Dual fluorescence probe for detecting nitroreductase and hydrogen sulfide, and manufacturing method therefor

Abstract

The present invention relates to a dual fluorescence probe for detecting nitroreductase and hydrogen sulfide, and a manufacturing method therefor. The dual fluorescence probe according to the present invention can detect both nitroreductase and hydrogen sulfide, which are indicators of a hypoxic environment, and can provide low background fluorescence noise and selective, high fluorescence signals, thus enabling normal cells and tumor cells in a hypoxic microenvironment to be accurately distinguished and detected through improved hypoxia detection effects and imaging capabilities.

Description

Dual fluorescent probe for detecting nitroreductase and hydrogen sulfide and method for preparing the same

[0001] The present invention relates to a dual fluorescent probe for detecting nitroreductase and hydrogen sulfide, a method for producing the same, and a method for detecting hypoxia and diagnosing cancer using the fluorescent probe of the present invention.

[0002]

[0003] Hypoxia, a condition in which intracellular and intercellular oxygen concentrations are lower (typically less than 3%) than the normal ambient oxygen concentration (21% oxygen), is commonly observed in various diseases such as ischemic anemia, vascular disease, and cancer. In mammalian cells, hypoxia-inducible factor-1α (HIF-1α) is expressed as a key transcription factor in response to low oxygen levels, activating the expression of various hypoxia-responsive genes, such as angiogenic growth factors and vascular endothelial growth factors. This establishes a hypoxic microenvironment distinct from normoxia, leading to changes in cellular metabolism and behavior. For example, hypoxia is a key regulator of tumor growth, triggering transcriptional programs that promote tumor cell proliferation, migration, and invasion, and is associated with increased resistance to radiotherapy and chemotherapy. Consequently, the degree of hypoxia reflects an unfavorable prognosis in cancer treatment, making hypoxia an important target for the diagnosis and treatment of various diseases.

[0004] Meanwhile, nitroreductase (NTR) is known to catalyze the reduction of the nitro group of aromatic substrates to amines. This reduction reaction is particularly activated in cancer hypoxia, which is closely associated with cancer treatment resistance and poor prognosis. Consequently, nitroreductase has been recognized as a potential biomarker for hypoxic tumors and an important target for prodrug development.

[0005] However, relying solely on nitroreductases may not provide a comprehensive understanding of the complex hypoxic microenvironment within tumors. Recent studies have revealed abnormal increases in hydrogen sulfide (H2S) levels during tumor hypoxia, and H2S has been implicated in tumor pathological effects, proliferation, and apoptosis. Specifically, H2S plays a crucial role in HIF-1α- and vascular endothelial growth factor-dependent angiogenesis, as well as in mitochondrial respiration-regulated energy homeostasis. Therefore, the ability to further investigate hypoxia-related bioreductive species, particularly changes associated with cancer hypoxia, could provide novel insights into fundamental aspects of hypoxia in both physiological and pathological contexts.

[0006] In recent years, numerous bioprobes have been developed to deeply investigate hypoxia in living systems. An attractive approach currently pursued in supramolecular and medicinal chemistry is the development of fluorescent probes that provide both quantitative analysis of hypoxia-related biomarkers and fluorescence imaging. To date, numerous chemical probes have been developed to detect nitroreductase activity, demonstrating the ability to distinguish between cancer and normal cells, thereby indicating the degree of hypoxia in various biological models. Chemical probes that detect hydrogen sulfide have also been developed and applied to various pathological models, including cancer cells. However, dual-fluorescent probes capable of detecting both nitroreductase and hydrogen sulfide remain unknown.

[0007] As a prior study on a fluorescent probe for detecting hypoxia, Korean Patent Publication No. 10-2024-0020584 discloses an AzNap compound containing a naphthalimide group, and discloses that the compound is activated in a hypoxic environment and can target cancer cells or cancer stem cells, and Korean Patent Publication No. 10-2018-0115992 discloses a fluorescent probe for detecting hydrogen sulfide that can selectively detect hydrogen sulfide.

[0008] However, there are no known prior studies that have disclosed a dual fluorescent probe capable of detecting both nitroreductase and hydrogen sulfide.

[0009] Accordingly, the inventors of the present invention have developed a dual-locked fluorescent probe that reacts to both nitroreductase and hydrogen sulfide. This dual-locked fluorescent probe provides low background fluorescence noise and highly selective fluorescence signals in hypoxic environments, demonstrating a very high signal-to-noise ratio (SNR) and demonstrating hypoxia detection and imaging capabilities compared to single-probing systems. Therefore, the dual-locked fluorescent probe of the present invention is expected to facilitate a better understanding of the complexities associated with hypoxic microenvironments and to more accurately distinguish between normal and diseased cells.

[0010]

[0011] One object of the present invention is to provide a compound capable of detecting nitroreductase and / or hydrogen sulfide.

[0012] Another object of the present invention is to provide a method for producing the compound.

[0013] Another object of the present invention is to provide a detection probe comprising the compound.

[0014] Another object of the present invention is to provide a method for detecting hypoxia using the compound.

[0015] Another object of the present invention is to provide a method for diagnosing cancer using the compound.

[0016]

[0017] One aspect of the present invention to achieve the above purpose provides a compound represented by the following chemical formula 1 or a salt thereof:

[0018] [Chemical Formula 1]

[0019]

[0020] (In the above chemical formula 1, p is 1 or 2, and q is an integer from 1 to 3.)

[0021] Another aspect of the present invention is, as shown in the following reaction scheme 1,

[0022] A method for producing a compound represented by Chemical Formula 1 is provided, comprising the step of producing a compound of Chemical Formula 1 by reacting a compound of Chemical Formula A and a compound of Chemical Formula B:

[0023] [Reaction Formula 1]

[0024]

[0025] (In the above reaction formula 1, p and q are defined according to the chemical formula 1 described above.)

[0026] Another aspect of the present invention provides a detection probe capable of detecting at least one of hydrogen sulfide and nitroreductase, comprising a compound represented by the above chemical formula 1 or a salt thereof.

[0027] Another aspect of the present invention provides a method for detecting intracellular hypoxia, comprising the following steps:

[0028] A step (step 1) of injecting the compound of claim 1 or a salt thereof into a cell to be detected; and

[0029] Step 2: Detecting intracellular fluorescence.

[0030] Another aspect of the present invention provides a method for diagnosing cancer, comprising the following steps:

[0031] A step of injecting the compound of claim 1 or a salt thereof into a cell to be diagnosed (step 1); and

[0032] Step 2: Detecting intracellular fluorescence.

[0033]

[0034] The dual fluorescent probe according to the present invention can detect both nitroreductase and hydrogen sulfide, which are indicators of a hypoxic environment, and can provide low background fluorescence noise and a selective, high fluorescence signal. This dual detection system exhibits enhanced hypoxia detection and imaging capabilities, enabling accurate differentiation and detection of normal and tumor cells in hypoxic microenvironments.

[0035]

[0036] Figure 1 illustrates the fluorescence change mechanism of the dual fluorescent probe (DNNC) of the present invention.

[0037] Figure 2a shows the UV / Vis absorption spectra of DNNC, Naph, NNC, and DN (1 μM each).

[0038] Figure 2b shows the fluorescence spectra of DNNC, Naph, NNC, and DN (each 1 μM).

[0039] Figure 2c is a diagram showing the fluorescence response of DNNC, NNC, and DN in the absence and presence of NaHS and / or NTR.

[0040] Figure 3a shows the absorption spectrum of DNNC (1 μM) according to the NaHS concentration (0-900 μM).

[0041] Figure 3b shows the fluorescence spectrum of DNNC (1 μM) according to the NaHS concentration (0-900 μM).

[0042] Figure 3c shows a fluorescence intensity plot of DNNC (1 μM) according to NaHS concentration (0-900 μM).

[0043] Figure 4a shows the absorption spectrum of DNNC (1 μM) according to NTR concentration (0-10 μM).

[0044] Figure 4b shows the fluorescence spectrum of DNNC (1 μM) according to NTR concentration (0-10 μM).

[0045] Figure 4c shows a fluorescence intensity plot of DNNC (1 μM) according to NTR concentration (0-10 μM).

[0046] Figure 5a shows the absorption spectrum of DNNC (1 μM) when various concentrations of NTR (0-10 μg / mL) were sequentially added in the presence of NaHS (500 μM).

[0047] Figure 5b shows the fluorescence spectrum of DNNC (1 μM) when various concentrations of NTR (0-10 μg / mL) were sequentially added in the presence of NaHS (500 μM).

[0048] Figure 5c shows a fluorescence intensity plot of DNNC (1 μM) when various concentrations of NTR (0-10 μg / mL) were sequentially added in the presence of NaHS (500 μM).

[0049] Figure 6a shows the absorption spectrum of DNNC (1 μM) when various concentrations of NaHS (0-800 μM) were sequentially added in the presence of NTR (5 μg / mL).

[0050] Figure 6b shows the fluorescence spectrum of DNNC (1 μM) when various concentrations of NaHS (0-800 μM) were sequentially added in the presence of NTR (5 μg / mL).

[0051] Figure 6c shows a fluorescence intensity plot of DNNC (1 μM) when various concentrations of NaHS (0-800 μM) were sequentially added in the presence of NTR (5 μg / mL).

[0052] Figure 6d shows the normalized UV / Vis absorption (dotted line) and fluorescence (solid line) spectra of Naph (black) and DNNC (red).

[0053] Figure 7a shows the change in fluorescence over time of DNNC (1 μM) when NaHS (500 μM) is added, and Figure 7b shows the change in fluorescence over time of DNNC (1 μM) when NTR (5 μg / mL) is sequentially added to the solution of Figure 7a.

[0054] Figure 7c shows the change in fluorescence over time of DNNC (1 μM) when NTR (5 μg / mL) and NADH (500 μM) were added, and Figure 7d shows the change in fluorescence over time of DNNC (1 μM) when NaHS (500 μM) was sequentially added to the solution of Figure 7c.

[0055] Figure 8 shows the change in fluorescence intensity over time when H2S (500 μM), NTR (5 μg / mL), and NADH (500 μM) were added simultaneously.

[0056] Figure 9 shows the fluorescence response to various biocomponents (100 μM each; 1 mM for GSH and glucose); (1) DNNC, (2) Na + , (3) K + , (4) Mg 2+ , (5) Ca 2+ , (6) Cu 2+ , (7) Zn 2+ , (8) Fe 2+ , (9) Fe 3+ , (10) Co 2+ , (11) ·O2 - , (12) ·OH, (13) ·O t Bu, (14) H2O2, (15) HOOt Bu, (16) ClO - , (17) Cys, (18) Hcy, (19) GSH, (20) Na2CO3, (21) ascorbic acid, (22) glucose, (23) NADH, (24) NTR, (25) NaHS, (26) NTR+NADH+NaHS.

[0057] Figure 10 shows the change in fluorescence intensity according to pH in the absence and presence of H2S and NTR.

[0058] Figure 11 shows the HPLC profiles of DNNC (black), Naph (blue), and DNNC+NaHS+NTR (red).

[0059] Figures 12a to 12c show confocal microscopy fluorescence images of DNNC (10 μM), NNC (10 μM), and DN (10 μM) in HeLa cells under normoxic and hypoxic conditions (scale bar = 50 μm), and the lower images are overlays of fluorescence and non-fluorescent phase contrast images.

[0060] Figure 12d shows the quantification of the fluorescence intensity changes of DNNC (10 μM), NNC (10 μM), and DN (10 μM) in HeLa cells according to oxygen concentration.

[0061] Figure 13 shows confocal microscopy fluorescence images of DNNC in the control group, AOAA and PAG treatment group (AOAA / PAG 1 mM), or Dic treatment group (Dic 400 μM) (scale bar = 50 μm).

[0062] Figure 14 shows the quantification of the fluorescence intensity of DNNC (10 μM) in various cancer cell lines under normoxic and hypoxic conditions.

[0063] Figure 15 shows the results of cytotoxicity analysis of DNNC in HeLa cells.

[0064] Figure 16a shows a Z-stack fluorescence image of DNNC in HeLa cell spheroids.

[0065] Figure 16b shows the evaluation of the viability of 3D spheroids (scale bar = 100 μm).

[0066] Figure 17a shows the protein expression of H2S generating enzyme and HIF-1α in various cancer cell lines under normoxic conditions.

[0067] Figure 17b shows the protein expression of H2S generating enzyme and HIF-1α in various cancer cell lines under CoCl2-induced hypoxia.

[0068]

[0069] Hereinafter, the present invention will be described in detail.

[0070] One aspect of the present invention provides a compound represented by the following chemical formula 1 or a salt thereof.

[0071] [Chemical Formula 1]

[0072]

[0073] In the above chemical formula 1, p is 1 or 2, q is an integer from 1 to 3, and in another specific example, p is 1 and / or q is 1.

[0074] In the present specification, the compound of formula 1 may be provided in the form of a salt, which is a diagnostically acceptable salt, and may typically be provided in the form of a salt ionically bonded with an inorganic acid or organic acid that can ionically bond with nitrogen. Examples of the inorganic acid include hydrochloric acid, hydrofluoric acid, nitric acid, sulfuric acid, etc., and examples of the organic acid include formic acid, acetic acid, trifluoroacetic acid, etc.

[0075] In a specific example, the compound of the above chemical formula 1 may be represented by the following chemical formula.

[0076]

[0077] In this specification, the compound of the above chemical formula is referred to as DNNC.

[0078] The compound of the above chemical formula 1 can function as a probe capable of simultaneously detecting hydrogen sulfide (H2S) and nitroreductase, which are indicators of a hypoxic environment in vivo. In the present invention, the term "probe" is also referred to as a "sensor" and is defined as something capable of detecting or imaging a target in vivo / ex vivo.

[0079] The compound of formula 1 according to the present invention, exemplarily represented by DNNC in the present specification, comprises a naphthalimide skeleton, which is a representative intramolecular charge transfer (ICT)-based fluorophore that emits strong fluorescence, and a nitrobenzyl carbamate (NC) group sensitive to nitroreductase (NTR) and a nitrobenzoxadiazole (NBD) group sensitive to hydrogen sulfide (H2S) as key components that lock fluorescence emission. Here, the NC group weakens the ICT phenomenon of naphthalimide, thereby inducing fluorescence attenuation, and the NBD group acts as a fluorescence quencher through photoinduced electron transfer (PET). As a result of this dual locking strategy, DNNC does not exhibit background fluorescence, but reacts with hydrogen sulfide to dissociate the nitrobenzoxadiazole (NBD) group, and reacts with nitroreductase to dissociate the nitrobenzyl carbamate (NC) group, thereby exhibiting green fluorescence (Fig. 1).

[0080] The compound of chemical formula 1 according to the present invention reacts with both hydrogen sulfide and nitroreductase, thereby exhibiting stronger fluorescence than a probe that can detect only one of them. In other words, the compound of chemical formula 1 according to the present invention functions as a dual fluorescent probe.

[0081]

[0082] Another aspect of the present invention is:

[0083] As shown in the following reaction scheme 1,

[0084] A method for producing a compound represented by Chemical Formula 1 is provided, comprising the step of producing a compound of Chemical Formula 1 by reacting a compound of Chemical Formula A and a compound of Chemical Formula B:

[0085] [Reaction Formula 1]

[0086]

[0087] In reaction formula 1, p and q are defined according to chemical formula 1.

[0088] The above manufacturing method is not limited to one embodiment of the present invention presented as an example, and can be performed by modifying the solvent, reactant, temperature conditions, etc. under general organic chemical knowledge.

[0089] The above reaction is carried out using an organic solvent, such as DMF or DMSO, which is not particularly limited, and may be carried out in the presence of a base, an amine base, or an inorganic base. The amine base preferably includes a tertiary amine, such as triethylamine or pyridine. The reaction temperature is carried out at room temperature, but is not particularly limited, and the reaction time can be appropriately selected by a person skilled in the art to a degree sufficient to complete the reaction, and for example, the reaction may be carried out for 6 hours or more.

[0090]

[0091] Another aspect of the present invention is:

[0092] A method for detecting intracellular hydrogen sulfide, nitroreductase or hypoxia is provided, comprising the following steps:

[0093] A step (step 1) of injecting the compound of claim 1 or a salt thereof into a cell to be detected; and

[0094] Step 2: Detecting intracellular fluorescence.

[0095] Cells, especially cancer cells, experience hypoxia, which increases the presence of hydrogen sulfide and nitroreductase within the cell. Therefore, detecting these compounds can help diagnose hypoxia and, by extension, whether the cell is cancerous.

[0096] Another aspect of the present invention is that cancer cells are accompanied by hypoxia.

[0097] A method for diagnosing cancer is provided, comprising the following steps:

[0098] A step (step 1) of injecting the compound of claim 1 or a salt thereof into a cell to be diagnosed; and

[0099] Step 2: Detecting intracellular fluorescence.

[0100] Fluorescence detection can be accomplished through a variety of methods. These include obtaining fluorescence images of cells using the naked eye or an optical microscope, or using a spectrophotometer to obtain wavelength-dependent absorption and fluorescence spectra. Meanwhile, detection of hydrogen sulfide and nitroreductase can be performed qualitatively or quantitatively. For detection, absorption or fluorescence spectra are prepared in advance according to the presence and concentration of each, allowing for the estimation of presence and quantitative concentration.

[0101] Qualitative detection or diagnosis can be achieved by detecting fluorescence changes after intracellular injection based on information about fluorescence changes of DNNCs.

[0102] The compound represented by the above chemical formula 1 can increase the fluorescence intensity by 6 times or more, 10 times or more, and preferably 12 times or more under hypoxic conditions compared to normal oxygen conditions.

[0103] The above cancer may be one or more selected from the group consisting of brain cancer, pituitary adenoma, acoustic neuroma, uveal malignant melanoma, meningioma, pharyngeal cancer, laryngeal cancer, tongue cancer, thyroid cancer, breast cancer, lung cancer, thymoma, thymic cancer, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, renal pelvis / ureter cancer, penile cancer, testicular (testicular) tumor, cervical cancer, ovarian cancer, vulvar cancer, skin cancer, malignant melanoma (skin), basal cell carcinoma, precursor of skin cancer, intraepidermal carcinoma, spinous cell carcinoma, mycosis fungoides, malignant bone tumor (osteosarcoma), soft part sarcoma, chondrosarcoma, and malignant fibrous histiocytoma, but is not limited thereto.

[0104] The above fluorescence may be green fluorescence exhibiting maximum intensity at a central wavelength of 540-555 nm, specifically 547 nm, and may exhibit fluorescence over a range of 495 to 600 nm.

[0105]

[0106] In specific embodiments and experimental examples of the present invention,

[0107] In the present invention, a fluorescent probe DNNC using a dual locking system for naphthalimide fluorescence and fluorescent probes DN and NNC using a single locking system were prepared, and through analysis of the absorption and fluorescence properties of the synthesized DNNC, Naph, NNC, and DN, it was confirmed that the NC group caused a blue shift to fix the naphthalimide emission at 550 nm, and the NBD group suppressed the naphthalimide emission intensity. In addition, it was confirmed that the dual locking system achieved an excellent signal-to-noise ratio due to a very low background signal compared to the single locking system (Figs. 2a to 2c).

[0108] In the present invention, the reaction of the synthesized DNNC with H2S or NTR was analyzed, and it was confirmed that the fluorescence intensity increased at 475 nm and 547 nm, respectively (Figs. 3a to 3c and Figs. 4a to 4c), and it was confirmed that a fluorescence signal was generated at 547 nm when reacting simultaneously with H2S and NTR (Figs. 5a to 5c and Figs. 6a to 6d). In addition, considering the reaction rate of DNNC with H2S and / or NTR, the fluorescence-based kinetics was analyzed, and it was confirmed that a unique fluorescence intensity appeared at 547 nm regardless of the reaction order due to the coexistence of NTR and H2S (Figs. 7a to 7d and Fig. 8). Furthermore, to confirm the selectivity of DNNC for H2S and NTR, the fluorescence response to various biologically abundant species including metal ions, reactive oxygen species, thiols, and other substrate acids was analyzed, confirming that DNNC can selectively detect H2S and NTR without interference of pH changes in various biological components and systems (Figs. 9 and 10). Furthermore, HPLC analysis verified that in the presence of both H2S and NTR, the NBD and NC groups of DNNC dissociated to form NNC and DN, which were ultimately converted to Naph, exhibiting strong green fluorescence (Fig. 11).

[0109] In the present invention, it was confirmed through confocal fluorescence imaging that DNNC can effectively detect hypoxia by H2S and NTR reactions in various cancer cell lines (Figs. 12a to 12d and 13, Fig. 14), and that there was no cytotoxicity (Fig. 15).

[0110] In addition, the present invention formed HeLa cancer cell spheroids to mimic in vivo hypoxic tumor tissue, and confirmed that DNNC can effectively visualize hypoxic regions without cytotoxicity (Figs. 16a and 16b).

[0111] Furthermore, the present invention analyzed the expression of H2S generating proteins and hypoxia-inducible factor (HIF-1α) in various cell lines under normoxic and hypoxic conditions, demonstrating that the biological model presented in the present invention effectively represents hypoxic conditions and accurately reflects specific pathological conditions characterized by simultaneous overexpression of H2S and NTR activities (Figs. 17a and 17b).

[0112]

[0113] Hereinafter, the present invention will be described in detail through examples and experimental examples.

[0114] However, the following examples and experimental examples are only intended to specifically illustrate one aspect of the present invention, and the present invention is not limited to the following examples and experimental examples.

[0115]

[0116] Materials and equipment

[0117] All chemical reagents for synthesis and analysis, including chloride salts of metal ions, thiols (Cys, Hcy, GSH, and NaHS), and PBS buffer, were purchased from TCI (TCI, Tokyo, Japan), Sigma-Aldrich (Aldrich, St. Louis, MO, USA), and Alfa (Alfa, Heysham, LA3 2XY, United Kingdom). All solvents used in the synthesis and spectroscopic experiments were of HPLC grade without purification.

[0118] UV / Vis absorption and fluorescence spectra were obtained on a UV-2600 (Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) and RF-6000 (Shimadzu Corporation, Kyoto, Kyoto Prefecture, Japan) spectrophotometer, respectively.

[0119] HPLC analysis was performed using a Thermo Scientific Acclain TMThe analysis was performed on a Shimadzu HPLC (Shimadzu LC 6AD) using a 120 C18 (3 μm, 120 Å, 2.1Υ150 mm) column. The flow rate was 0.5 mL / min, and buffer A (water containing 0.1% v / v trifluoroacetic acid) and buffer B (acetonitrile containing 0.1% v / v trifluoroacetic acid) were used as mobile phases.

[0120] NMR spectra were performed on a Bruker (500 MHz) instrument.

[0121]

[0122] Statistical analysis

[0123] To analyze statistical significance, a statistically significant sample size was selected for all quantitative results to allow for significant differences between the control and treatment groups. All experiments were performed three times in triplicate, with three independent experiments, and the results are presented with the standard error of the mean. To determine statistical significance, SAS software (version 8.2, Cary, NC, USA) with one-way and two-way analysis of variance (ANOVA) functions was used. If the ANOVA results indicated a significant difference, a two-tailed Student's t-test was applied using SAS or Origin software packages (version 9.4, Northampton, MA, USA). Significant P values ​​(P < 0.05) are indicated by an asterisk.

[0124]

[0125] <Example 1> Synthesis of compound DNNC

[0126]

[0127] Synthesis of compounds d, c, and b

[0128] Compounds d, c and b were synthesized according to procedures reported in the literature (Zhang, C.; Wang, R.; Cheng, L.; Li, B.; Xi, Z.; Yi, L.Sci. Rep.2016, 6, 30148. DOI: 10.1038 / srep30148).

[0129] Synthesis of compound a

[0130] Compound b (0.06 g, 0.15 mmol) and 4-Dimethylaminopyridine (DMAP) (0.08 g, 0.65 mmol) were dissolved in dry dichloromethane (DCM) (20 mL) using an ice-salt bath and stirred for 10 min. Then, phosgene solution (15 wt% in toluene, 1 mL, 0.9 mmol) (Caution: toxic) was added dropwise over 30 min and stirred for another 30 min at room temperature. After the reaction was complete, the reaction mixture was bubbled with N2 gas to remove excess phosgene. Then, 4-nitrobenzyl alcohol (0.21 g, 1.37 mmol) dissolved in DCM was added to the reaction mixture and stirred overnight. Upon completion of the reaction, the mixture was poured into water and extracted three times with DCM. The collected organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using DCM / ethyl acetate (10:0 to 10:1, v / v) as the eluent. The collected compound was dried to obtain compound a (0.07 g, 85%).

[0131] HR-ESI-MS [MH]- calc. 573.1991 m / z, [MH]- obs. 573.1990 m / z. 1H NMR (CDCl3, 500 MHz): δ 1.49 (s, 9H); 1.61 (d, J = 13 Hz, 2H); 2.67-2.69 (m, 2H); 2.79-2.82 (m, 2H); 4.25 (d, J = 12.0 Hz, 2H); 5.12-5.17 (m, 1H); 5.39 (s, 2H); 7.59 (d, J = 8.5 Hz, 2H); 7.75-7.78 (m, 1H); 8.03 (s, 1H); 8.21 (d, J = 8.5 Hz, 2H); 8.28 (d, J = 8.5 Hz, 1H); 8.31 (d, J = 8.5 Hz, 1H); 8.54 (d, J = 8.5 Hz, 1H); 8.59 (d, J = 7.5 Hz, 1H) ppm. 13C NMR (CDCl3, 125 MHz): δ 164.4, 163.9, 157.7, 152.9, 147.9, 142.7, 138.7, 132.4, 131.4, 128.9,128.7, 126.8, 126.2, 123.9, 123.7, 123.1, 118.5, 117.4, 79.7, 66.2, 51.7, 28.5, 28.2 ppm.

[0132] Synthesis of compound NNC

[0133] The reaction mixture of compound a (0.1 g, 0.17 mmol) in DCM was added to trifluoroacetic acid (TFA) (10 mL) and stirred for 3 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure and dried to obtain compound NNC (0.09 g, 99%).

[0134] HR-ESI-MS [M+H]+ calc. 475.1612 m / z, [M+H]+ obs. 475.1631 m / z. 1H NMR (DMSO-d6, 500 MHz): δ 1.85 (d, J = 12.5 Hz, 2H); 2.49 (s, 1H); 2.85-2.9 (m, 2H); 3.03-3.08 (m, 2H); 3.38-3.4 (m, 2H); 5.14-5.18 (m, 1H); 5.41 (s, 2H); 7.76 (d, J = 8.5 Hz, 2H); 7.81-7.84 (m, 1H); 8.17 (d, J = 8.5 Hz, 1H); 8.26 (d, J = 8.5 Hz, 2H); 8.44 (d, J = 8.5 Hz, 1H); 8.48 (d, J = 7.0 Hz, 1H); 8.72 (d, J = 8.5 Hz, 1H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.9, 163.3, 153.7, 147.1, 144.2, 140.4, 131.7, 131.0, 129.3, 128.6, 128.4, 126.4, 123.7, 123.6, 122.6, 118.2, 117.5, 65.3, 48.3, 43.4, 25.1 ppm.

[0135] Synthesis of compound DNNC

[0136] Compounds NNC (0.09 g, 0.19 mmol), NBD-Cl (0.03 g, 0.15 mmol), and TEA (Triethylamine) (0.07 mL, 0.53 mmol) were dissolved in dry DMF (Dimethylformamide) (10 mL) and stirred overnight. After the reaction was completed, the reaction mixture was poured into water, and the saturated powder was filtered to obtain DNNC (0.023 g, 23%).

[0137] [M-H]- calc. 636.1484 m / z, [M-H]- obs. 636.1476 m / z. 1H NMR (DMSO-d6, 500 MHz): δ 1.99 (d, J = 9.5 Hz, 2H); 2.73-2.79 (m, 2H); 3.72 (t, J = 12.0 Hz, 2H); 4.95 (s, 2H); 5.42-5.45 (m, 3H); 6.74 (d, J = 9.5 Hz, 1H); 7.76 (d, J = 9.0 Hz, 2H); 7.85 (t, J = 8.0 Hz, 1H); 8.19 (d, J = 8.5 Hz, 1H); 8.28 (d, J = 9.0 Hz, 2H); 8.45-8.53 (m, 3H); 8.72 (d, J = 8.5 Hz, 1H); 10.5 (s, 1H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 163.9, 163.3, 153.7, 147.2, 145.1, 145.0, 144.8, 144.2, 140.5, 136.5, 131.8, 131.0, 129.2, 128.6, 128.4, 126.5, 123.8, 123.6, 122.7, 120.9, 118.3, 117.6, 103.5, 65.3, 49.7, 49.2, 27.7 ppm.

[0138]

[0139] <제조예 1> 화합물 DN의 합성

[0140]

[0141] The reaction mixture of compound b (0.68 g, 1.72 mmol) in DCM (20 mL) was added to TFA (20 mL) and stirred for 3 h. After the reaction was completed, the reaction mixture was evaporated under reduced pressure and used in the next reaction without further purification. After that, the compound and TEA (0.85 mL, 8.4 mmol) were dissolved in dry DMF (20 mL), and NBD-Cl (0.35 g, 1.75 mmol) was slowly added and stirred overnight. After the reaction was completed, the reaction mixture was poured into water, and the saturated powder was filtered to obtain compound DN (0.31 g, 37%).

[0142] HR-ESI-MS [M+H]+ calc. 475.1612 m / z, [M+H]+ obs. 475.1631 m / z. 1H NMR (DMSO-d6, 500 MHz): δ 1.92-1.96 (m, 2H); 2.76-2.84 (m, 2H); 3.69 (t, J = 10.5, 2H); 4.94 (s, 2H); 5.43-5.48 (m, 1H); 6.70 (d, J = 9.0, 1H); 6.82 (d, J = 8.5, 1H); 7.49 (s, 2H); 7.62 (t, J = 7.8, 1H); 8.14 (d, J = 8.5 Hz, 1H); 8.36 (d, J = 7.5 Hz, 1H); 8.48 (d, J = 9.0 Hz, 1H); 8.60 (d, J = 8.5 Hz, 1H) ppm. 13C NMR (DMSO-d6, 125 MHz): δ 164.7, 163.7, 153.2, 145.5, 145.4, 145.2, 136.8, 134.5, 131.5, 130.2, 129.8, 124.4, 122.5, 121.2, 119.6, 108.7, 108.1, 103.9, 50.3, 49.1, 28.3 ppm.

[0143]

[0144] <Manufacturing Example 2> Synthesis of compound Naph

[0145]

[0146] Compound Naph (Zhang, C.; Wang, R.; Cheng, L.; Li, B.; Xi, Z.; Yi, L.Sci. Rep.2016, 6, 30148. DOI: 10.1038 / srep30148) was synthesized according to procedures reported in the literature.

[0147]

[0148] <Experimental Example 1> Analysis of absorption and fluorescence characteristics

[0149] Stock solutions of synthetic compounds were prepared in DMSO, and various pH buffer solutions were prepared according to procedures reported in the literature (MH Lee, J. Fluoresc. 2016, 26, 807-811). Stock solutions of chloride salts of metal ions were prepared in water. Stock solutions of reactive oxygen species (ROS) were prepared using procedures reported in the literature (D. Oushiki, H. Kojima, T. Terai, M. Arita, K. Hanaoka, Y. Urano, T. Nagano, J. Am. Chem. Soc. 2010, 132, 2795-2801). Briefly, H2O2, tert-butyl hydroperoxide (HOO t Bu) and hypochlorite (NaOCl) were delivered in 35%, 70% and 11-14% aqueous solutions, respectively. 10 mM Fe(ClO4)2 and 10 mM H2O2 or HOO t By the reaction of Bu, each hydroxyl radical ( · OH) and tert-butoxy radical ( t BuO · ) was generated. The stock solution of thiol was prepared in 100 mM pH 7.4 PBS buffer.

[0150] Samples for absorption and emission measurements were placed in quartz cuvettes (4 mL volume) using PBS solution (100 mM, pH 7.4) containing 30% (v / v) DMSO, and then incubated at 37°C for 2 h before measurement. The excitation and emission slit widths were set to 3 and 5 nm, respectively, and excitation was provided at 420 nm. Fluorescence quantum yields (Φ) of DNNC, NNC, DN, and Naph f ) is a reference substance (Fluorescein; Φ in 0.1 M NaOH f = 0.90) was measured.

[0151] <Experimental Example 1-1> Analysis of absorption and fluorescence characteristics of synthesized DNNC, Naph, NNC, and DN

[0152] Before testing the activity of DNNC against H2S and NTR, the absorption and fluorescence properties were investigated and compared with those of reference compounds Naph, NNC, and DN, which lack all or each of the locking groups. As shown in Figures 2a and 2b, DNNC dissolved in PBS solution exhibited an orange color and absorption bands around 350 and 500 nm, corresponding to the naphthalimide and NBD moieties, respectively. It was confirmed that DNNC exhibited almost no fluorescence intensity under naphthalimide excitation.

[0153] In contrast, Naph, which lacks a double locking system, exhibited naphthalimide absorption around 430 nm and strong fluorescence intensity around 550 nm. NNC, a single locking system containing an NC group, exhibited naphthalimide absorption around 350 nm and blue-shifted fluorescence emission around 478 nm. DN, which uses an NBD group and a single locking system, exhibited a broad absorption band in the 350–550 nm range and relatively weak fluorescence emission around 550 nm. The absorption and fluorescence characteristics of DNNC and the reference compound are also shown in Table 1.

[0154] Compoundsλ Abs (nm)Absorption coefficients(x10) 4 M -1 cm -1 )λ Em (nm)Quantum yieldsDNNC360, 4921.891, 2.099-0.085Naph4340.9925480.503NNC3701.0854780.516DN360, 430, 4970.793, 1.268, 2.1075570.051

[0155] Additionally, it was confirmed that the fluorescence intensities of DNNC, NNC, and DN at 547 nm increased 72-fold, 5-fold, and 4-fold, respectively, in the presence of NaHS and / or NTR (Fig. 2c).

[0156] From the above results, two results were clearly confirmed: i) the NC group induces a blue shift, fixing the naphthalimide emission at 550 nm, and the NBD group suppresses the intensity of the naphthalimide emission. ii) Compared to the single-locking system, the dual-locking system achieves an excellent signal-to-noise ratio due to a very low background signal.

[0157] <Experimental Example 1-2> Analysis of the reaction of synthesized DNNC with H2S or NTR

[0158] The responses of DNNC to H2S and NTR were analyzed by monitoring the changes in absorption and fluorescence, respectively. In the presence of NaHS, an H2S source, DNNC exhibited an immediate increase in fluorescence intensity at 475 nm accompanied by a change in absorption (Figs. 3a and 3b). The DNNC solution visually changed from orange to colorless. On the other hand, in the presence of NTR enzyme and its cofactor NADH, DNNC exhibited an increase in fluorescence intensity at 547 nm (Figs. 4a and 4b). These changes varied depending on the concentration of H2S or NTR, and a linear relationship between the reactive fluorescence intensity and the concentrations of H2S and NTR was confirmed, indicating a minimum detectable level of 1.5 μM for H2S and 0.87 μg / mL for NTR, respectively (Figs. 3c and 4c).

[0159] <Experimental Example 1-3> Analysis of the simultaneous reaction of synthesized DNNC with H2S and NTR

[0160] Based on the above results, the ability of DNNC for simultaneous detection of H2S and NTR was analyzed. When various concentrations of NTR ranging from 0 to 10 μg / mL were added to DNNC in the presence of excess NaHS, fluorescence was significantly enhanced with an increase in the absorption band at 420 nm and a wavelength shift from 475 nm to 547 nm as the NTR concentration increased (Figs. 5a and 5b). Furthermore, minimal detection was achieved at 3.4 ng / mL NTR (Fig. 5c). Conversely, when NaHS ranging from 0 to 800 μM was introduced into DNNC in the presence of excess NTR, an increase in fluorescence at 547 nm was observed with a corresponding absorption shift as the NaHS concentration increased (Figs. 6a and 6b). Furthermore, minimal detection was achieved at 1.1 μM H2S (Fig. 6c).

[0161] When the above UV / Vis absorption and fluorescence spectra were normalized and displayed, it was confirmed that the spectrum of DNNC reacted with NTR and H2S was consistent with that of its product, Naph (Fig. 6d).

[0162] These results demonstrate that DNNC exhibits high sensitivity to H2S and NTR and is efficiently converted to Naph, resulting in corresponding changes in both absorption and fluorescence. Consequently, DNNC generates a distinct and measurable fluorescence signal at 547 nm, facilitating the simultaneous detection of H2S and NTR.

[0163] <Experimental Example 1-4> Fluorescence-based dynamic analysis of synthesized DNNC

[0164] Fluorescence-based kinetics were analyzed considering the reaction rates for H2S and / or NTR. When NaHS was added in excess, DNNC showed an immediate increase in fluorescence intensity at 478 nm over time, reaching a peak at 120 min (Fig. 7a). Sequential addition of NTR resulted in a marked increase in fluorescence intensity at 547 nm, a decrease in fluorescence at 478 nm, and saturation at 30 min (Fig. 7b). However, conversely, excessive addition of NTR resulted in a rapid increase in fluorescence intensity at 547 nm within 5 min (Fig. 7c), and sequential addition of NaHS resulted in a gradual increase in fluorescence at 547 nm, reaching a peak at 120 min (Fig. 7d).

[0165] Additionally, the fluorescence response of DNNC was monitored over time under conditions in which H2S and NTR were simultaneously present. As a result, as shown in Fig. 8, DNNC exhibited an immediate increase in fluorescence at 547 nm and a kinetic pattern dependent on H2S.

[0166] These analyses revealed that DNNC exhibits distinct kinetics toward NTR and HS, with the coexistence of NTR and HS resulting in a unique fluorescence intensity at 547 nm, independent of the reaction order. This suggests that DNNC's responses to NTR and HS may interfere minimally with each other, allowing DNNC to provide preferentially readable fluorescence in pathological situations where both factors are overexpressed.

[0167] <Experimental Example 1-5> Selectivity Analysis of Synthesized DNNC for H2S and NTR

[0168] To confirm the selectivity of DNNC for H2S and NTR, metal ions (e.g., Na + , K + , Mg 2+ , Ca 2+ , Cu 2+ , Zn 2+ , Fe 2+ and Fe 3+ ), including a variety of biologically abundant species including reactive oxygen species (e.g., ·O2 - , ·OH,t-BuO - , H2O2,t-BuOOH and ClO - ), thiols (e.g., GSH, Cys, and Hcy), and other substrate acids such as Na2CO3, glucose, and ascorbic acid were analyzed. As a result, it was confirmed that a significant increase in fluorescence at 547 nm occurred only in the simultaneous presence of H2S and NTR, and no fluorescence change was detected in the other biological species tested (Fig. 9).

[0169] Furthermore, to investigate the effect of pH on the fluorescence response of DNNC to H2S and NTR, the fluorescence intensity at 547 nm was monitored at various pH values ​​ranging from 2 to 11. As a result, DNNC readily showed an increase in fluorescence intensity in response to H2S and NTR within the biologically relevant pH range of 4 to 9, and was particularly effective at pH 7 to 8 (Fig. 10). However, no significant changes were detected in the absence of H2S and NTR.

[0170] Based on this, it is expected that DNNC will be able to selectively detect H2S and NTR without interference from pH changes in various biological components and biological systems.

[0171]

[0172] <Experimental Example 2> HPLC Analysis

[0173] HPLC analysis was performed to verify the hypothesis that DNNC is converted to Naph through cleavage of NBD and NC groups by H2S and NTR. The LC peak was analyzed by detecting absorption at 420 nm (λ ex = 420 nm), all data were measured after incubation for 2 h at 37°C in a PBS solution (100 mM, pH 7.4) containing 30% (v / v) DMSO. In addition, NADH (500 μM) was used as a cofactor for NTR activity.

[0174] As shown in the mechanism in Figure 1, the NBD group is sensitive to nucleophilic attack by H2S, whereas the NC group is sensitive to nitroreduction by NTR. In the HPLC analysis results, actual DNNC and Naph eluted at 12.3 and 9.1 minutes, respectively, but in the presence of H2S and NTR, the initial peak of DNNC disappeared and a new peak appeared at 9.1 minutes, consistent with Naph (Figure 11).

[0175] The above results clearly show that in the presence of both H2S and NTR as proposed in the present invention, DNNC dissociates into NBD and NC groups to form NNC and DN, which are ultimately converted to Naph with strong green fluorescence.

[0176]

[0177] <Experimental Example 3> Cell culture and confocal microscopy observation

[0178] Human cervical cancer cells (HeLa), human gastric adenocarcinoma cells (AGS), and human pancreatic cancer cells (Panc1) were cultured in Dulbecco's modified Eagle's medium (DMEM), breast cancer cells (MCF-7) and pancreatic cancer cells (LNCap-LN3) were cultured in Minimum Essential Media (MEM), and human prostate cancer cells (DU145), human hepatocarcinoma cells (HepG2), human liver cancer cells (Huh-7), human breast cancer cells (MDA-MB-231), human lung cancer cells (A549), human breast cancer cells (T47D), and human colon cancer cells (HCT116) were cultured in RPMI 1640 medium supplemented with 10% Gibco® fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin. Two days before microscopic observation, cells were plated on cover glass-bottom dishes at 10 5 Cells were seeded individually and maintained at 37°C in a humidified atmosphere containing 5% (v / v) CO2. All cells were purchased from the Korean Cell Line Bank (Seoul, South Korea). DMEM, MEM, RPMI 1640, FBS, trypsin 0.25%-EDTA, and penicillin-streptomycin used in cell culture media were purchased from Gibco (Grand Island, NY, USA). Transparent adhesive confocal dishes (diameter = 35 mm) were purchased from SPL (Phocheon-si, Korea).

[0179] For confocal microscopy images, cells were harvested after incubation with DNNC, NNC, and DN (10 μM) for 30 min. To perform CoCl2-induced hypoxia experiments, cells were pretreated with CoCl2 (200 μM) for 24 h and then incubated with DNNC, NNC, or DN (each 10 μM) for 30 min, and then imaged. To perform experiments on the inhibition of endogenous activity of H2S and NTR, cells were pretreated with AOAA / PAG (each 1 mM) or dicoumarol (400 μM) for 1 h and then incubated with CoCl2 for 24 h. Afterwards, cells were incubated with DNNC (10 μM) for 30 min before imaging.

[0180] <Experimental Example 3-1> Confirmation of DNNC's fluorescence response to H2S and NTR in cancer cell lines

[0181] We analyzed whether DNNC with a dual locking system effectively detects intracellular NTR and H2S using normoxic and hypoxic HeLa cells. This is consistent with the previously discussed fact that NTR and H2S are overexpressed under hypoxic conditions. Comparative experiments with NNC and DN with a single locking system were performed using a 405 nm excitation laser and a 500-800 nm emission bandpass filter. The results showed that DNNC showed an approximately 12-fold increase in fluorescence intensity under hypoxic conditions compared to normoxic conditions, whereas NNC and DN showed relatively small increases of approximately 1.4-fold and 1.2-fold, respectively (Figs. 12a to 12c).

[0182] In addition, to analyze the intracellular NTR and H2S detection ability of DNNC according to oxygen concentration, DN, NNC, and DNNC were treated in normoxic (21% O2), 3% hypoxic, and 1% hypoxic HeLa cells, and then fluorescence intensity was measured. As a result, fluorescence intensity increased as oxygen concentration decreased, and in particular, the fluorescence intensity of DNNC was confirmed to have increased significantly (Fig. 12d).

[0183] These results suggest that DNNC with a dual locking system can effectively detect hypoxia by providing a high signal-to-noise ratio in response to the simultaneous presence of H2S and NTR.

[0184] In addition, to confirm whether the observed fluorescence was due to H2S and NTR, inhibition studies were performed using H2S inhibitors aminooxyacetic acid (AOAA) and N-propargyl glycine (PAG) and NTR activity inhibitor dicoumarol (Dic). As a result, the fluorescence response of DNNC was significantly reduced in cells treated with the inhibitors, confirming that the fluorescence observed in cells treated with DNNC was due to the intracellular H2S and NTR reaction (Fig. 13).

[0185] <Experimental Example 3-1> Fluorescence response analysis of DNNC in various cancer cell lines

[0186] To further investigate the levels of H2S and NTR activity in cancer cells, various cancer cell lines were treated with DNNC (10 μM) for 30 min under both normoxic and hypoxic conditions, and fluorescence responses were observed. Various cancer cell lines were used, including breast (MCF7, MDA-MB-231, T47D), prostate (DU145, LNCap / LN3), liver (HepG2, Huh7), gastrointestinal (AGS, HCT116), pancreatic (Panc1), lung (A549), and cervical (HeLa) cancer cells.

[0187] As a result, we confirmed that under normoxic conditions, eight cell lines (MDA-MB-231, T47D, LNCap / LN3, HepG2, Huh7, AGS, HCT116, and Panc1) showed strong fluorescence, whereas four cell lines (MCF7, DU145, A549, and HeLa) showed very weak fluorescence (Fig. 14). This heterogeneity is thought to be caused by differences in the levels of inherent H2S and NTR activities under normoxic conditions. In addition, variability in the levels of H2S and NTR activities among cancer cell lines of the same origin was observed, as evidenced by the fluorescence response of DNNC. On the other hand, under hypoxic conditions, most cell lines showed increased fluorescence intensity compared to normoxic conditions. In particular, HeLa and Huh7 showed noticeably higher fluorescence intensities. Among breast cancer cell lines, T47D under hypoxia showed the highest fluorescence compared to MCF7 and MDA-MB-231, and among prostate cancer cell lines, LNCap / LN3 showed higher fluorescence than DU145 under hypoxic conditions.

[0188] Consequently, the above results demonstrate the broad applicability of DNNC for cell imaging in various cancer cell lines, and confirm that DNNC can provide insight into the levels of H2S and NTR expression and the degree of hypoxia.

[0189]

[0190] <Experimental Example 4> MTT Analysis

[0191] Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 1.0Υ10 4Breast (MCF7, MDA-MB-231, T47D), prostate (DU145, LNCap / LN3), liver (HepG2, Huh7), digestive (AGS, HCT116), pancreatic (Panc1), lung (A549), and cervical (HeLa) cancer cells were treated with different concentrations of DNNC in 96-well plates at 37°C for 6 or 24 h, respectively. Then, MTT solution dissolved in serum-free medium (5 mg / mL) was added to each well and incubated for an additional 3 h. During the incubation, water-insoluble formazan was generated, and the amount of formazan was measured by adding DMSO to each well and observing the absorbance at 540 nm using a Spectra Max i3x microplate reader (Molecular devices, San Jose, CA). MTT was purchased from Sigma-Aldrich (St. Louis, MO) and used as received without further purification.

[0192] DNNC at various concentrations (0-40 μM) was applied to various cancer cell lines under normoxic and hypoxic conditions and cultured for 6 hours. Cell viability was evaluated using the MTT assay. As a result, most of the tested cancer cell lines survived even at a concentration of 40 μM DNNC, confirming that DNNC is not cytotoxic (Fig. 15).

[0193]

[0194] <Experimental Example 5> Tumor spheroid formation and staining

[0195] HeLa cells (1x10 5) were seeded on ultra-low attachment dishes (Corning) to form tumor spheroids. On the second day of tumor spheroid formation, DNNC (10 μM) or the control (DMSO 5%) were treated with fresh culture medium and cultured for 24 h under normoxic conditions (5% CO2 and 21% O2) or 3% hypoxic conditions (3% O2, 5% CO2, and the remainder 92% N2). Fluorescence images were taken on the third day of tumor formation using a confocal laser microscope (Carl Zeiss LSM 700) equipped with Ex 420 nm and Ex 550 nm filters. All images were acquired at 405 nm excitation and 500–800 nm emission through a band-pass filter.

[0196] Dead / live cells in tumor spheroids were assessed on day 2 of spheroid formation using 5 μM propidium iodide (dead cells: PI, red, Ex 530 nm / Em 620 nm) and 5 μM Calcein AM (live cells: green, Ex 485 nm / Em 535 nm) (Invitrogen). Tumor spheroids were treated with DNNC (10 μM) or control (DMSO 5%) for 24 h at 37°C under normoxia or 3% hypoxia, respectively. Then, a PI / Calcein AM mixture solution was added and incubated for 30 min to 1 h. For quantitative analysis, nucleus count staining was performed on a rotator at room temperature for 1 to 2 h using 0.5 μg / ml DAPI. Fluorescence images were captured using a confocal laser microscope (Carl Zeiss LSM 700).

[0197] To strengthen the practical feasibility of DNNC for cancer imaging, we analyzed the fluorescence response using HeLa cell spheroids, which mimic hypoxic cervical tumors in vivo. DNNC-treated tumor spheroids exhibited intense fluorescence within the hypoxic interior, as confirmed using z-stack images (Fig. 16a). In contrast, tumor spheroids without DNNC treatment exhibited no fluorescence signature. Furthermore, Calcein-AM / PI staining demonstrated that confocal imaging of tumor spheroids with DNNC could be achieved without cytotoxic effects under both normoxic and hypoxic conditions (Fig. 16b).

[0198] The use of 3D spheroids, which represent a more accurate tumor-mimicking microenvironment compared to 2D cell culture, is essential for observing the expression levels of H2S and NTR in real-world applications, and the above results demonstrate that DNNC can detect hypoxic conditions in response to H2S and NTR at the tumor level as well as the cellular level.

[0199]

[0200] <Experimental Example 6> Western Blot Analysis

[0201] To measure endogenous CBS, CSE, MST-3, and HIF-1a expression, normal cell lines (human fibroblasts, BJ, and WI38) as well as various cancer cell lines [breast (MDA-MB-231, MCF7, and T47D), gastrointestinal (HCT116 and AGS), prostate (DU145 and LNCap / LN3), liver (HepG2 and Huh7), pancreatic (Panc1), lung (A549), and cervical (HeLa)] were analyzed. Briefly, cells (2Y10 6 ) were seeded in 100 mm culture dishes and cultured until the cell density was more than 80% for maintenance.

[0202] Cells were seeded in 100 mm culture dishes and cultured to 80% confluency (approximately 1 week) under normoxia and CoCl2 (200 μM) treatment. The cultured cells were washed three times with ice-cold PBS and lysed in RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 30 mM NaCl, 0.5% Triton X-100] containing protease inhibitor cocktail (Sigma). The supernatant was collected by centrifugation, and the total protein concentration was measured using a Bradford protein assay kit (Bio-Rad Laboratories).

[0203] A total of 30 μg of collected protein was loaded onto each well of an SDS-PAGE and electrotransferred to a nitrocellulose membrane (Merk Millipore, Burlington, MA, USA). The membrane was then incubated with primary antibodies (CBS: Santa Cruz Biotechnology sc-133154, CSE: Santa Cruz Biotechnology sc-374249, 3-MST: Santa Cruz Biotechnology sc-1374326, HIF-1a: Santa Cruz Biotechnology sc-13515, GAPDH: Santa Cruz Biotechnology sc-47724) diluted in 5% BSA overnight at 4°C. The next day, the membranes were washed with Tris-buffered saline 0.1% Tween-20 (TBS-T) and incubated with anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, Santa Cruz Biotechnology sc-2357) for 2 h at room temperature. Enhanced chemiluminescence reagent (WesterGlow MAX, Biomax) was used to detect protein bands according to the manufacturer's instructions.

[0204] Analysis of the expression of H2S-generating proteins and hypoxia-inducible factor (HIF-1α) in various cell lines under normoxic and hypoxic conditions revealed that under normoxic conditions, cancer cell lines (LNCap / LN3, Huh7, and HeLa) showed higher expression of H2S-generating proteins, such as cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), compared to normal cell lines (BJ and WI38), while no HIF-1α expression was observed (Fig. 17a). However, under hypoxic conditions, all cell lines tested showed a marked increase in the expression of H2S-generating proteins, and the expression levels of HIF-1α were also significantly elevated (Fig. 17b).

[0205] These results demonstrate that the biological model effectively represents hypoxic conditions and accurately reflects specific pathological conditions characterized by simultaneous overexpression of H2S and NTR activities.

Claims

1. A compound represented by the following chemical formula 1 or a salt thereof: [Chemical Formula 1] The above p is 1 or 2, and q is an integer from 1 to 3.

2. In paragraph 1, A compound or a salt thereof, wherein both p and q are 1.

3. In paragraph 1, The compound is a compound having the following structure, or a salt thereof: .

4. In paragraph 1, The above compound is a compound or a salt thereof capable of detecting hydrogen sulfide and nitroreductase.

5. A detection probe capable of detecting at least one of hydrogen sulfide and nitroreductase, including the compound of paragraph 1 or a salt thereof.

6. As shown in the following reaction scheme 1, A method for producing a compound represented by Chemical Formula 1, comprising the step of producing a compound of Chemical Formula 1 by reacting a compound of Chemical Formula A and a compound of Chemical Formula B: [Reaction Formula 1] 7. A method for detecting intracellular hypoxia, comprising the following steps: A step (step 1) of injecting the compound of claim 1 or a salt thereof into a cell to be detected; and Step 2: Detecting intracellular fluorescence.

8. A method for diagnosing cancer, comprising the following steps: A step (step 1) of injecting the compound of claim 1 or a salt thereof into a cell to be diagnosed; and Step 2: Detecting intracellular fluorescence.

9. In paragraph 8, A method for diagnosing cancer, wherein the cancer is at least one selected from the group consisting of breast cancer, brain cancer, prostate cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, lung cancer, thyroid cancer, gallbladder cancer, bile duct cancer, kidney cancer, and cervical cancer.

10. In paragraph 7, A method for detecting hypoxia, wherein the fluorescence is green fluorescence in the range of 495 to 600 nm.

11. In paragraph 8, A method for diagnosing cancer, wherein the fluorescence is green fluorescence in the range of 495 to 600 nm.

Images