KR102332454B1 - Chemiluminescent probe compound for detecting cancer cell and a cancer cell detection chemiluminescent sensor comprising the same - Google Patents

Abstract

.The present invention relates to a light emitting probe compound for detecting cancer cells represented by the following [Formula 1] and a light emitting sensor for detecting cancer cells comprising the same:
[Formula 1]

.

Description

A luminescent probe compound for detecting cancer cells and a luminescent sensor for detecting cancer cells comprising the same

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

Stimulus-responsive synthetic constructs are receiving more and more attention due to their applicability to applications such as sensing and drug delivery (Non-Patent Document 1). Bioreactive substances that not only respond to, but actually amplify, specific pathophysiological signals can play an important role not only in medicine but also in biological research. Such a system enables improved differentiation between diseased and healthy tissues, thereby improving diagnosis and treatment of diseases (Non-Patent Document 2). A system that relies on fluorescence (non-patent document 3), photoacoustic (non-patent document 4), and magnetic resonance (non-patent document 5) imaging in cancer research was developed as a stimulus-response probe specific for cancer biomarkers. These systems and the detection techniques on which they depend have substantially improved the routinely performed tumor imaging experiments. Nevertheless, achieving high specificity and fidelity in differentiation between cancerous lesions and normal healthy tissues remains an important challenge, and in many cases remains unmet medical needs, such as surgical dissection. In this regard, chemiluminescence holds great promise as it offers several advantages over existing fluorescence-based approaches while maintaining the simplicity of optical imaging. These advantages include better penetration depth, higher signal-to-noise ratio, reduced interference by autofluorescence, light scattering and photobleaching, and the need for an external excitation light source is also an important advantage.

On the other hand, NAD(P)H:quinone oxidoreductase-1 (NQO1) is an enzyme expressed throughout the cell, and its expression is upregulated in response to stress. This enzyme acts as a former reductase for various toxins, such as quinone xenobiotics and superoxide anions, while consuming either NADH or NADPH together. In view of this stress-induced upregulation, it is not surprising that the enzyme is overexpressed in various cancerous tissues (especially in liver carcinoma overexpression at a rate of up to 50-fold). Furthermore, it has been theorized that NQO1 upregulation may be an early event in which normal cells are pathologically transformed into cancer cells (Non-Patent Document 6).

A. Roda, M. Guardigli, Anal. Bioanal. Chem. 2012, 402, 69-76. N. Hananya, O. Green, R. Blau, R. Satchi-Fainaro, D. Shabat, Angew. Chem. Int. Ed. 2017, 56, 11793 ? 11796; Angew. Chem. 2017, 129, 11955-11958. H. Kobayashi, P. L. Choyke, Acc. Chem. Res. 2011, 44, 83-90. Q. Q. Miao, K. Y. Pu, Bioconjugate Chem. 2016, 27, 2808-2823. D. J. Ye, A. J. Shuhendler, P. Pandit, K. D. Brewer, S. S. Tee, L. N. Cui, G. Tikhomirov, B. Rutt, J. H. Rao, Chem. Sci. 2014, 5, 3845-3852. T. Cresteil and A. K. Jaiswal, Biochem. Pharmacol. 1991, 42, 1021-1027.

An object of the present invention is to provide a light emitting probe compound and a light emitting sensor having high selectivity for cancer cells overexpressing the NQO1 enzyme.

The present invention in order to solve the above problems,

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

[Formula 1]

.

.

According to the present invention, the cancer cell may be one in which NAD(P)H:quinone oxidoreductase-1 (NQO1) is overexpressed.

According to the present invention, the compound may be reduced by NQO1 to emit chemiluminescence.

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

The luminescent probe compound according to the present invention responds with excellent selectivity to cancer cells in which NQO1 is overexpressed, and has excellent photophysical properties and low cytotoxicity, thereby enabling effective imaging of cancer cells.

1(A) shows the results of a time-dependent chemiluminescence study of probe 1 (10 μM) upon incubation with ^{NQO1 (40 μgmL −1 ) and NAD(P)H (100 μM).} The intensity change at 515 nm was monitored as a function of time, and the inset represents the fundamental emission spectrum. (B) is a bar graph showing the selectivity of probe 1 for NQO1 in the presence of various analytes such as amino acids, biothiol, and reductase: CPR: cytochrome P450 reductase, GSR: glutathione-disulfide reductase, NRD: nitroreductase. (C) shows a time-lapse image of probe 1 over 10 min.
FIG. 2A shows an in vitro chemiluminescence image and signal intensity quantification of probe 1 incubated with A549 cells and H596 cells. (B) shows the time-dependent chemiluminescence intensity of probe 1 incubated with A549 cells. After 6 hours of incubation in the presence (yellow) or absence (orange) of 50 μM dicoumarol, A549 cells were treated with 10 μM of Probe 1 and chemiluminescence was measured for 2 hours. Chemiluminescence intensity was obtained using a microplate reader. (C) shows the signal-to-noise intensity ratio of probe 1 compared to _{Q Me NN.} NQO1-positive A549 cells were treated with 10 M probe 1 or Q _Me NN and the chemiluminescence (CL) of probe 1 or _{fluorescence intensity (FL) of Q Me} NN was measured as a function of time using a microplate reader.
3 shows in vivo and ex vivo images of xenograft models of A549 and H596 cells recorded using a non-invasive bioluminescence imaging system after treatment with probe 1. (A) Tumor-bearing nude mice (A549- or H596-cell-derived xenografts) were injected with probe 1 (40 μg in 100 μL PBS, 10% DMSO) or vehicle (control), and in vivo obtained 10 min later. This is a chemiluminescent image. (B) In vitro chemiluminescence images recorded 10 min after direct injection of probe 1 (40 μg in 100 μL PBS, 10% DMSO) into tumors excised from A549- and H596-cell-derived xenografts. Chemiluminescence images of probe 1 were obtained using a bioluminescence imaging system. For in vitro imaging studies, tumor masses were harvested from tumor-bearing mice 28 days after initial cell inoculation.
Figure 4 shows the luminescence intensity of probe 1 (10 μM) with NAD(P)H (100 μM) as a cofactor under conditions of various concentrations of NQO1 enzyme (λ _em = 515 nm, data recorded after 150 s of equilibration) .
Figure 5 shows the RP-HPLC chromatogram of probe 1 (10 μM) recorded before incubation with NQO1 (40 μg/mL) and NADH (100 μM) in PBS (red line: probe 1, blue line: benzo 8 (decomposed), black line: benzoated derivatives (synthetic)).
Figure 6 shows the cell viability of probe 1 in A549, H596 and WI-38 cells. After treatment of A549 (NQO1-positive, cancer), H596 (NQO1-negative, cancer) and WI-38 (NQO1-negative, normal lung) cells with probe 1 at 0, 1, 5, 10, 30 and 50 μM Incubated for 24 hours. Data values are expressed as mean±SD (n = 3). *p <0.05.
7 is a graph showing the protein expression of NQO1 in H596, A549 and WI-38 cells, and is a Western blot result showing the NQO1 expression level in H596, A549 and WI-38 cells.
8 shows the chemiluminescence image and signal intensity quantification results for probe 1 incubated with A549 cells for 2 hours in the presence or absence of 50 μM dicoumarol. Chemiluminescence intensities were obtained using a multiluminescence microplate reader.
9 shows in vitro images of NQO1-negative (H596 and WI-38) cells incubated with probe 1 in the presence or absence of dicoumarol. (A) shows the results of chemiluminescence image and signal intensity quantification of H596 cells incubated with probe 1. Chemiluminescence images were obtained using a bioluminescence imaging system. (B) shows the chemiluminescence intensity of H596 cells incubated with probe 1. After incubation for 6 h in the presence or absence of 400 μM dicoumarol, H596 cells were treated with 10 μM Probe 1 for 2 h. (C) shows the chemiluminescence intensity of WI-38 cells incubated with probe 1. After incubation for 6 h in the presence or absence of 400 μM dicoumarol, WI-38 cells were treated with 10 μM Probe 1 for 2 h. Chemiluminescence intensities were obtained using a multi-luminescence microplate reader.
10 shows the results of a time-dependent fluorescence study of _{Q Me} NN (10 μM) upon incubation with NQO1 (40 μg/mL) and NADH (100 μM).

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 of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Experimental method

Materials, methods and apparatus

Unless otherwise stated, reactions were carried out at room temperature. As the solvent, an analytical reagent from Duksan Pure Chemical Co., Ltd. was used. Thin layer chromatography (TLC) was performed on silica gel plates Merck 60 F254. Column chromatography purification was performed using silica gel 60 (particle size 0.040-0.063 mm) as a stationary phase. HPLC analysis was performed using a YL9101S (YL-Clarity) instrument equipped with a reversed-phase column (C18, 5mm, Waters). The eluent used in the analysis was a water-acetonitrile gradient (0-20 min; acetonitrile 70-100%) at a flow rate of 5.0 mL/min. A UV-vis detector (365 nm) was used. Fluorescence and UV-Vis absorption spectra were recorded with Shimadzu RF-5301PC and Agilent 8453 spectrophotometers, respectively. ESI mass spectral analysis was performed using LC/MS-2020 series (Shimadzu). DMSO and dicoumarol used for spectral and biological studies were purchased from Sigma-Aldrich (St. Louis, Mo, USA). H ₂ O was deionized. RPMI1640 medium and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan-si, South Korea) and Gibco BRL Life Technologies (ThermoFisher Scientific, Waltham, MA, USA), respectively. Terrell ^TM liquid (isoflurane) is manufactured by Piramal Critical Care, Inc. (Bethlehem, PA, USA), Matrigel™ was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Chemiluminescence images of microplates and mice (in vitro and in vivo) were obtained using a Spectral Lago X optical imaging system (Spectral Instruments Imaging; Tucson, AZ, USA) and an IVIS Spectrum In Vivo Imaging System (PerkinElmer; Waltham, MA, USA). was obtained using

compound synthesis

According to the following synthetic route, a compound of [Formula 1] (represented by compound 1) according to the present invention was synthesized.

[synthetic route]

Synthesis of compound 2

Compound 4 (200mg, 0.6mmol) and methy(triphenylphosphoranylidene)acetate (220mg, 0.66mmol) were dissolved in DCM (2ml). The mixture was stirred at room temperature for 15 min while monitoring by TLC (Hex: EtOAc 90:10). After reaction completion the reaction mixture was diluted with EtOAc (100 mL), washed with 0.5M HCl (50 mL) and brine (50 mL). The residue was purified by column chromatography on silica gel (Hex: EtOAc 85:15) to obtain a pale yellow solid compound 2 (211 mg, yield 91%).

¹ H NMR (400 MHz, CDCl ₃ ) δ7.94 (d, J = 16.2 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.61 ( d, J = 16.2 Hz, 1H), 6.28 (s, 1H), 3.84 (s, 3H), 3.35 (s, 3H), 3.27 (d, J = 4.9 Hz, 1H), 2.12 (s, 1H), 2.02-1.66 (m, 12H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ232.43, 206.72, 173.51, 167.74, 150.65, 139.23, 136.60, 132.96, 126.80, 123.82, 123.70, 121.97, 121.54, 119.77, 95.27, 57.19, 51.89, 39.19, 38.89, 39. 37.09, 32.95, 32.03, 29.78, 28.37, 24.44.MS (ES-): m/z calc. for C ₂₂ H ₂₅ ClO ₄ : 388.14; found: 387.4 [MH] ^- .

Synthesis of compound 3

N-(4-(hydroxymethyl)phenyl)-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl) at 0 °C To a mixture of butanamide (1.35 g, 3.98 mmol) and carbon tetrabromide (3.96 g, 11.9 mmol) in methylene chloride (30 ml) was added PhP (1.30 g, 4.77 mmol) and stirred at room temperature for 3 h. Purification by silica chromatography using heptane/ethyl acetate as an eluent gave compound 3 (yield 72%).

probe Synthesis of compound 1

Compound 2 (50 mg, 0.13 mmol) and K ₂ CO ₃ (35 mg, 0.26 mmol) were dissolved in DMF (1 mL) and stirred for 10 min before compound 3 (66 mg, 0.13 mmol) was added to the mixture . The reaction mixture was stirred at room temperature for 2 h and the reaction progress was monitored by TLC analysis (Hex/EtOAc: 90:10). When the reaction was complete, the volatiles were evaporated under reduced pressure. A mixture of THF, H ₂ O and NaOH (10 mg, 0.26 mmol) was then added. The resulting mixture was stirred at 80 °C for an additional 1 h. After confirming complete hydrolysis based on TLC analysis (Hex:EtOAc, 60:40), the mixture was diluted with EtOAc (100 mL) and washed with 0.5M HCl (50 mL). The organic layer was separated, washed with brine, dried over Na ₂ SO ₄ and evaporated under reduced pressure. The crude residue was then dissolved in DCM (20 mL) and then methylene blue (~1 mg) was added. Oxygen was bubbled into the solution while irradiating yellow light. The reaction was monitored by RP-HPLC (gradient of ACN in water, 0.1% TFA). After completion of the reaction (about 20 min), the mixture was concentrated under reduced pressure, and the product was purified by RP-HPLC (gradient of ACN in water, 0.1% TFA) to give probe compound 1 as a pale green solid (57 mg, yield). 59%).

¹ H NMR (400 MHz, CDCl ₃ ) δ8.00 (d, J = 16.2 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.57 ( d, J = 7.8 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 6.52 (d, J = 16.1 Hz, 1H), 4.97 (s, 2H), 3.23 (s, 3H), 3.15 ( s, 3H), 3.02 (s, 1H), 2.75 (s, 2H), 2.31 (d, J = 12.5 Hz, 1H), 2.07 (s, 3H), 1.98 (m, 6H), 1.93 (s, 3H) ), 1.87-1.54 (m, 9H), 1.46 (d, J = 10.9 Hz, 1H), 1.29 (s, 6H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ191.3, 187.8, 172.3, 170.9, 154.7, 154.4, 144.4, 143.6, 140.2, 138.0, 136.5, 135.9, 135.5, 131.3, 130.3, 129.4, 127.8, 125.7, 121.1, 111.8, 96.5, 77.5, 77.2, 76.9, 75.7, 49.8, 47.8, 38.2, 37.3, 36.7, 34.0, 33.7, 32.8, 32.3, 31.6, 29.8, 28.6, 26.2, 25.9, 14.2, 12.8, 12.2, 2.0. MS (ES-): m/z calc. for C ₄₃ H ₄₈ ClNO ₉ : 757.3; found: 756.6 [MH] ^- .

LOD determination

The detection limit was determined from a linear regression of the luminescence intensity for the NQO1 enzyme in the range 0-1 (μg/mL) (see Equation 1 below).

[Equation 1]

y = mX + C as

I = 43.450 [NQO1] + 1.0937

Hence LOD=3.3 x 0.6695/43.450=0.051 (μg/mL) using formula LOD = 3.3 x σ/m

m = slope of calibration curve (43.450)

σ = standard deviation of blank readings (0.6695)

cell culture

A549 (human lung epithelial adenocarcinoma) and H596 (human lung adenosquamous carcinoma) cells were grown in RPMI1640 with FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin in a humid atmosphere containing _{5% CO 2 at 37° C.} .

Assessing cell viability

Approximately 2x10 ⁴ cells were seeded in 96 well microplates (SPL Life Science) and incubated for 24 hours. After incubation, cells were treated with DMSO as a control or with probe compound 1 for 24 h. To assay the cell viability of cells in the presence and absence of probe compound 1, the CytoTox96® non-radioactive cytotoxicity assay kit (Promega) was used according to the manufacturer's instructions. Fluorescence levels were measured using a SPECTRA MAX GEMINI EM microplate reader (Molecular Devices). The wavelength was set to 490 nm. Cell viability assays were performed in triplicate and viability (%) was expressed as a percentage of the measured absorbance relative to control cells.

western blotting analysis

The protein expression level of NQO1 was measured by Western blotting. Briefly, the adherent cells of the cell line were washed 3 times with cold PBS and scraped to collect the cell pellet. After removal of PBS, a radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors provided by the manufacturer (Up-State) was added to the cell pellet to obtain protein lysates. BCA analysis was performed to measure the protein concentration of each cell line, and then, the protein (30 μg/lane) of each cell line was loaded on SDS-PAGE to separate the protein into bands. The separated protein band was transferred to a PVDF membrane (Merck Millipore). The membranes were incubated with NQO1 antibody (Santa Cruz Biotechnology) at 1/1000, or GAPDH antibody (Santa Cruz Biotechnology) at 1/1000 or diluted overnight at 4°C. The resulting membrane was washed with Tris-buffered saline containing Tween-20 (TBS-T), and then incubated with an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 hours at room temperature. . To detect immunoreactive protein bands, enriched chemiluminescent reagents (Luminate, Merck Millipore) were used according to the manufacturer's instructions.

in vitro (In vitro) Chemiluminescence imaging

A549 and H596 cells were seeded in 24-well plates. After incubation for 24 h, cells were incubated for 6 h in the presence or absence of the NQO1 inhibitor, 50 μM dicoumarol (1.04 mM NaOH). Then, 10 μM of probe 1 in RPMI1640 medium was added to each well, and a chemiluminescence image was obtained using a bioluminescence imaging system.

in vivo (In in vivo ) chemiluminescence imaging

Specific pathogen-free, 5-week-old female BALB/c nude mice (Japan SLC, Inc., Hamamatsu, Shizuoka, Japan) were housed under controlled temperature (22±2 °C) conditions and allowed to undergo a light/dark cycle for 12 h. did. Mice were maintained in a Korea University animal facility under specific pathogen-free conditions and received humane care according to the guidelines of the Korea University Animal and Animal Care Committee. ^{A549 and H596 cells (5x10 5} cells/100 μL) in serum-free medium containing 50% Matrigel™ were injected subcutaneously. Mice were randomized into two groups (n=5 per group) for each cell line. On day 28, mice ^{were anesthetized with Terrell™} liquid (isoflurane) and injected intratumorally, followed by bioluminescence by injection of a solution of Probe 1 (40 μg) in 100% PBS, 10% DMSO for 10 min for 10 min. Images were obtained. For control, mice were treated with 100 μL PBS, 10% DMSO vehicle. In vivo chemiluminescence images of probe 1 and negative control were obtained using a Spectral Lago X optical imaging system.

ex-vibo (Ex in vivo ) chemiluminescence imaging

Ex vivo imaging studies using probe compound 1 were performed using the aforementioned tumor xenograft model. On day 28 post-inoculation, mice were sacrificed and tumor tissue was immediately removed from the body. Then, probe compound 1 (40 μg/100 μL) in 10% DMSO, PBS was directly injected into the tumor mass. Similar injections were performed using DMSO/PBS (1:9) vehicle for the control group. In vitro chemiluminescence images were obtained using a Spectral Lago X imaging system.

Results and Discussion

In the present invention, a novel chemiluminescent compound (Probe 1) that enables differential detection of NQO1 activity in various cancer cells was synthesized, and the ability of Probe 1 to detect NQO1 in various cancer cells was tested. The chemical structure and NQO1-mediated chemiluminescence mechanism of probe 1 are shown in [Synthetic route] and [chemiluminescence mechanism below], respectively.

[Mechanism of chemiluminescence of probe 1]

Compound 1 according to the present invention consists of an acrylic acid-substituted phenoxy-dioxetane covalently bonded to a trimethyl-locking quinone via a para-aminobenzyl alcohol self-condensing linker. The phenoxy-dioxetane moiety is designed to produce chemiluminescence: phenolates are formed under physiological conditions, which spontaneously undergo a chemiluminescent process to produce green light. For probe 1, the essential free phenolate is produced upon NQO1-mediated reduction of the quinone, followed by intramolecular lactonation and removal of the benzylaniline tether.

Next, the chemiluminescent emission of probe 1 as a function of time was tested in the presence of NQO1. Unless otherwise indicated, all cell-free experiments were performed at 37°C in PBS (pH 7.4, 0.5% DMSO). Probe 1 itself is non-emissive. However, a typical chemiluminescence reaction was observed at 515 nm with a 130-fold increased signal intensity in the presence of NAD(P)H (100 μM) when treated with NQO1 (40 μgmL ^{− 1) ( FIG. 1A ).} The minimum limit of detection (LOD) of probe 1 for NQO1 ^{was found to be 51 ngmL −1} ( FIG. 4 ). As expected, a sharp increase in signal intensity was observed during the first 5 min, followed by a slow decrease in chemiluminescence output (Fig. 1C).

Next, the selectivity of Probe 1 for NQO1 was tested in the presence of other biologically abundant amino acids, bio-thiols and reductases, which are expected to result in cleavage of the central thther and release of the active phenolate form. As a result of the test, as shown in FIG. 1B , probe 1 showed chemiluminescence only in the presence of NQO1, and did not show chemiluminescence when exposed to other putative chemical or biological triggers.

Next, in order to confirm the chemiluminescence mechanism of Probe 1, a substance produced from Probe 1 was analyzed in the presence of NQO1 and NAD(P)H using high performance liquid chromatography (HPLC) (FIG. 5). Probe 1 itself showed a peak at t = 13 min in the HPLC chromatogram (C18 column; 70-100% gradient of acetonitrile in water, 0.1% TFA), and was incubated with NQO1 and NAD(P)H for 30 min. Later, the peak corresponding to hydroxy benzoate (compound 5) was exclusively observed (see chemiluminescence mechanism of probe 1). Through these results, as shown in the chemiluminescence mechanism above, probe 1 acts as an effective substrate for the mixture consisting of NQO1 and NAD(P)H, which is efficient to generate a final chemical species corresponding to the observed green luminescence. It was confirmed that it undergoes enzymatic reduction and rapid degradation.

Next, the ability of Probe 1 to generate an NQO1-mediated chemiluminescence response was further investigated in an in vitro model. For these studies, A549 (human lung epithelial adenocarcinoma), H596 (human lung adenocarcinoma) and WI-38 (normal lung) cell lines were selected. Under standard culture conditions (cell viability assay), probe 1 showed no significant cytotoxicity up to concentrations of 50 μM. These results were shown in all cell lines tested, and through this, it was possible to confirm the safety of the probe according to the present invention (FIG. 6). Next, the protein expression level of NQO1 was measured in these cell lines by Western blot analysis ( FIGS. 2B and 7 ). First, as previously reported (EA Bey, MS Bentle, KE Reinicke, Y. Dong, CR Yang, L. Girard, JD Minna, WG Bornmann, JM Gao, DA Boothman, Proc. Natl. Acad. Sci. USA 2007 , 104, 11832-11837), NQO1 levels were found to be substantially different between the A549, H596 and WI-38 cell lines, and approximately 10-fold higher in A549 cells. These differences allowed us to test whether NQO1 plays a role in mediating the level of chemiluminescence generated by probe 1 in vitro.

Next, A549, H596 and WI-38 cells were incubated with probe 1 at a concentration of 10 μM for 10 min and imaged using a bioluminescence imager. As shown in Figures 2A and 8, a strong signal was observed from the A549 cell where a maximum of a 34-fold increase in the release response was observed. The chemiluminescence signal intensity increased during the first 5 min of the assay, then persisted for about 10 min, and then slowly decreased for 40-60 min (Fig. 2B). In contrast, no such release response was observed for H596 and WI-38 cells with low NQO1 levels (or NQO1-negative) ( FIGS. 2A and 9 ). These results are consistent with the results of overexpression of NQO1 levels in A549 cells deduced from western blotting experiments, suggesting that this enzyme plays a very important role in the activation of probe 1. This was also evidenced by the result that the chemiluminescence intensity was significantly decreased in the experiment in which A549 cells were pre-incubated with dicoumarol (chemical NQO1 inhibitor) and then treated with probe 1 (FIGS. 2B, 8-9).

_{Luminescent emission of probe 1 was compared with Q Me} NN (GG Dias, A. King, F. de Moliner, M. Vendrell, EN da Silva, Chem. Soc), a conventional naphthalimide-based probe that has been used to monitor NQO1 activity in A549 cells. Rev. 2018, 47, 12-27) compared with the fluorescence response (FIG. 10). As shown in Fig. 2C, the chemiluminescent probe 1 according to the present invention generated a significantly higher signal-to-noise ratio than the _{fluorescent probe Q Me NN over 120 min.} Taken together, these results suggest that probe 1 may be useful for imaging of NQO1-overexpressed cancerous lesions, and can differentiate NQO1-positive tumors from normal tissues or tumors with low NQO1-expression levels.

To test the ability of probe 1 to visualize NQO1 activity in vivo, isolated xenografts derived from the A549 and H596 cell lines were grown subcutaneously in the right flank of BALB/c nude mice. Probe 1 (40 μg in 100 μL PBS, containing 10% DMSO) was injected intratumorally. In vivo chemiluminescence images were collected at 515-575 nm using a bioluminescence imaging system. As shown in Figure 3A, A549-tumor bearing mice showed strong chemiluminescent emission across the tumor area. No discernible signal was observed for xenografts from the corresponding NQO1-negative H596-cell line. Ex vivo image analysis of A549 tumors was also performed. As a result, the chemiluminescent signal was present only in the A549 tumor, whereas no prominent signal was observed in the various controls ( FIG. 3B ).

In summary, the present invention provides a novel chemiluminescence probe 1 that generates a light emission reaction that is easy to visualize following NQO1 enzymatic reduction. In the test described above, probe 1 showed an almost instantaneous response when treated with a combination of NQO1 and NAD(P)H at physiological pH. Compared with the blank control, under these conditions, probe 1 showed up to 130-fold improvement in luminescence intensity. This reaction is due to bio-reduction of the quinone moiety followed by cyclization to release the active chemiluminescent phenoxydioxetane. In addition, from the result that the chemiluminescence reaction did not occur with respect to the general bioanalytes, it was confirmed that the enzymatic reduction is decisive for the function of Probe 1. In addition, several in vitro and in vivo studies confirmed the ability of Probe 1 to discriminate between human lung cancers with different levels of NQO1 activity. In particular, probe 1 was found to produce a strong response in A549 cells and mouse xenografts in which the NQO1 enzyme was expressed at high levels. In contrast, no significant response was seen in the NQO1-negative H596 cell line and in xenograft mouse tumors. Through these results, it was confirmed that probe 1 according to the present invention can be useful for optical monitoring of NQO1 activity in a preclinical environment. In addition, Probe 1 according to the present invention is expected to be useful for early diagnosis of NQO1-overexpressed lung cancer, surgical or clinical lesion identification, and the like.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (4)

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

. According to claim 1,
The cancer cell is a luminescent probe compound for detecting cancer cells, characterized in that NAD(P)H:quinone oxidoreductase-1 (NQO1) is overexpressed. According to claim 1,
The compound is a luminescent probe compound for detecting cancer cells, characterized in that it is reduced by NQO1 to emit chemiluminescence. A light emitting sensor for detecting cancer cells comprising the light emitting probe compound according to claim 1 .

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