KR102105937B1 - Fluorescent probe compound for detecting cancer cell and a cancer cell detection fluorescence sensor comprising the same - Google Patents

Abstract

The present invention relates to a fluorescent probe compound for detecting cancer cells represented by chemical formula 1, and to a fluorescent sensor for detecting cancer cells comprising the same. The fluorescent probe compound reacts with excellent selectivity to the cancer cells.

Description

Fluorescent probe compound for detecting cancer cell and a cancer cell detection fluorescence sensor comprising the same}

The present invention relates to a fluorescent probe compound for detecting cancer cells and a fluorescent sensor for detecting cancer cells comprising the same.

Cancer is a major life-threatening disease worldwide and has been reported by the World Health Organization (WHO) to kill 7 million cancers each year and to account for the enormous social and economic pressure on health care systems. However, in the case of undergoing surgery and chemotherapy in combination with careful monitoring of chemotherapy, it is estimated that 30% of cancer patients diagnosed at an early stage of cancer progression can avoid death (Non-Patent Document 1). For example, the 5-year survival rate in patients with local metastasis or pre-metastatic colorectal cancer exceeds 90%, but in the case of distant metastasis, it rapidly decreases to less than 10% (Non-Patent Document 2).

In order to solve this problem, various imaging techniques have been developed to find disease specific probes that can selectively identify disease biomarkers (Non-Patent Document 3). Among them, the fluorescence imaging method has attracted much attention due to its high selectivity and sensitivity, relatively low measurement cost, and ease of operation (Non-Patent Document 4). However, non-specific bio-distribution and inefficient removal of fluorescent probes from non-target organs are obstacles to obtaining a high signal-to-noise ratio after administration (Non-Patent Document 5). In this regard, the development of an inductive fluorescence contrast agent that can be selectively retained at the target site can improve the signal-to-noise ratio by providing sufficient time to remove the probe from the non-target site. It can be the best way.

Cyclooxygenase (COX), also known as prostaglandin-endoperoxide synthase (PTGS) enzyme, is classified as a family of two isozymes, COX-1 and COX-2, of which COX-2 is an inflammatory reaction. Prostaglandin biosynthesis and plays an important role in the central nervous system (Non-Patent Document 6). According to clinical data, it has been reported that COX-2 is overexpressed in all stages of cancer, from the initial pre-cancerous stage to metastatic stage (non-patent document 7), and particularly, high levels of COX-2 overexpression occur in colorectal, stomach, and pancreatic cancer. Is known. Therefore, COX-2 is considered as a potential candidate for a high-efficiency next-generation tumor target unit (Non-Patent Document 8).

Detection and imaging of COX-2 can guide early signs (and symptoms) as well as solutions for cancer treatment. Although several fluorescent probes targeting COX-2 have been developed (non-patent document 9), the development of COX-2 specific cancer biomarkers is still in its infancy.

(Non-Patent Document 1) R.T. Greenlee, T. Murray, S. Bolden, P.A. Wingo, Cancer statistics, 2000, Ca-Cancer J Clin 50 (2000) 7-33.

(Non-Patent Document 2) A.J. Dannenberg, S.M. Lippman, J.R. Mann, K. Subbaramaiah, R.N. DuBois, Cyclooxygenase-2 and epidermal growth factor receptor: Pharmacologic targets for chemoprevention, J Clin Oncol 23 (2005) 254-66.

(Non-Patent Document 3) C.H. Contag, M.H. Bachmann, Advances in in vivo bioluminescence imaging of gene expression, Annu Rev Biomed Eng 4 (2002) 235-60.

(Non-patent document 4) C. Denkert, K.J. Winzer, B.M. Muller, W. Weichert, S. Pest, M. Kobel, et al., Elevated expression of cyclooxygenase-2 is a negative prognostic factor for disease free survival and overall survival in patients with breast carcinoma, Cancer 97 (2003) 2978-87 .

(Non-patent document 5) T. Toyokuni, J.S.D. Kumar, J.C. Walsh, A. Shapiro, J.J. Talley, M.E. Phelps, et al., Synthesis of 4- (5- [F-18] fluoromethyl-3-phenylisoxazol-4-yl) -benzenesulfonamide, a new [F-18] fluorinated analogue of valdecoxib, as a potential radiotracer for imaging cyclooxygenase -2 with positron emission tomography, Bioorg Med Chem Lett 15 (2005) 4699-702.

(Non-Patent Document 6) Z. Xu, S. Choudhary, O. Voznesensky, M. Mehrotra, M. Woodard, M. Hansen, et al., Overexpression of Cox-2 in human osteosarcoma cells decreases proliferation and increases apoptosis, Cancer Res 66 (2006) 6657-64.

(Non-patent document 7) T.A. Samad, K.A. Moore, A. Sapirstein, S. Billet, A. Allchorne, S. Poole, et al., Interleukin-1 beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity, Nature 410 (2001) 471-5 .

(Non-Patent Document 8) M.J. Uddin, B.C. Crews, A.L. Blobaum, P.J. Kingsley, D.L. Gorden, J.O. McIntyre, et al., Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents, Cancer Res 70 (2010) 3618-27.

(Non-Patent Document 9) A. Bhardwaj, J. Kaur, F. Wuest, E.E. Knaus, Fluorophore-Labeled Cyclooxygenase-2 Inhibitors for the Imaging of Cyclooxygenase-2 Overexpression in Cancer: Synthesis and Biological Studies, Chemmedchem 9 (2014) 109-16.

The present invention is to provide a fluorescent probe compound for detecting cancer cells having high selectivity to cancer cells and a fluorescent sensor for detecting cancer cells comprising the same.

The present invention to solve the above problems,

It provides a fluorescent probe compound for detecting cancer cells represented by the following [Formula 1]:

[Formula 1]

.

.

According to the present invention, the cancer cells may be cyclooxygenase-2 (Cyclooxygenase-2, COX-2) overexpressed.

In addition, the present invention provides a fluorescent sensor for detecting cancer cells comprising the fluorescent probe compound.

The fluorescent probe compound according to the present invention responds to cancer cells with excellent selectivity, and has excellent photophysical properties and low cytotoxicity, so that cancer cells can be effectively imaged.

1 shows (A) UV-visible absorption and (B) emission spectra of a 10 μM IQ-1 (compound represented by Formula 1) solution in PBS (10 mM, pH 7.4). (C) Other species (1: IQ-1 alone, 2: Asn, 3: Asp, 4: Gln, 5: Glu, 6: Gly, 7: Ile, 8: Met, 9: Pro, 10: Ser , 11: Tyr, 12: Trp, 13: Cys, 14: Hcy, 15: GSH, 16: H ₂ O ₂ , 17: Mg ^{2 +} , 18: Na ⁺ ) IQ-1 fluorescence intensity at 555 nm Indicates. All living species were equal to 100 equivalents in 10 mM PBS buffer at room temperature (pH 7.4). The excitation and emission slit widths were set to 10 nm and excited at 430 nm. Spectra were recorded after incubation of IQ-1 with other species for 1 hour.
2 shows the viability of IQ-1 treated cells. (A) RAW 264.7 and (B) HeLa cells were incubated with 0, 2.5, 5, 10, 20 and 50 μM IQ-1 for 24 hours. P values represent the mean ± standard error of three identical independent experiments; * p <0.05.
3 shows fluorescence images of normal and cancer cell lines. All cells were treated with IQ-1 (5 μM) for 2 hours. (1: RAW 246.7, 2: fibroblasts, 3: OVCAR 3, 4: HepG2, 5: HeLa cells) (magnification: x63; λ _ex : 488 nm; λ _em : 500-600 nm).
4 shows fluorescence images of cell lines (RAW 264.7 cells and fibroblasts) induced by LPS inflammation. Cells were incubated with LPS for 12 hours and then treated with IQ-1. Specifically, (A) is RAW 264.7 cultured with LPS, and (B) is a confocal microscope image of fibroblasts cultured with LPS. (C) shows normalized fluorescence intensity of RAW 264.7 cells and inflamed RAW 264.7 cells, and (D) fibroblasts and inflamed fibroblasts after LPS treatment. (Magnification: x63; λ _ex : 488 nm; λ _exem : 500-600 nm). P values represent the mean ± standard error of three identical independent experiments; * p <0.05.
5 (A) is a confocal microscopic image showing the COX-2 expression level of HeLa cells treated with a COX-2 inhibitor (IMC = Indomethacin, Ace = Aceclofenac), (B) fluorescence intensity, (C) Western blot Results are shown. (Magnification: x63; λ _ex : 488 nm; λ _ex : 500-600 nm). P values represent the mean ± standard error of three identical independent experiments; * p <0.05.
FIG. 6 shows the fluorescence intensity of IQ-1 (10 μM) at 555 nm at various pH values (λ _ex 430 nm, slit: 10 nm / 10 nm.) Spectra incubate IQ-1 under various pH conditions for 1 hour. It was recorded after.
Figure 7 shows the results of measuring the dose-dependent fluorescence intensity (0, 1, 2.5, 5, 10 μM) of HeLa cells cultured for 2 hours with IQ-1.
Figure 8 shows the results of measuring the time (0, 30, 60, 120 minutes) dependent fluorescence intensity of Hela cells treated with 5μM IQ-1.
Figure 9 shows the results of comparing the fluorescence intensity of IQ-1 in cancer cell lines (OVCAR3, HepG2, HeLa).

Hereinafter, the present invention will be described in more detail through examples, but the following examples are only to aid understanding of the present invention and are not intended to limit the scope of the present invention.

Experiment method

Materials, methods and equipment

All reagents and solvents were purchased from Aldrich, TCI, Samcheong and Alfa aesar and used without further purification. Column chromatography purification was performed using silica gel 60 (70-230 mesh) as the stationary phase. Analytical thin layer chromatography was performed using 60 silica gel (0.25 mm thick coated sheet). Mass spectra were recorded with an IonSpecHiRes ESI Shimadzu LC / MS-2020 mass spectrometer and LC / MS-8050 mass spectrometer. ¹ H and ¹³ C NMR spectra were collected using a Bruker 500 MHz spectrometer using CDCl ₃ and TMS. All fluorescence and UV / Vis absorption spectra were recorded on RF-5301PC and S-3100 spectrophotometers, respectively. Probe stock solution (500 μM) in DMSO, probe solutions at 10 μM concentration were prepared in PBS buffer with 2% DMSO. In all experiments, the excitation wavelength was 430 nm and the excitation and emission slit width was 10 nm.

Compound synthesis

Compound (IQ-1) represented by [Chemical Formula 1] according to the present invention was synthesized in the following synthetic route.

[Synthetic route]

(i) EDCI, DMAP, DMF, L1, (ii) Hexamethylenetetramine, TFA, 3M HCl, (iii) Propargyl bromide, K ₂ CO ₃ , DMF, (iv) 2-cyclohexen-1-one, imidazole, THF: H ₂ O = 1: 1 (v / v), (v) CuSO ₄ · 5H ₂ O, sodium ascorbate, THF / H ₂ O, (vi) Q1, piperidine, ethanol, reflux.

Synthesis of L1, Q1 and compound 5

Compounds L1, Q1 and Compound 5 used in the [Synthetic Route] are conventionally described in S. Zhang, JL Fan, ZY Li, NJ Hao, JF Cao, T. Wu, et al., A bright red fluorescent cyanine dye for live-cell nucleic acid imaging, with high photostability and a large Stokes shift, J Mater Chem B 2 (2014) 2688-93., C. Romuald, E. Busseron, F. Coutrot, Very Contracted to Extended co-Conformations with or Without Oscillations in Two- and Three-Station [c2] Daisy Chains, J Org Chem 75 (2010) 6516-31.).

IMC -N ₃ Synthesis of

Indomethacin (627 mg, 1.827 mmol, 1 eq), N - (3-Dimethylaminopropyl) - N '-ethylcarbodiimide hydrochloride (EDCI, 312 mg, 2.010 mmol, 1.1 eq) and 4-dimethylaminopyridine (DMAP, 245 mg , 2.010 mmol, 1.1 eq) was dissolved in 0 ° C DMF (12 mL). After 30 minutes, L1 (285 mg, 2.010 mmol, 1.1 eq) was added to the solution. The reaction mixture was stirred at room temperature overnight. Monitored by TLC, the solvent was removed and washed with 1N HCl solution, and the organic layer was dried over anhydrous Na ₂ SO ₄ . The main product was then purified by column chromatography (ethyl acetate / dichloromethane, 1:50, v / v). Subsequent recrystallization with ethyl ether gave compound IMC-N ₃ (510 mg) as a pale yellow solid (68.3%).

¹ H NMR (500 MHz, CDCl ₃ ): δ 7.696-7.687 (t, 1H), 7.678-7.669 (t, 1H), 7.521-7.513 (t, 1H), 7.503-7.495 (t, 1H), 6.901- 6.896 (d, 1H), 6.887-6.869 (d, 1H), 6.733-6.710 (dd, 1H), 5.642-5.620 (t, 1H), 3.839 (s, 3H), 3.657 (s, 2H), 3.236- 3.197 (m, 4H), 2.406 (s, 3H), 1.547-1.204 (m, 8H). ¹³ C NMR (500 MHz, CDCl ₃ ): 169.767, 168.347, 156.301, 139.612, 136.325, 133.567, 131.238, 131.190, 131.164, 130.916, 130.324, 129.233, 129.125, 115.120, 112.934, 112.281, 100.887, 67.970, 55745 , 55.681, 51.259, 39.452, 32.276, 29.431, 28.677, 26.302, 13.258 ppm. ESI-MS: the calculated value (calcd) for C ₂₅ H ₂₈ ClN ₅ O ₃ ([M + H] ⁺ ): 482.19, C ₂₅ H ₂₈ ClN ₅ O ₃ ([M + Na] ⁺ ): 504.19, C ₂₅ H ₂₈ ClN ₅ O ₃ ([M + K] ⁺ ): 521.19, found 219.10, 231.00. found 504.20.

Synthesis of Compound 4

A suspension was prepared by mixing compound 5 (2 g, 0.011 mol, 1 eq) and potassium carbonate (1.679 g, 0.0121 mol, 1.1 eq) in DMF (50 mL) at 60 ° C. After 1 hour, a solution of DMF (20 ml) in which propargyl bromide (1.174 ml, 0.0105, 0.95 eq) was dissolved was slowly added and stirred for 12 hours. After that, all solvents were removed and washed with dichloromethane and 1N HCl (20 ml x 3). The organic layer was concentrated and dried in _over anhydrous Na ₂ SO _4. Purification via column chromatography (dichloromethane: hexane = 9: 1, v / v) gave compound 4 (400 mg) as a white powder (16.5%).

¹ H NMR (500 MHz, CDCl ₃ ): δ 11.895 (s, 1H), 10.287 (s, 1H), 9.921 (s, 1H), 8.091 (s, 1H), 4.789 (s, 2H), 2.588-2.578 (t, 1H), 2,257 (s, 3H). ¹³ C NMR (500 MHz, CDCl ₃ ): 195.935, 188.198, 165.942, 164.555, 133.633, 123.643, 120.928, 117.606, 77.785, 62.425, 25.614, 8.622 ppm. ESI-MS: the calculated value (calcd) for C ₁₂ H ₁₀ O ₄ ([M + H] ⁺ ): 219.06, C ₁₂ H ₁₀ O ₄ ([M + Na] ⁺ ): 231.06, C ₁₂ H ₁₀ O ₄ ([M + K] ⁺ ): 256.06, found 219.10, 231.00.

Synthesis of Compound 3

2-cyclohexen-1-one (422 μl, 6.548 mmol, 1.5 eq) and imidazole (297 mg, 6.548 mmol, 1.5) in a solution of compound 4 (635 mg, 4.365 mmol, 1 eq) on THF (5 mL) eq). After mixing with distilled water (5 mL), the mixture was stirred at room temperature for 2 days. After monitoring the completion of the reaction by TLC, the solvent was removed and washed with ethyl acetate and 1N HCl. The organic layer is anhydrous Na ₂ SO ₄ Dried over and concentrated in vacuo. Purification through column chromatography (ethyl acetate: dichloromethane = 1:50, v / v) afforded Compound 3 (331 mg) as a pale yellow powder (yield 35.8%).

¹ H NMR (500 MHz, CDCl ₃ ): δ 10.271 (s, 1H), 7.659 (s, 1H), 7.415 (s, 1H), 5.117-5.083 (m, 1H), 4.718-4.714 (t, 2H) , 2.658-2.640 (m, 3H), 2.45-2.37 (m, 1H), 2.21 (s, 3H), 2.181-2.081 (m, 2H), 1.78-1.70 (m, 1H). ¹³ C NMR (500 MHz, CDCl ₃ ): 196.903, 188.864, 161.841, 159.892, 130.413, 130.138, 128.009, 124.792, 119.861, 118.673, 77.633, 77.417, 75.469, 62.435, 38.766, 29.756, 17.946, 9.192 ppm. ESI-MS: the calculated value (calcd) for C ₁₈ H ₁₆ O ₄ ([M + H] ⁺ ): 297.32, C ₁₈ H ₁₆ O ₄ ([M + Na] ⁺ ): 319.32, C ₁₈ H ₁₆ O ₄ ([M + K] ⁺ ): 335.32, found 297.20.

Synthesis of Compound 2

Compound 3 (50 mg, 0.169 mmol, 1 eq) was dissolved in THF (3 mL), IMC-N ₃ (81 mg, 0.169 mmol, 1 eq), CuSO ₄ ^. After mixing with 5H ₂ O (4.4 mg, 0.0188 mmol, 0.3 eq), sodium ascorbate (3.6 mg, 0.0188 mmol, 0.3 eq) and distilled water (1.5 mL), the mixture was stirred for 12 hours. All solvent was removed and washed with ethyl acetate / 1N HCl. The organic layer is anhydrous Na ₂ SO ₄ Dried over and concentrated in vacuo. Purification via column chromatography (methanol: dichloromethane = 1:50, v / v) gave compound 2 (80 mg) as a yellow solid (60.9%).

¹ H NMR (500 MHz, CDCl ₃ ): δ 9.939 (s, 1H), 7.661-7.765 (t, 1H), 7.643-7.635 (t, 1H), 7.623 (s, 1H), 7.542 (s, 1H) , 7.487-7.479 (t, 1H), 7.496-7.461 (t, 1H), 7.371-7.367 (d, 1H), 6.896-6.891 (d, 1H), 6.863-6.845 (d, 1H), 6.693-6.670 ( dd, 1H), 5.796-5. 772 (t, 1H), 5.124-5.118 (d, 2H), 5.105-5.067 (m, 1H), 4.303-4.274 (t, 2H), 3.806 (s, 3H), 3.638 (s, 2H), 3.209-3.169 (q, 2H), 2.605-2.534 (m, 2H), 2.441-2.393 (m, 1H), 2.378 (s, 3H), 2.158-2.103 (m, 1H), 1.835-1.807 (t, 2H), 1.759-1.718 (m, 1H), 1.428-1.401 (t, 2H), 1.237-1.223 (t, 3H). ¹³ C NMR (500 MHz, CDCl ₃ ): 198.017, 188.563, 169.907, 168.356, 162.339, 160.105, 156.229, 142.811, 139.583, 136.362, 133.567, 131.205, 130.914, 130.374, 129.992, 129.234, 128.613, 124.193, 123.240, , 118.429, 115.129, 112.948, 112.199, 100.912, 75.474, 68.324, 55.773, 50.147, 39.177, 38.771, 32.289, 30.065, 29.735, 20.177, 25.845, 25.766, 17.945, 13.330, 9.180, 0.020 ppm. ESI-MS: the calculated value (calcd) for C ₄₄ H ₄₆ ClN ₅ O ₆ ([M + H] ⁺ ): 776.31, C ₄₄ H ₄₆ ClN ₅ O ₆ ([M + Na] ⁺ ): 798.31, C ₄₄ H ₄₆ ClN ₅ O ₆ ([M + K] ⁺ ): 814.31, found 800.30.

Synthesis of IQ-1

A solution of compound 2 (60 mg, 0.0703 mmol, 1 eq), Q1 (23 mg, 0.0703 mmol, 1 eq) and piperidine (7 μL, 0.0703 mmol, 1 eq) in ethanol (6 mL) for 2 hours During reflux. After monitoring the completion of the reaction by TLC, the solvent was removed and washed with dichloromethane and distilled water. The organic layer was concentrated and anhydrous Na ₂ SO ₄ Phases. The residue was purified by column chromatography (3-5% methanol in dichloromethane) to give the compound IQ-1 (31 mg) as a brown powder (38.0%).

¹ H NMR (500 MHz, CDCl ₃ ): δ 9.843-9.830 (d, 1H), 8.623-8.606 (d, 1H), 8.340-8.329 (d, 1H), 8.156-8.094 (m, 3H), 7.941- 7.912 (t, 1H), 7.802-7.770 (d, 1H), 7.641-7.624 (m, 3H), 7.449-7.425 (m, 3H), 7.023-7.019 (d, 1H), 6.787-6.769 (d, 1H) ), 6.664-6.640 (t, 1H), 6.577-6.554 (m, 1H), 5.102-4.985 (m, 5H), 4.416-4.387 (t, 2H), 3.791 (s, 3H), 3.693 (s, 2H) ), 3.225-3.187 (q, 2H), 2.4554-2.407 (m, 1H), 2.354 (s, 3H) 2.311 (s, 3H), 2.177-2.120 (m, 1H), 1.880-1.794 (m, 3H) , 1.750-1.721 (t, 3H), 1.480-1.444 (m, 2H), 1.256 (s, 3H). ¹³ C NMR (500 MHz, CDCl ₃ ): 207.040, 197.126, 170.285, 168.348, 159.826, 157.993, 156.063, 153.402, 147.311, 142.264, 139.222, 138.416, 137.562, 136.072, 135.174, 133.805, 131.184, 130.787, 13086 , 129.104, 126.126, 124.663, 123.066, 120.387, 118.953, 118.141, 117.651, 117.080, 114.857, 113.836, 111.894, 101.477, 75.321, 67.371, 56.057, 52.820, 50.120, 39.224, 38.795, 32.279, 31.919, 309.699 , 29.354, 28.922, 25.963, 25.594, 22.684, 17.945, 15.419, 14.117, 13.677, 9.633 ppm. ESI-MS: the calculated value (calcd) for C ₅₅ H ₅₆ ClIN ₆ O ₆ ([M + H] ⁺ ): 1059.30, C ₅₅ H ₅₆ ClIN ₆ O ₆ ([M + Na] ⁺ ): 1082.30, C ₅₅ H ₅₆ ClIN ₆ O ₆ ([M + K] ⁺ ): 1097.30, found 931.30.

Cell culture

Human cervical cancer (HeLa), human hepatocellular carcinoma (HepG2), human ovarian cancer (OVCAR3), and rat monocyte / macrophage (Raw 264.7) cell lines were purchased from the Korean Cell Line Bank and human skin fibroblasts were purchased from Invitrogen. All cells were cultured in high glucose DMEM (Hyclone) supplemented with 10% fetal bovine serum (Cell Signaling Technology) and 1% penicillin-streptomycin (Hyclone). All cultured cells were maintained at 37 ° C in a humid environment containing 5% carbon dioxide.

cell Survival rate  analysis

Approximately 2 × 10 ⁴ cells were dispensed in 96 well microplates (SPL Life Science) and cultured for one day. After incubation, cells were treated with DMSO and probe IQ-1 for 24 hours. To analyze the cell viability of the probe in the cell, cell viability analysis was performed using the CytoTox96® non-radioactive cytotoxicity assay kit (Promega) according to the manufacturer's instructions. Fluorescence levels were analyzed using a SPECTRA MAX GEMINI EM microplate reader (Molecular Devices). The wavelength was set to 490 nm. Cell viability was expressed as a percentage of absorbance compared to control wells and treated wells.

Confocal Imaging  analysis

2 × 10 ⁵ cells were placed in a 35 mm glass bottom confocal dish (SPL Life Science) and stabilized for 1 day. When the cells reached 80% confluency, they were treated with probe IQ-1 (5 μM on DMSO) for 2 hours at 37 ° C., 5% CO ₂ . Fluorescence images were taken under the same conditions using a confocal laser scanning microscope (Carl-Zeiss LSM 700 Exciter). Fluorescence channels were excited with a Si laser at 405 nm and emission was collected with a 420-500 nm band pass filter.

With LPS  Induced inflammation and COX-2 inhibition and induction testing

To investigate the inflammatory effect, Raw 264.7 and human fibroblasts were pretreated for 12 hours in DMEM containing 1 μg / ml LPS (O111: B4, Sigma). After pre-incubation in LPS-containing medium, cells were washed 3 times with PBS and treated with 5 μM of probe IQ-1 dissolved in DMSO for 2 hours. To test the inhibitory effect of COX-2, HeLa cells were incubated with 500 μM of indomethacin (Tokyo Company Industry) or aceclofenac (Tokyo Company Industry) for 12 hours. After pre-incubation with COX-2 inhibitor containing medium, cells were washed 3 times with PBS and treated with 5 μM probe IQ-1 dissolved in DMSO for 2 hours. Cells were washed 3 times with PBS.

Western Blotting  analysis

COX-2 expression at the protein level was confirmed by Western blotting. Briefly, the attached cells were washed 3 times with cold PBS and scraped to collect the cell pellet. After the PBS was removed, a radioactive immunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor (Up-state) provided by the manufacturer was added to the cell pellet to obtain a protein lysate. Bradford analysis was performed to measure the protein concentration of each cell line, and the protein of each cell line (30 μg / lane) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (SDS-) to separate the protein into several bands. PAGE). The separated protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Merk Millipore). Membranes were incubated overnight at 4 ° C. with COX-II antibody (Cell signaling) diluted to 1/1000 and GAPDH antibody (Santa Cruz Biotechnology) diluted to 1/1000. The resulting membranes were washed 3 times with Tris-buffered saline Tween-20 (TBS-T), and 2 hours at room temperature with an anti-rabbit horseradish peroxidase (HRP) -binding secondary antibody (Santa Cruz Biotechnology). Cultured for a while. To detect the immunoreactive protein band, an enhanced chemiluminescent reagent (Luminate, Merk Millipore) was used according to the manufacturer's instructions.

Statistical analysis

Statistical significance of the treatment values was analyzed by comparison with control values using Student's t-test. Data represent mean ± SE, and * p <0.05 was considered statistically significant.

Results and Discussion

The compound according to the present invention, Probe IQ-1, was synthesized according to the above [synthetic route]. Briefly, compound 4 was obtained through the reaction of compound 5 with propargyl bromide, which was cyclized with 2-cyclohexen-1-one to produce compound 3. Compound 2 was synthesized through a click reaction of azide-tailed indomethacin derivative IMC-N3 with compound 3, and finally, Knoevenagel condensation reaction yielded compound IQ-1 in which the quinolinium moiety was successfully conjugated. The presence of the synthesized compound was confirmed by mass spectrometry, ¹ H NMR / ¹³ C NMR.

Next, the spectral properties of IQ-1 were examined under physiological conditions (10 mM phosphate buffer solution (PBS), pH 7.4) simulation. The probe solution showed an absorption peak at 430 nm (Figure 1A) and a strong emission peak at the center of 555 nm (Figure 1B). To evaluate the applicability of IQ-1 under physiological conditions, the change in fluorescence spectrum of IQ-1 was examined by exposing it to various biologically relevant analytes in aqueous solution (10 mM PBS buffer, pH 7.4). As shown in FIG. 1C, amino acids (Asn, Asp, Gln, Glu, His, Ile, Met, Pro, Ser, Tyr, Trp), thiol-containing amino acids (Cys, Hcy, GSH), highly reactive oxides (H ₂ O ₂ ) and when typical metal ions (Mg ^{2 +} , Na ⁺ ) were added to the aqueous IQ-1 solution (10 μM), no large fluorescence change was induced even at a very high concentration (0.01 M). In addition, these results were confirmed once again through pH-dependent fluorescence intensity results (FIG. 6). Through these results, it was confirmed that the probe IQ-1 according to the present invention can be used under physiologically common conditions.

Next, the cell viability of IQ-1 treated cells was tested to see if this probe could be used in biological systems. As shown in FIG. 2, RAW 264.7 (normal cell line) and HeLa cells (cancer cell line) treated with IQ-1 at various concentrations showed very low cytotoxicity, indicating that IQ-1 indicates cancer cells in biological conditions. It was confirmed that it can be usefully used as a fluorescent probe.

Next, the COX-2 targeting ability of IQ-1 in living cells was evaluated through a series of In Vitro experiments. IQ-1 was incubated with normal cells (RAW 264.7 and fibroblasts) and carcinoma cell lines (OVCAR, HepG2 and HeLa) for 2 hours. As shown in Figure 3, bright green fluorescence was observed in IQ-1 of carcinoma (ovary, HepG2 and HeLa) cell lines with COX-2 overexpression, whereas normal cells with lower COX-2 expression (RAW 264.7 and fibroblasts) Fluorescence was hardly observed. Furthermore, in HeLa cells, fluorescence was shown to be dependent on the concentration of COX-2 (Figure 7) and probe incubation time (Figure 8). Through these results, it was confirmed that 5 μM IQ-1 has excellent cell permeability and is useful as an indicator for distinguishing cancer cell lines from normal cell lines.

Next, the applicability of IQ-1 to detect intracellular COX-2 inflammatory cells caused by cellular oxidative damage was evaluated. First, RAW 264.7 and fibroblast cell lines were prepared as control cells. 4A, it was confirmed that the fluorescence of probe IQ-1 in the cell line was enhanced because the amount of COX-2 was increased by LPS-induced oxidative stress when cultured for 12 hours with lipopolysaccharide (LPS), and the fibroblasts were also identical. It was confirmed that the experiment showed a similar pattern (Fig. 4B). In addition, as shown in FIGS. 4C and 4D, it was confirmed that the fluorescence intensity increased 6-fold after LPS treatment, thereby confirming that IQ-1 can be usefully used for monitoring oxidative inflammation in a living system.

In addition, COX-2 inhibition experiments were performed to confirm the origin of selectivity for cancer cells over normal cells. The COX-2 enzyme is known to be overexpressed in HeLa cells, and it was also confirmed that IQ-1 treatment increased fluorescence in HeLa cell lines (FIG. 9). HeLa cells were selected as control cells. For inhibition studies, HeLa cells were incubated with 500 μM of indomethacin (IMC) and aceclofenac (Ace), respectively. As shown in FIGS. 5A and 5B, it was confirmed that fluorescence intensity was significantly reduced in HeLa cells when the inhibitor was treated due to depletion of intracellular COX-2 levels before adding IQ-1. In addition, Western blotting analysis confirmed that COX-2 levels were down-regulated in the presence of inhibitors (more 5C). Through this, it was confirmed that the indomethacin-inducing probe IQ-1 showed a clear dependency on the COX-2 expression level.

In conclusion, the present invention provides a novel indomethacin-linked fluorescent probe compound (IQ-1) represented by [Formula 1] capable of effectively imaging cancer cells by having high selectivity for cancer cells. Through the various experiments described above, it was confirmed that the compound according to the present invention, IQ-1, has no cytotoxicity, and that the fluorescence intensity in various human cells depends on the concentration of COX-2. Fluorescence is significantly increased in overexpressed cancer cells, and amplified fluorescence can be observed in cell lines inflamed by LPS with high COX-2 levels, while COX-2 inhibitors such as indomethacin and aceclofenac are used in combination. When it was confirmed that the fluorescence intensity decreases. Through these results, it was confirmed that the compound IQ-1 according to the present invention has superior targeting and selectivity to cancer cells in proportion to the level of COX-2, and the compound according to the present invention selectively bioimages cancer cells. It can be used as a tool that can be used, and is expected to be widely used for efficient diagnosis and treatment monitoring in precision medicine.

Claims (3)

Fluorescent probe compound for detecting cancer cells represented by the following [Formula 1]:
[Formula 1]

. According to claim 1,
The cancer cell is a cyclooxygenase-2 (Cyclooxygenase-2, COX-2) fluorescent probe compound for detecting cancer cells, characterized in that overexpressed. A fluorescent sensor for detecting cancer cells comprising the fluorescent probe compound according to claim 1.

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