 8.0 Hz, 1H), 1.14 (d, J = 6.5 Hz, 4H). 1 H NMR ((CD 3 ) 2 SO, 400 MHz): δ 14.05 (s, 1H), 13.27 (s, 1H), 7.91 (d, J = 4.3 Hz, 3H), 7.85 - 7.72 (m, 12H), 7.68-7.61 (m, 9H), 7.47 (s, 1H), 7.24 (d, J = 8.1 Hz, 1H), 7.19 (s, 1H), 5.46 (s, 1H), 5.31 (d, J = 9.9 Hz) , 2H), 5.26 (s, 1H), 4.96 (t, J = 4.1 Hz1H), 4.87 (t, J = 5.9 Hz, 1H), 4.80 (d, J = 6.1 Hz, 1H), 4.57 (d, J) = 5.9 Hz, 2H), 4.01 - 3.79 (m, 13H), 3.78 - 3.66 (m, 3H), 3.47 (d, J = 29.5 Hz, 5H), 2.97 (d, J = 5.0 Hz, 2H), 2.25 - 2.07 (m, 2H), 1.89 (td, J = 9.6, J = 2.5 Hz, 1H), 1.52 (d, J = 8.0 Hz, 1H), 1.14 (d, J = 6.5 Hz, 4H).

13C NMR ((CD3)2SO, 100 MHz): δ 214.3, 186.8, 186.7, 161.1,156.5, 155.4, 154.9, 153.7, 146.9, 139.2, 136.6, 135.9, 134.9, 134.5, 134.1, 130.2, 130.1, 128.7, 120.2, 120.1, 119.9, 119.0, 111.1, 110.1, 108.9, 100.9, 100.6, 75.4, 70.2, 69.0, 64.2, 62.7, 57.2, 56.8, 56.5, 47.9, 47.6, 37.0, 32.6, 30.5, 30.3, 22.9, 17.6, 17.4. 13 C NMR ((CD 3 ) 2 SO, 100 MHz): δ 214.3, 186.8, 186.7, 161.1,156.5, 155.4, 154.9, 153.7, 146.9, 139.2, 136.6, 135.9, 134.9, 134.5, 134.1, 130.2, 130.1, 128.7, 120.2, 120.1, 119.9, 119.0, 111.1, 110.1, 108.9, 100.9, 100.6, 75.4, 70.2, 69.0, 64.2, 62.7, 57.2, 56.8, 56.5, 47.9, 47.6, 37.0, 32.6, 30.5, 30.3, 22.9, 17.6, 17.4.

MS (FAB+,DMSO and Glycerol) : calcd. For [M]+ 1101.3422 found 1101.3428.MS (FAB+, DMSO and Glycerol): calcd. For [M] + 1101.3422 found 1101.3428.

실시예 2. 5-메톡시-2-나이트로-4-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-트라이하이드록시-6-(하이드록시메틸)테트라하이드로-2H-파이렌-2-일)옥시)에톡시)에톡시)벤질 ((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바메이트의 제조Example 2. 5-Methoxy-2-nitro-4-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydro Roxymethyl)tetrahydro-2H-pyren-2-yl)oxy)ethoxy)ethoxy)benzyl ((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S) ,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydro Preparation of tetrasen-1-yl)oxy)tetrahydro-2H-pyren-4-yl)carbamate

Figure pat00023
Figure pat00023

둥근바닥 플라스크에 중간화합물 (2R,3R,4S,5R,6R)-2-(아세톡시메틸)-6-(2-(2-(4-(((((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바모일)옥시)메틸)-2-메톡시-5-나이트로펜옥시)에톡시)에톡시)테트라하이드로-2H-파이렌-3,4,5-트라일트라이아세테이트 119 mg을 넣고 디클로로메탄/메탄올 (1:4) 15 mL에 용해시킨뒤 소듐메톡사이드 16 mg을 상온에서 넣어준 뒤 2시간 동안 반응시켰다. 출발물질이 사라지는 것을 TLC로 확인 후 감압 농축하였다. 얻어진 농축물을 컬럼 크로마토그래피(용리액: 다이클로로메테인/메탄올= 10/1.5, v/v)로 정제하여 5-메톡시-2-나이트로-4-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-트라이하이드록시-6-(하이드록시메틸)테트라하이드로-2H-파이렌-2-일)옥시)에톡시)에톡시) 벤질((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바메이트을 제조하였다.Intermediate compound (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(4-(((((2S,3S,4S,6R) -3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-di Oxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyren-4-yl)carbamoyl)oxy)methyl)-2-methoxy- After adding 119 mg of 5-nitrophenoxy) ethoxy) ethoxy) tetrahydro-2H-pyrene-3,4,5-triyltriacetate and dissolving it in 15 mL of dichloromethane/methanol (1:4) After adding 16 mg of sodium methoxide at room temperature, it was reacted for 2 hours. The disappearance of the starting material was confirmed by TLC, and then concentrated under reduced pressure. The resulting concentrate was purified by column chromatography (eluent: dichloromethane/methanol = 10/1.5, v/v) to 5-methoxy-2-nitro-4-(2-(2-(((2R) ,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyren-2-yl)oxy)ethoxy)ethoxy)benzyl(( 2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10- Methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyren-4-yl)carbamate was prepared.

수율: 40 %Yield: 40%

1H NMR ((CD3)2SO, 400 MHz): δ 14.03 (s, 1H), 13.26 (s, 1H), 7.91 (s, 1H), 7.90 (s, 1H), 7.70 (s, 1H), 7.65 (t, J = 4.8 Hz, 1H), 7.24-7.20 (m, 2H), 5.46 (s, 1H), 5.28 (d, J = 4.3 Hz, 2H), 5.24 (d, J = 2.4 Hz, 1H), 4.98 (d, J = 4.9 Hz, 1H), 4.95 (t, J = 4.8 Hz, 1H), 4.92 (d, J = 4.7 Hz, 1H), 4.89 (d, J = 4.9 Hz, 1H), 4.85 (t, J = 5.9 Hz, 1H), 4.79 (d, J = 5.8 Hz, 1H), 4.58 (d, J = 5.9 Hz, 2H), 4.49 (t, J = 5.9 Hz, 1H), 4.21-4.14 (m, 4H), 3.99 (s, 3H), 3.95-3.85 (m, 4H), 3.80 - 3.70 (m, 3H), 3.69 - 3.56 (m, 5H), 3.48 - 3.40 (m, 2H), 3.15 - 2.90 (m, 6H), 2.25 - 2.09 (m, 2H), 1.89 (td J = 11.2, 5 Hz, 1H), 1.51 (dd, J = 8.3, 4 Hz, 1H), 1.14 (d, J = 6.4 Hz, 3H). 1 H NMR ((CD 3 ) 2 SO, 400 MHz): δ 14.03 (s, 1H), 13.26 (s, 1H), 7.91 (s, 1H), 7.90 (s, 1H), 7.70 (s, 1H) , 7.65 (t, J = 4.8 Hz, 1H), 7.24-7.20 (m, 2H), 5.46 (s, 1H), 5.28 (d, J = 4.3 Hz, 2H), 5.24 (d, J = 2.4 Hz, 1H), 4.98 (d, J = 4.9 Hz, 1H), 4.95 (t, J = 4.8 Hz, 1H), 4.92 (d, J = 4.7 Hz, 1H), 4.89 (d, J = 4.9 Hz, 1H) , 4.85 (t, J = 5.9 Hz, 1H), 4.79 (d, J = 5.8 Hz, 1H), 4.58 (d, J = 5.9 Hz, 2H), 4.49 (t, J = 5.9 Hz, 1H), 4.21 -4.14 (m, 4H), 3.99 (s, 3H), 3.95-3.85 (m, 4H), 3.80 - 3.70 (m, 3H), 3.69 - 3.56 (m, 5H), 3.48 - 3.40 (m, 2H) , 3.15 - 2.90 (m, 6H), 2.25 - 2.09 (m, 2H), 1.89 (td J = 11.2, 5 Hz, 1H), 1.51 (dd, J = 8.3, 4 Hz, 1H), 1.14 (d, J = 6.4 Hz, 3H).

13C NMR ((CD3)2SO, 100 MHz): δ 214.3, 186.8, 186.7, 161.2, 156.5, 155.4, 154.9, 154.0, 147.2, 139.4, 136.6, 135.9, 135.0, 134.5, 128.6, 120.3, 120.1, 119.4, 111.1, 111.00, 110.98, 109.6, 103.4, 100.8, 77.3, 77.2, 75.4, 73.8, 70.5, 70.26, 70.25, 69.1, 68.9, 68.5, 68.3, 67.2, 64.2, 62.7, 61.5, 57.0, 56.8, 47.6, 37.0, 32.5, 30.3, 17.5. 13 C NMR ((CD 3 ) 2 SO, 100 MHz): δ 214.3, 186.8, 186.7, 161.2, 156.5, 155.4, 154.9, 154.0, 147.2, 139.4, 136.6, 135.9, 135.0, 134.5, 128.6, 120.3, 120.1, 119.4, 111.1, 111.00, 110.98, 109.6, 103.4, 100.8, 77.3, 77.2, 75.4, 73.8, 70.5, 70.26, 70.25, 69.1, 68.9, 68.5, 68.3, 67.2, 64.2, 62.7, 61.5, 57.0, 56.8, 47.6, 37.0, 32.5, 30.3, 17.5.

MS (FAB+,m-NBA and Glycerol) : calcd. For [M]- 1018.3067 found 1018.3068.MS (FAB+,m-NBA and Glycerol): calcd. For [M] - 1018.3067 found 1018.3068.

실시예 3. (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-다이메틸-2,3,9,9a-테트라하이드로옥사졸[3,2-a]인돌-9a-일)바이닐)페녹시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에톡시)-6-(하이드록시메틸)테트라하이드로-2H-파이렌-3,4,5-트라이올의 제조Example 3. (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-dimethyl-2,3, 9,9a-tetrahydrooxazole[3,2-a]indol-9a-yl)vinyl)phenoxy)methyl)-2-methoxy-5-nitrophenoxy)ethoxy)ethoxy)-6- Preparation of (hydroxymethyl)tetrahydro-2H-pyrene-3,4,5-triol

Figure pat00024
Figure pat00024

둥근바닥 플라스크에 중간화합물 (2R,3R,4S,5R,6R)-2-(아세토메틸)-6-(2-(2-(4-((4-((E)-2-(9,9-다이메틸-2,3,9,9a-테트라하이드로옥사졸[3,2-a]인돌-9a-릴)바이닐)펜옥시)메틸)-2-메톡시-5-나이트로펜옥시)에톡시)에톡시)테트라하이드로-2H-파이렌-3,4,5-트라일 트라이아세테이트 23 mg을 넣고 메탄올 lmL에 용해시킨뒤 물에 용해시킨 소듐메톡사이드 7.5 μL를 상온에서 넣어준 뒤 20 분 동안 반응시켰다. TLC로 확인 결과 출발물질이 사라졌을 때 감압 농축하였다. 얻어진 농축물을 컬럼 크로마토그래피(용리액: 다이클로로메테인/메탄올= 10/1, v/v)로 정제하여 (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-다이메틸-2,3,9,9a-테트라하이드로옥사졸[3,2-a]인돌-9a-일)바이닐)페녹시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에톡시)-6-(하이드록시메틸)테트라하이드로-2H-파이렌-3,4,5-트라이올을 제조하였다. Intermediate compound (2R,3R,4S,5R,6R)-2-(acetomethyl)-6-(2-(2-(4-((4-((E)-2-(9, 9-dimethyl-2,3,9,9a-tetrahydrooxazole [3,2-a] indole-9a-yl) vinyl) phenoxy) methyl) -2-methoxy-5-nitrophenoxy) Ethoxy)ethoxy)tetrahydro-2H-pyrene-3,4,5-triacetate 23 mg, dissolved in 1mL of methanol, 7.5 μL of sodium methoxide dissolved in water at room temperature, 20 reacted for a minute. When the starting material disappeared as a result of TLC, it was concentrated under reduced pressure. The resulting concentrate was purified by column chromatography (eluent: dichloromethane/methanol = 10/1, v/v) to (2R,3R,4S,5S,6R)-2-(2-(2-(4) -((4-((E)-2-(9,9-dimethyl-2,3,9,9a-tetrahydrooxazole[3,2-a]indol-9a-yl)vinyl)phenoxy) Methyl)-2-methoxy-5-nitrophenoxy)ethoxy)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyrene-3,4,5-triol was prepared.

수율: 87 %Yield: 87%

1H NMR (CD3OD, 400MHz): δ 7.83 (s, 1H), 7.43 (d, J = 8.8, 2H), 7.35 (s, 1H), 7.15 (td, J = 7.6, 1.2, 1H), 7.09 (dd, J = 7.6, 1.2, 1H), 7.01 (d, J = 8.8, 2H), 6.96 (td, J = 7.2, 0.8, 1H), 6.87-6.81 (m, 2H), 6.19 (d, J = 16.0, 1H), 5.46 (s, 2H), 4.33 (d, J = 7.6, 1H), 4.25 (t, J = 4.4, 2H), 4.07-4.04 (m, 1H), 3.94-3.91 (m, 5H), 3.88 (dd, J = 11.6, 2.0, 1H), 3.82-3.73 (m, 5H), 3.70-3.65 (m 1H), 3.56 (q, J = 7.6, 1H), 3.47-3.36 (m, 2H), 3.30-3.27 (m, 2H), 3.21 (t, J = 8.8, 1H), 1.42 (s, 3H), 1.15 (s, 3H). 1 H NMR (CD 3 OD, 400 MHz): δ 7.83 (s, 1H), 7.43 (d, J = 8.8, 2H), 7.35 (s, 1H), 7.15 (td, J = 7.6, 1.2, 1H), 7.09 (dd, J = 7.6, 1.2, 1H), 7.01 (d, J = 8.8, 2H), 6.96 (td, J = 7.2, 0.8, 1H), 6.87-6.81 (m, 2H), 6.19 (d, J = 16.0, 1H), 5.46 (s, 2H), 4.33 (d, J = 7.6, 1H), 4.25 (t, J = 4.4, 2H), 4.07-4.04 (m, 1H), 3.94-3.91 (m , 5H), 3.88 (dd, J = 11.6, 2.0, 1H), 3.82-3.73 (m, 5H), 3.70-3.65 (m 1H), 3.56 (q, J = 7.6, 1H), 3.47-3.36 (m) , 2H), 3.30-3.27 (m, 2H), 3.21 (t, J = 8.8, 1H), 1.42 (s, 3H), 1.15 (s, 3H).

13C NMR (CD3OD, 100 MHz): δ 158.11, 154.24, 150.46, 147.26, 139.72, 139.25, 131.66, 129.96, 128.83, 128.74, 127.80, 127.21, 123.66, 121.93, 121.49, 114.74, 114.32, 111.91, 110.04, 109.88, 109.73, 103.06, 76.56, 73.70, 70.34, 70.22, 69.20, 68.80, 68.36, 66.79, 63.14, 61.40, 55.49, 49.57, 48.47, 27.47, 19.38. 13 C NMR (CD 3 OD, 100 MHz): δ 158.11, 154.24, 150.46, 147.26, 139.72, 139.25, 131.66, 129.96, 128.83, 128.74, 127.80, 127.21, 123.66, 121.93, 121.49, 114.32, 111.91. , 109.88, 109.73, 103.06, 76.56, 73.70, 70.34, 70.22, 69.20, 68.80, 68.36, 66.79, 63.14, 61.40, 55.49, 49.57, 48.47, 27.47, 19.38.

MS (FAB+,Glycerol) : calcd. For [M+H]+ 739.3078 found 739.3085.MS (FAB+, Glycerol): calcd. For [M+H] + 739.3078 found 739.3085.

실시예 4. 4,5-다이메톡시-2-나이트로벤질-4-(아이소퀴놀린-5-일설포닐)-1,4-다이아제페인-1-카복실레이트의 제조Example 4. Preparation of 4,5-dimethoxy-2-nitrobenzyl-4-(isoquinolin-5-ylsulfonyl)-1,4-diazepain-1-carboxylate

Figure pat00025
Figure pat00025

둥근바닥 플라스크에 (4,5-다이메톡시-2-나이트로페닐)메탄올 426 mg, N,N-다이아이소프로필에틸아민 (N,N-Diisopropylethylamine, DIPEA) 1.05 mL 을 다이클로로메테인 (DCM) 4 mL를 가하여 0 oC 에서 용해시키고, 30 분 반응시켰다. 이후 4-나이트로페닐 클로로포메이트 484 mg 을 다이클로로메테인 (DCM) 2 mL를 가하여 상온에서 용해시킨 후 3시간 동안 상온에서 반응시켰다. TLC로 확인 결과 출발물질이 사라졌을 때 감압 농축하여 활성화된 카보네이트를 제조하였다. 이후 파수딜 (Fasudil) 98 mg 과 N,N-다이아이소프로필에틸아민 52 μL을 다이클로로메테인 (DCM) 0.5 mL 를 가하여 0 oC 에서 용해시키고, 활성화된 카보네이트 114 mg 을 다이클로로메테인 (DCM) 0.3 mL 를 가하여 0 oC 에서 넣어주고 6 시간 반응시켰다. TLC로 확인 결과 출발물질이 사라졌을 때 감압 농축하였다. 얻어진 농축물을 컬럼 크로마토그래피(용리액: 디클로로메탄/메탄올 = 10/1, v/v)로 정제하여 4,5-다이메톡시-2-나이트로벤질4-(아이소퀴놀린-5-일설포닐)-1,4-다이아제페인-1-카복실레이트을 제조하였다.In a round-bottom flask, 426 mg of (4,5-dimethoxy-2-nitrophenyl)methanol, 1.05 mL of N,N-diisopropylethylamine (N,N-Diisopropylethylamine, DIPEA) was added to dichloromethane (DCM ) was added 4 mL , dissolved at 0 o C, and reacted for 30 minutes. Then, 484 mg of 4-nitrophenyl chloroformate was dissolved at room temperature by adding 2 mL of dichloromethane (DCM), followed by reaction at room temperature for 3 hours. As a result of TLC, when the starting material disappeared, the activated carbonate was prepared by concentration under reduced pressure. Then, 98 mg of Fasudil and 52 μL of N,N-diisopropylethylamine were dissolved in 0.5 mL of dichloromethane (DCM) at 0 o C, and 114 mg of activated carbonate was dissolved in dichloromethane ( DCM) 0.3 mL was added , put at 0 o C, and reacted for 6 hours. When the starting material disappeared as a result of TLC, it was concentrated under reduced pressure. The resulting concentrate was purified by column chromatography (eluent: dichloromethane/methanol = 10/1, v/v) to 4,5-dimethoxy-2-nitrobenzyl4-(isoquinolin-5-ylsulfonyl) -1,4-diazepain-1-carboxylate was prepared.

수율: 98 %Yield: 98%

1H NMR (CDCl3, 400MHz): δ 9.37 (s, 1H), 8.71 (d, J = 6.4 Hz, 1H), 8.40 (d, J = 6.0 Hz, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.75-7.67 (m, 2H), 6.97 (s, 1H), 5.46 (s, 2H), 3.98 (s, 3H), 3.97 (s, 3H), 3.70-3.60 (m, 4H), 3.51-3.40 (m, 4H), 2.05-1.94 (m, 2H). 1 H NMR (CDCl 3 , 400 MHz): δ 9.37 (s, 1H), 8.71 (d, J = 6.4 Hz, 1H), 8.40 (d, J = 6.0 Hz, 1H), 8.33 (d, J = 7.6 Hz) , 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.75-7.67 (m, 2H), 6.97 (s, 1H), 5.46 (s, 2H), 3.98 (s, 3H), 3.97 (s, 3H), 3.70-3.60 (m, 4H), 3.51-3.40 (m, 4H), 2.05-1.94 (m, 2H).

실시예5. 4,5-다이메톡시-2-나이트로벤질((R)-1-((1R,4R)-1-메틸-4-(피리딘-4-일카바모일)싸이클로헥실)에틸)카바메이트의 제조Example 5. of 4,5-dimethoxy-2-nitrobenzyl((R)-1-((1R,4R)-1-methyl-4-(pyridin-4-ylcarbamoyl)cyclohexyl)ethyl)carbamate Produce

Figure pat00026
Figure pat00026

둥근바닥 플라스크에 (4,5-다이메톡시-2-나이트로페닐)메탄올 426 mg, N,N-다이아이소프로필에틸아민 (N,N-Diisopropylethylamine, DIPEA) 1.05 mL 을 다이클로로메테인 (DCM) 4 mL를 가하여 0 oC 에서 용해시키고, 30 분 반응시켰다. 이후 4-나이트로페닐 클로로포메이트 484 mg 을 다이클로로메테인 (DCM) 2 mL를 가하여 상온에서 용해시킨 후 3시간 동안 상온에서 반응시켰다. TLC로 확인 결과 출발물질이 사라졌을 때 감압 농축하여 활성화된 카보네이트를 제조하였다. 이후 Y-27632 96 mg 과 N,N-다이아이소프로필에틸아민 52 μL을 다이메틸포름아마이드 (DMF) 0.5 mL 를 가하여 0 oC 에서 용해시키고, 활성화된 카보네이트 114 mg 을 다이메틸포름아마이드 (DMF) 0.3 mL 를 가하여 0 oC 에서 넣어주고 34 oC 에서 8 시간 반응시켰다. TLC로 확인 결과 출발물질이 사라졌을 때 감압 농축하였다. 얻어진 농축물을 컬럼 크로마토그래피(용리액: 디클로로메탄/메탄올 = 10/1, v/v)로 정제하여 4,5-다이메톡시-2-나이트로벤질((R)-1-((1R,4R)-1-메틸-4-(피리딘-4-일카바모일)싸이클로헥실)에틸)카바메이트를 제조하였다.In a round-bottom flask, 426 mg of (4,5-dimethoxy-2-nitrophenyl)methanol, 1.05 mL of N,N-diisopropylethylamine (N,N-Diisopropylethylamine, DIPEA) was added to dichloromethane (DCM ) was added 4 mL , dissolved at 0 o C, and reacted for 30 minutes. Then, 484 mg of 4-nitrophenyl chloroformate was dissolved at room temperature by adding 2 mL of dichloromethane (DCM), followed by reaction at room temperature for 3 hours. As a result of TLC, when the starting material disappeared, the activated carbonate was prepared by concentration under reduced pressure. Then, 96 mg of Y-27632 and 52 μL of N,N-diisopropylethylamine were dissolved in 0.5 mL of dimethylformamide (DMF) at 0 o C, and 114 mg of activated carbonate was dissolved in dimethylformamide (DMF). 0.3 mL was added, put at 0 o C, and reacted at 34 o C for 8 hours. When the starting material disappeared as a result of TLC, it was concentrated under reduced pressure. The resulting concentrate was purified by column chromatography (eluent: dichloromethane/methanol = 10/1, v/v) to 4,5-dimethoxy-2-nitrobenzyl ((R)-1-((1R, 4R)-1-methyl-4-(pyridin-4-ylcarbamoyl)cyclohexyl)ethyl)carbamate was prepared.

수율: 35 %Yield: 35%

1H NMR (CDCl3, 400MHz): δ 8.50 (d, J = 6.4 Hz, 2H), 7.73 (s, 1H), 7.58 (s, 1H), 7.53 (d, J = 6.4 Hz, 2H), 7.01 (s, 1H), 5.55-5.46 (m, 2H), 4.74 (d, J = 9.2 Hz, 1H), 4.00 (s, 3H), 3.98 (s, 3H), 3.68-3.59 (m, 1H), 2.27-2.17 (m, 1H), 2.10-1.86 (m, 5H), 1.45-1.33 (m, 2H), 1.18 (d, J = 6.8 Hz, 3H), 1.16-1.6 (m, 2H). 1 H NMR (CDCl 3 , 400 MHz): δ 8.50 (d, J = 6.4 Hz, 2H), 7.73 (s, 1H), 7.58 (s, 1H), 7.53 (d, J = 6.4 Hz, 2H), 7.01 (s, 1H), 5.55-5.46 (m, 2H), 4.74 (d, J = 9.2 Hz, 1H), 4.00 (s, 3H), 3.98 (s, 3H), 3.68-3.59 (m, 1H), 2.27-2.17 (m, 1H), 2.10-1.86 (m, 5H), 1.45-1.33 (m, 2H), 1.18 (d, J = 6.8 Hz, 3H), 1.16-1.6 (m, 2H).

시험예 1. 생체 외 빛 조사에 따른 UV , Flu 데이터 변화관찰Test Example 1. Observation of changes in UV and Flu data according to in vitro light irradiation

실시예 3에서 얻어진 화합물 10μmol를 다이메틸설폭사이드 1 mL에 용해시켜 10 mM이 되도록 준비하였다. 이 중 4μL를 PBS 완충용액(pH 7.4, 1X) 2mL에 혼합하여 실시예 3의 화합물이 20μM의 농도가 되도록 준비하였다. 이후, 740 nm의 빛을 조사하여 UV 비저블 스펙트로포토미터(UV-Visible Spectrophotometer)와 형광 스펙트로미터(Fluorescence Spectrometer)를 사용하여 24시간 동안 UV와 Flu 데이터의 변화를 관찰하였다. (도 1A, 도 1B)10 μmol of the compound obtained in Example 3 was dissolved in 1 mL of dimethyl sulfoxide to prepare 10 mM. Of these, 4 μL was mixed with 2 mL of PBS buffer (pH 7.4, 1X) to prepare the compound of Example 3 to have a concentration of 20 μM. Thereafter, by irradiating light at 740 nm, changes in UV and Flu data were observed for 24 hours using a UV-Visible Spectrophotometer and a Fluorescence Spectrometer. (FIG. 1A, FIG. 1B)

실시예 1과 실시예 2에서 얻어진 화합물도 동일한 방법으로 수행하여 각각의 농도가 10μM, 20μM 가 되도록 준비하여 365 nm의 빛을 조사하여 형광 스펙트로미터(Fluorescence Spectrometer)로 24 시간 동안의 Flu 데이터의 변화를 관찰하였다. (도 1C, 도 1D)The compounds obtained in Example 1 and Example 2 were prepared in the same manner so that their respective concentrations were 10 μM and 20 μM, and 365 nm light was irradiated to change the Flu data for 24 hours with a Fluorescence Spectrometer. was observed. (Fig. 1C, Fig. 1D)

실시예 4에서 얻어진 화합물도 동일한 방법으로 수행하여 농도가 20 μM 가 되도록 준비하여 365 nm의 빛을 조사하여 UV 스펙트로미터로 6 시간 동안의 UV 데이터의 변화를 관찰하였다. (도 1E)The compound obtained in Example 4 was prepared in the same manner to have a concentration of 20 μM, and the change of UV data for 6 hours was observed with a UV spectrometer by irradiating 365 nm light. (Fig. 1E)

도 1A 내지 도 1E을 참조하면 12 시간 혹은 6 시간 만에 빛에 의한 구조 변형이 거의 종결되어 형광 변화가 포화되는 것을 확인할 수 있었다. 따라서 실시예 1 내지 4에 의해 제조되는 화합물은 빛에 의해 작동하는 광활성 형광체 조성물로 사용될 수 있음을 확인하였다.Referring to FIGS. 1A to 1E , it was confirmed that the structural transformation due to light was almost completed after 12 hours or 6 hours, so that the fluorescence change was saturated. Therefore, it was confirmed that the compounds prepared in Examples 1 to 4 can be used as photoactive phosphor compositions operated by light.

시험예 2. 실시예 1 화합물의 세포 내에서 바이오마커 작동 실험Test Example 2. Experiments on the operation of biomarkers in cells of the compound of Example 1

Figure pat00027
[실시예 1]
Figure pat00027
[Example 1]

바이오마커로서 마이토콘드리아를 추적 할 수 있는 트라이페닐 포스핀 작용기를 도입한 화합물인 실시예 1을 준비하여 진행하였다. 자궁경부암에서 유래된 헬라 (HeLa) 세포의 마이토콘드리아의 형광을 공초점레이저현미경을 사용하여 관찰하였다. 위에 4개의 세포 사진은 빛을 조사하기 전의, 아래의 사진4개는 빛을 15분 동안 조사한 뒤 의 사진이다. 왼쪽에서부터 Bright field 사진, Rhodamine 123 5μM을 처린한 사진, 실시예 1의 화합물을 처리한 사진, 그리고 마지막으로 3개의 사진을 겹친 사진이다. 위의 사진들은 빛을 조사하기 전의 사진은 분자구조에 의한 형광 감소 및 도입한 바이오마커에 의해 정상적으로 마이토콘드리아를 추적을 관찰 하였으며, 세포의 사진이 정상적인 것을 관찰하였다. 반면 아래의 사진들은 특정한 영역대 파장의 빛을 15분 동안 조사 한 뒤 세포사진을 관찰하였을 때 조사하기 전보다 세포의 사진이 비정상적인 것을 확인하였으며, 약물의 방출에 의한 세포사멸에 의해 마이토 콘드리아의 모양 또한 비정상적인 것을 관찰 하였다. 본 시험예를 토대로 실시예 1의 화합물이 목적대로 정상적으로 마이토콘드리아를 추적하며, 빛에 작동으로 인한 약물 방출이 세포 사멸을 유발하는 것을 관찰하였다 (도 2).Example 1, a compound in which a triphenyl phosphine functional group capable of tracking mitochondria as a biomarker was introduced, was prepared and proceeded. The fluorescence of mitochondria of HeLa cells derived from cervical cancer was observed using a confocal laser microscope. The top 4 cell photos are before light irradiation, and the bottom 4 photos are after light irradiation for 15 minutes. From the left, a bright field photograph, a photograph treated with Rhodamine 123 5 μM, a photograph treated with the compound of Example 1, and finally three photographs are superimposed. In the above photos, before light irradiation, the fluorescence reduction due to molecular structure and normal tracking of mitochondria by the introduced biomarker were observed, and the photos of cells were observed to be normal. On the other hand, in the photos below, when the photo of the cell was observed after irradiating light of a specific wavelength for 15 minutes, it was confirmed that the photo of the cell was abnormal than before the irradiation. The shape was also observed to be abnormal. Based on this test example, it was observed that the compound of Example 1 normally traces mitochondria as intended, and that drug release due to operation in light induces cell death ( FIG. 2 ).

시험예 3. 실시예 1 화합물의 세포 내의 정밀 작동 실험Test Example 3. Precise operation of the compound of Example 1 in cells

바이오마커로서 마이토콘드리아를 추적 할 수 있는 트라이페닐 포스핀 작용기를 도입한 화합물인 실시예 1을 준비하여 진행하였다. 자궁경부암에서 유래된 헬라 (HeLa) 세포의 마이토콘드리아의 형광을 공초점 레이저현미경을 사용하여 관찰하였다. 왼쪽의 그림부터 첫 번째는 미토콘드리아를 추적하는 Rhodamine 123 5μM을 처리한 것이고 두번째 그림은 화학식1로 표시되는 표제 화합물 3에 바이오 마커로서 트라이페닐 포스핀 작용기를 도입한 화합물을 5μM 를 헬라세포에 처리한 후 빛을 쬐여준 후 하루 지난 사진이고, 세 번째 사진은 같은 조건에서 3일이 지난 후의 사진이고, 맨 오른쪽 그림은 같은 조건에서 빛을 조사하지 않은 세포의 3일이 지난 후의 사진이다. 사진으로 보았을 때 첫 번째 사진에 비해 왼쪽에서 3번째 사진의 세포의 모양이 뚜렷하게 보인다. 이와 대조군으로 4번째 사진과 비교하였을 때 빛에 의한 약물의 방출을 확인 할 수 있다 (도 3).Example 1, a compound in which a triphenyl phosphine functional group capable of tracking mitochondria as a biomarker was introduced, was prepared and proceeded. The fluorescence of mitochondria of HeLa cells derived from cervical cancer was observed using a confocal laser microscope. From the left picture, the first is a treatment with 5 μM of Rhodamine 123, which traces mitochondria, and the second picture shows that HeLa cells were treated with 5 μM of a compound that introduced a triphenyl phosphine functional group as a biomarker to the title compound 3 represented by Formula 1. The photo is one day after exposure to light, the third photo is the photo after three days under the same conditions, and the far right photo is the photo after three days of cells that were not irradiated with light under the same conditions. When viewed from the photo, the shape of the cells in the third photo from the left is clearly visible compared to the first photo. As a control, when compared with the fourth picture, the release of the drug by light can be confirmed (FIG. 3).

시험예 4. 실시예 1 화합물의 세포 독성 실험Test Example 4. Cytotoxicity test of the compound of Example 1

화학식 1로 표시되는 표제화합물 3에 바이오마커로서 트라이페닐 포스핀 작용기를 도입한 화합물인 실시예 1을 준비하여 진행하였다. 자궁경부암에서 유래된 헬라 (HeLa) 세포의 마이토콘드리아의 세포 증식 및 사멸도 평가를 진행하였다. 왼쪽의 막대 그래프들은 특정한 영역대의 빛을 조사하지 않았을 때와 빛을 조사한 후 3일이 지난 세포의 세포 증식 및 사멸도 평가를 나타낸 자료이다. 왼쪽의 막대 그래프인 대조군은 빛을 조사하지 않았을 경우 세포의 사멸이 일어나지 않는 반면 화학식 1로 표시되는 표제 화합물 3 에 바이오 마커로 트라이페닐 포스핀 작용기를 도입한 화합물인 실시예 1은 세포 사멸이 일어나는 것을 관찰하였다. 이 실험을 통해 실제 암세포 내에서 빛의 조사에 의한 약물 방출로 인하여 세포 사멸이 일어나는 것을 간접적으로 확인하였다 (도 4).Example 1, a compound in which a triphenyl phosphine functional group was introduced as a biomarker in the title compound 3 represented by Formula 1, was prepared and proceeded. Cell proliferation and apoptosis of mitochondria of HeLa cells derived from cervical cancer were evaluated. The bar graphs on the left show the evaluation of cell proliferation and apoptosis in a specific area when no light was irradiated and 3 days after light irradiation. The control group, the bar graph on the left, did not cause cell death when not irradiated with light, whereas Example 1, a compound in which a triphenyl phosphine functional group was introduced as a biomarker in the title compound 3 represented by Formula 1, caused apoptosis. that was observed. Through this experiment, it was indirectly confirmed that apoptosis occurs due to drug release by light irradiation in actual cancer cells (FIG. 4).

시험예 5. 실시예 2 화합물의 세포 독성 확인Test Example 5. Confirmation of cytotoxicity of the compound of Example 2

Figure pat00028
[실시예 2]
Figure pat00028
[Example 2]

쥐의 편평세포암종에서 유래된 SCC7 세포에서 마이토콘드리아의 세포 증식 및 사멸도 평가를 진행하였다. 막대 그래프들의 좌측의 y 축은 세포의 생존력을 x 축은 약물의 농도를 나타내고 있으며 좌측의 그래프는 SCC7 세포주에서 실험을 한 자료이다. 각 막대그래프 묶음에서 왼쪽에 있는 것은 실시예2의 약물을 처리하고 빛을 조사하지 않았을 때의 세포 활성을 나타내는 것이고, 가운데는 실시예2의 약물을 처리하고, 빛을 조사한 후 2일이 지난 세포 활성을 나타내는 것이며 오른쪽은 방출되는 약물인 독소루비신(doxorubicin)을 처리한 대조군의 세포 활성을 나타내는 자료이다. 독소루비신을 단독으로 처리하였을 때, 약물의 농도가 높아짐에 따라 세포 독성으로 인한 세포 사멸이 더욱 강력해지는 것을 관찰하였다. 반면, 대조군으로 빛을 조사하지 않은 실시예 2의 화합물의 경우 처리한 약물의 농도가 증가함에도 세포의 사멸이 일어나지 않는 것을 관찰하였다. 본 실험을 통하여 빛의 조사에 의한 약물 방출이 세포 사멸에 결정적인 역할을 한다는 것을 확인 할 수 있다 (도 5). In SCC7 cells derived from squamous cell carcinoma in mice, the cell proliferation and apoptosis of mitochondria were evaluated. The y-axis on the left of the bar graphs shows the viability of cells, the x-axis shows the concentration of drugs, and the graph on the left shows the data of the experiment in the SCC7 cell line. In each bar graph group, the one on the left shows the cell activity when the drug of Example 2 was treated and not irradiated with light, and the middle cell was treated with the drug of Example 2 and 2 days after irradiation with light The activity is shown, and the right side is data showing the cell activity of the control group treated with the released drug, doxorubicin. When doxorubicin was treated alone, it was observed that cell death due to cytotoxicity became stronger as the concentration of the drug increased. On the other hand, in the case of the compound of Example 2 that was not irradiated with light as a control, it was observed that cell death did not occur even when the concentration of the treated drug was increased. Through this experiment, it can be confirmed that drug release by light irradiation plays a decisive role in cell death (FIG. 5).

Claims (5)

하기 [화학식 1]로 표시되는 OPA 또는 TPA 구조체를 포함하는 활용한 광활성 형광체 화합물.
[화학식 1]
Figure pat00029

상기 [화학식1]에서, D (Drug)는 카바메이트 또는 카보네이트 또는 벤질 이써 형태로 연결된 항생제(antibiotics) 또는 억제제(inhibitors)를 포함하고,
상기 [화학식1]에서, BM (Biomarker)은 선형 또는 비선형의 C1 내지 C20의 알킬기, 선형 또는 비선형의 C1 내지 C20의 알킬기를 갖는 케톤기, 선형 또는 비선형의 C1 내지 C20의 알코올기, 선형 또는 비선형의 C1 내지 C20의 알킬기를 갖는 에스테르기,C5 내지 C24의 치환 또는 비치환된 아릴기, 및 C6 내지 C24의 아릴알킬기 또는 비선형의 C4 내지 C18의 알킬기를 갖는 아실기 그룹으로 이루어진 군에서 선택된 어느 하나와 결합된 바이오마커(Biomarker)를 포함한다.A photoactive phosphor compound utilizing the OPA or TPA structure represented by the following [Formula 1].
[Formula 1]
Figure pat00029

In the [Formula 1], D (Drug) includes antibiotics or inhibitors linked in the form of carbamate or carbonate or benzyl isomer,
In the above [Formula 1], BM (Biomarker) is a linear or nonlinear C1 to C20 alkyl group, a ketone group having a linear or nonlinear C1 to C20 alkyl group, a linear or nonlinear C1 to C20 alcohol group, linear or nonlinear Any one selected from the group consisting of an ester group having a C1 to C20 alkyl group, a C5 to C24 substituted or unsubstituted aryl group, and an acyl group having a C6 to C24 arylalkyl group or a nonlinear C4 to C18 alkyl group. and a biomarker bound to it. 제1항에 있어서,
상기 D는 [화학식 2] 또는 [화학식 3]과 같은 독소루비신 (Doxorubicin) 또는 [화학식 4]과 같은 파수딜 (Fasudil, Rho-kinase 억제제) 또는 [화학식 5]와 같은 Y-27632 (lac 억제제) 또는 캄토테신 (Camptothecin, CPT) 중 어느 하나인 광활성 형광체 화합물.
[화학식 2]
Figure pat00030

[화학식 3]
Figure pat00031

[화학식 4]
Figure pat00032

[화학식 5]
Figure pat00033
According to claim 1,
The D is [Formula 2] or [Formula 3], such as doxorubicin (Doxorubicin) or [Formula 4], such as Fasudil (Fasudil, Rho-kinase inhibitor) or [Formula 5] Y-27632 (lac inhibitor) or A photoactive phosphor compound which is any one of camptothecin (CPT).
[Formula 2]
Figure pat00030

[Formula 3]
Figure pat00031

[Formula 4]
Figure pat00032

[Formula 5]
Figure pat00033
 제1항에 있어서,
상기 BM은 (CH2)2O(CH2)2OAcGlu, (CH2)2O(CH2)2OGlu, (CH2)2O(CH2)2PPh3, Glucose, Galactose, 트라이페닐포스핀 (TPP, mitochondria marker), 모르폴린 (Morpholine, endosome marker), 엽산 (Folic acid) 및 바이오틴 (Biotin)으로 이루어진 군에서 선택된 어느 하나인 광활성 형광체 화합물.According to claim 1,
The BM is (CH 2 ) 2 O(CH 2 ) 2 OAcGlu, (CH 2 ) 2 O(CH 2 ) 2 OGlu, (CH 2 ) 2 O(CH 2 ) 2 PPh 3, Glucose, Galactose, triphenylphos A photoactive phosphor compound selected from the group consisting of pin (TPP, mitochondria marker), morpholine (Morpholine, endosome marker), folic acid, and biotin. 제1항에 있어서,
상기 [화학식 1]은 (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-다이메틸-2,3,9,9a-테트라하이드로옥사졸[3,2-a]인돌-9a-일)바이닐)페녹시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에톡시)-6-(하이드록시메틸)테트라하이드로-2H-파이렌-3,4,5-트라이올, 5-메톡시-2-나이트로-4-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-트라이하이드록시-6-(하이드록시메틸)테트라하이드로-2H-파이렌-2-일)옥시)에톡시)에톡시)벤질 ((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바메이트, (2-(2-(4-(((((2S,3S,4S,6R)-3-하이드록시-2-메틸-6-(((1S,3S)-3,5,12-트라이하이드록시-3-(2-하이드록시아세틸)-10-메톡시-6,11-다이옥소-1,2,3,4,6,11-헥사하이드로테트라센-1-일)옥시)테트라하이드로-2H-파이렌-4-일)카바모일)옥시)메틸)-2-메톡시-5-나이트로페녹시)에톡시)에틸)트라이페닐포스포늄 브로마이드,4,5-다이메톡시-2-나이트로벤질 4-(아이소퀴놀린-5-일설포닐)-1,4-다이아제페인-1-카복실레이트, 및 4,5-다이메톡시-2-나이트로벤질 ((R)-1-((1r,4R)-1-메틸-4-(피리딘-4-일카바모일)싸이클로헥실)에틸)카바메이트로 이루어진 군에서 선택된 어느하나인 것을 특징으로 하는 광활성 형광체 화합물.According to claim 1,
The [Formula 1] is (2R,3R,4S,5S,6R)-2-(2-(2-(4-((4-((E)-2-(9,9-dimethyl-2, 3,9,9a-tetrahydrooxazole [3,2-a] indol-9a-yl) vinyl) phenoxy) methyl) -2-methoxy-5-nitrophenoxy) ethoxy) ethoxy) - 6-(Hydroxymethyl)tetrahydro-2H-pyrene-3,4,5-triol, 5-methoxy-2-nitro-4-(2-(2-(((2R,3R,4S) ,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyren-2-yl)oxy)ethoxy)ethoxy)benzyl ((2S,3S, 4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6 ,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyren-4-yl)carbamate, (2-(2- (4-(((((2S,3S,4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12-trihydroxy-3-(2) -Hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetrasen-1-yl)oxy)tetrahydro-2H-pyrene-4 -yl) carbamoyl) oxy) methyl) -2-methoxy-5-nitrophenoxy) ethoxy) ethyl) triphenylphosphonium bromide, 4,5-dimethoxy-2-nitrobenzyl 4- ( Isoquinolin-5-ylsulfonyl)-1,4-diazepain-1-carboxylate, and 4,5-dimethoxy-2-nitrobenzyl ((R)-1-((1r,4R)- A photoactive phosphor compound, characterized in that it is any one selected from the group consisting of 1-methyl-4-(pyridin-4-ylcarbamoyl)cyclohexyl)ethyl)carbamate. 제1항 내지 제4항 중 어느 한 항의 광활성 형광체 화합물을 이용한 암세포 검출방법.
A method for detecting cancer cells using the photoactive phosphor compound of any one of claims 1 to 4.
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