Two-photon fluorescence dyes generating reactive oxygen species and uses thereof

Abstract

The present invention relates to a novel asymmetric pyrene derivative compound and its use, showing that it can further enhance ROS generation compared to other similar structural compounds, and exhibits impressive phototoxicity (IC50 0.12 μM) and excellent biocompatibility, making it useful for photodynamic therapy (PDT).

Description

Technology Field

[0001] The present invention provides a two-photon fluorescent dye comprising an asymmetric pyrene derivative that generates reactive oxygen species upon two-photon excitation, and uses thereof. Background Technology

[0002] Photodynamic therapy (PDT) utilizes photoactive agents (photosensitizers; PS) and has sparked significant interest in anticancer therapy research due to its substantial advantages over conventional chemotherapy and radiation therapy. PDT involves administering a harmless photosensitizer (PS) to target cells (or entire tissues) and then illuminating them with light of an appropriate wavelength to induce the formation of reactive oxygen species (ROS) and subsequent cytotoxicity. Over the past decade, numerous PSs have been developed for preclinical studies. However, photosensitizers such as chlorine, propyrine, and phthalocyanine have not been approved for clinical application. Unfortunately, the potential of PDT has not yet been fully realized due to the limitations of existing photosensitizers. For example, abnormal cell division and disordered cancer-associated vasculature lead to inadequate oxygen perfusion and resulting hypoxia in the tumor site. A hypoxic microenvironment can induce resistance to both traditional cancer therapies and oxygen stress-dependent PDT. Furthermore, most PS utilize light sources within the UV or visible spectrum, limiting their use to surface tissues such as skin and esophageal cancer while causing photodamage to normal surrounding tissues. Additionally, these clinically approved photosensitizers have disadvantages, such as being difficult to synthesize, having low tissue staining ability, exhibiting toxicity under cancer conditions, and being non-cancer-selective.

[0003] Small molecule photosensitizers are easy to synthesize and can be readily incorporated into biological specimens. A common strategy for designing small molecule photosensitizers is to incorporate heavy atoms. Heavy atom-based photosensitizers enhance spin-orbit coupling (SOC) between singlet and triplet states, thereby increasing intersystem crossing (ISC) efficiency. However, transition metals and halogens used as heavy atoms in PS are expensive and exhibit undesirable carcinogenic toxicity. Furthermore, transition metal-based PS can be activated by short-wavelength light sources and have the disadvantage of low biodegradability.

[0004] Generally, light sources within the near-infrared (NIR) region exhibit low tissue absorption and facilitate tissue penetration through light scattering. However, near-infrared photosensitizers are rare and often exhibit low reactive oxygen species (ROS) yields, water solubility, and photostability, while lacking biocompatibility and adequate biodegradability. Two-photon excitation (hereinafter referred to as TPE) utilizes near-infrared light as an excitation source suitable for deep tumor PDT. Furthermore, the secondary dependence of TPE enables PS activation at the focal point of the laser beam. Consequently, PS activity can be controlled three-dimensionally, minimizing damage to surrounding normal tissues. TPE-induced PDT is an alternative method that addresses the limitations of conventional PDT. Additionally, photosensitizers capable of generating reactive oxygen species in TPE via Type I PDT can reduce PDT dependence on oxygen levels. Therefore, there is a need for efficient two-photon Type I PS, and research on this is currently required. Prior art literature

[0005] 1. Republic of Korea Published Patent No. 10-2014-0106773. The problem to be solved

[0006] The object of the present invention is to provide a novel asymmetric pyrene derivative compound.

[0007] Another objective of the present invention is to provide a two-photon fluorescent dye comprising the above compound as an active ingredient.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the above compound as an active ingredient.

[0009] Another objective of the present invention is to provide a method for promoting cancer cell death comprising the steps of applying the two-photon fluorescent dye to a cell or tissue and irradiating the cell with light in the range of 700 nm to 950 nm. means of solving the problem

[0010] To achieve the above objective, the present invention provides an asymmetric pyrene derivative compound represented by the following chemical formula 1 or chemical formula 2.

[0011] [Chemical Formula 1]

[0012]

[0013] [Chemical Formula 2]

[0014]

[0015] In the above Chemical Formula 1 or Chemical Formula 2, R is or And, above, n is one of 1 to 12, and above, X may be the same or different, and is hydrogen or fluorine, and above, Y is hydrogen, (C1~C4)alkyl, (C1~C4)alkoxy, (C1~C4)alkylsulfonyl, halogen, trifluoromethyl (CF3), -OH, -SH, -SS-(C5~C10)aryl, -SO3 - , -COOH, -PPh3 + (Triphenylphosphine) and -NR'3 + It is one of the above, and R' may be the same or different, and is (C1~C4)alkyl, (C1~C4)alkoxy, halogen, and trifluoromethyl It could be one of them.

[0016] In addition, the present invention provides a two-photon fluorescent dye comprising the above compound as an active ingredient.

[0017] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the above compound as an active ingredient.

[0018] In addition, the present invention provides a method for promoting cancer cell death comprising the steps of applying the two-photon fluorescent dye to a cell or tissue and irradiating the cell with light in the range of 700 nm to 950 nm. Effects of the invention

[0019] The present invention relates to a novel asymmetric pyrene derivative compound, a two-photon fluorescent dye containing the same as an active ingredient, and the use thereof. The two-photon fluorescent dye has excellent reactive oxygen species generating ability and can be used for photodynamic therapy on cells, and thus can be usefully utilized in medical procedures. Brief explanation of the drawing

[0020] FIG. 1a shows 1,6-bis(benzo[d]thiazol-2-yl)pyrene (1,6-bis(benzo[d]thiazol-2-yl)pyrene; hereinafter referred to as Py1), 4,4'-pyrene-1,6-diylbis(ethene-2,1-diyl))bis(1-methylpyridin-1-ium); Figure 1b shows a schematic diagram of the synthesis of 4-(2-(6-(benzo[d]thiazol-2-yl)pyren-1-yl)vinyl)-1-methylpyridin-1-ium (hereinafter referred to as Py2) and 4-(2-(6-(benzo[d]thiazol-2-yl)pyren-1-yl)vinyl)-1-methylpyridin-1-ium (hereinafter referred to as Py3), and Figure 1b shows a schematic diagram of the PDT mechanism (a) and the structure of the novel pyren derivative compound and its advantages (b). Figure 2 shows the UV-vis absorption spectra of Py1, Py2, and Py3 in DMF (dimethylformamide) (a), the fluorescence emission spectra of Py1, Py2, and Py3 at λmax excitation (b), the two-photon absorption cross-sectional values ​​of Py1, Py2, and Py3 in DMF (c), and the normalized PL spectra of Py1 and Py2 in different parts of H2O in THF (Tetrahydrofuran) solvent at 420 nm (d). Figure 3 shows the difference in singlet oxygen generation upon white light irradiation in H2O (a) and the fluorescence intensity at 525 nm indicates whether reactive oxygen species (ROS) are generated by the probe upon white light irradiation (b). Figure 4 shows the optimization of the ground state (S0) and frontier molecular orbitals obtained from the density functional theory (DFT) calculations (M062X / 6-31g(d)) for Py1, Py2, and Py3 (a), and the mechanism of triplet state formation of Py1, Py2, and Py3 through intersystem crossing (ISC) and internal conversion (IC) and the calculated electronic excited state energies (b). Figure 5 shows the viability of BF10 cell lines Py1, Py2, and Py3 after irradiating with white light for 10 minutes under darkness (a) or normal oxygen deficiency or hypoxia (b). Figure 6 shows confocal fluorescence images of Py3-labeled BF10 cell lines in the absence or presence of MSH (Melanocyte-stimulating hormone). Figure 7 shows data confirming the photo-induced BF10 cell line excision test and the resulting ratio of dead cells. Figure 8 shows a photo-induced BF10 spheroid ablation test. Specific details for implementing the invention

[0021] The present invention will be described in more detail below.

[0023] The present invention provides an asymmetric pyrene derivative compound represented by the following chemical formula 1 or chemical formula 2.

[0024] [Chemical Formula 1]

[0025]

[0026] [Chemical Formula 2]

[0027]

[0028] In the above Chemical Formula 1 or Chemical Formula 2, R is or And, above, n is one of 1 to 12, and above, X may be the same or different, and is hydrogen or fluorine, and above, Y is hydrogen, (C1~C4)alkyl, (C1~C4)alkoxy, (C1~C4)alkylsulfonyl, halogen, trifluoromethyl (CF3), -OH, -SH, -SS-(C5~C10)aryl, -SO3 - , -COOH, -PPh3 + (Triphenylphosphine) and -NR'3 + It is one of the above, and R' may be the same or different, and is (C1~C4)alkyl, (C1~C4)alkoxy, halogen, and trifluoromethyl It could be one of them.

[0029] The above R may be methyl.

[0030] The above pyrene derivative compound may be 4-(2-(6-(benzo[d]thiazol-2-yl)pyrene-1-yl)vinyl)-1-methylpyridin-1-ium.

[0031] In addition, the present invention provides a two-photon fluorescent dye comprising the above compound as an active ingredient.

[0032] The above two-photon fluorescent dye can selectively kill cells or tissues by selectively generating reactive oxygen species (ROS) through two-photon excitation (TPE).

[0033] The above two-photon fluorescent dye can be photoactivated by light in the near-infrared (NIR) region, specifically in the range of 700 nm to 950 nm, and preferably in the range of 700 nm to 850 nm.

[0035] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising the above compound as an active ingredient.

[0036] The above-mentioned cancer treatment composition can be used for two-photon photodynamic therapy (TP-PDT).

[0037] The above cancer may be one or more selected from the group consisting of melanoma, skin cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, blood cancer, sarcoma, and brain cancer, but is not limited thereto.

[0038] The above cancer treatment composition may be administered via one or more routes selected from the group consisting of intravenous injection, intraperitoneal injection, intramuscular injection, intracranial injection, intratumoral injection, intraepithelial injection, skin penetrating delivery, esophageal administration, abdominal administration, arterial injection, intra-articular injection, and oral administration.

[0040] In addition, the present invention provides a method for promoting cancer cell death comprising the steps of applying the two-photon fluorescent dye to a cell or tissue and irradiating the cell with light in the range of 700 nm to 950 nm.

[0041] Not only has the action of the two-photon fluorescent dye containing the above compound as a photosensitizer been confirmed, but it can also selectively induce apoptosis in cancer cells upon light irradiation. Therefore, by applying it to the treatment of cancer or various diseases, an excellent therapeutic effect can be obtained in which it is harmless to normal cells and selectively causes necrosis in diseased cells.

[0043] Hereinafter, to aid in understanding the present invention, examples and the like will be described in detail. However, the following examples and the like are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples and the like. The examples and the like of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0045] [Synthesization Example] Synthesis of Py3 (Fig. 1a)

[0046] (1) Pyren -1,6- dicarbaldehydride ( pyrene -1,6- dicarbaldehyde ; Synthesis of Compound 1) hereinafter referred to as Compound 1

[0047] Compound 1 was synthesized according to a known synthesis method.

[0049] (2) 4-(2-(6- Formylpyrene -1-day)Vinyl)-1- Methylpyridine -1- Ium Synthesis of (4-(2-(6-formylpyren-1-yl)vinyl)-1-methylpyridin-1-ium; hereinafter referred to as Compound 2)

[0050] Compound 1 (1 g, 3.87 mmol) and 1,4-dimethylpyridin-1-ium (1,4-dimethylpyridin-1-ium; 0.73 g, 3.87 mmol) were mixed with MeOH and CH2Cl2 It was dissolved in a (1:1, 20 mL) mixed solution and stirred under reflux overnight. Afterward, the reaction mixture was slowly cooled to room temperature, and the dark red solid was filtered. After drying, the crude product was filtered at high temperature using methanol (4 times) to obtain Compound 2 (1.1 g) as a deep orange solid.

[0051] Yield: 66%. 1H NMR (DMSO-d6,600MHz): δ (ppm) 10.80 (s, 1H), 9.42 (d, J = 8.2 Hz, 1H), 9.05-9.10 (m, 2H), 8.91 (d, J = 5.5 Hz, 2H), 8.69 (d, J = 8.2 Hz, 1H), 8.60 (d, J = 8.2 Hz, 1H), 8.51 (q, J = 3.7 Hz, 2H), 8.49-8.45 (3H), 8.41 (d, J = 9.6 Hz, 1H), 7.84 (d, J = 15.1 Hz, 1H), 4.27 (s, 3H); 13 C-NMR (151 MHz, DMSO-d6)δ 194.30, 152.73, 145.67, 136.72, 135.05, 131.83, 131.80, 131.66, 131.15, 131.02, 129.76, 128.82, 128.43, 127.69, 127.36, 126.85, 126.22, 125.36, 124.67, 124.35, 124.30, 124.26, 47.55 HRMS (ESI + ):m / zfoundfor[C 25 H 18 NO] + :cald. 348.1383. found 348.1370.

[0053] (3) Synthesis of Py1

[0054] Compound 1 (1 g, 3.87 mmol) and 2-aminobenzenethiol (1.21 g, 9.68 mmol) were mixed in DMF (20 mL) in which PTSA (p-Toluenesulfonic acid; 66 mg) was dissolved and stirred overnight at 80 °C. Afterward, the reaction mixture was slowly cooled to room temperature and the solid was filtered. After drying, the crude product was hot-filtered with methanol (4 times) to obtain Py1 (1.45 g) as a green solid.

[0055] Yield: 78%. HRMS (ESI +):m / zfoundfor[C 30 H 16 N2S2] + :cald. 469.0855. found469.0807.

[0057] (4) Synthesis of Py2

[0058] Compound 1 (1 g, 3.87 mmol) and 1,4-dimethylpyridin-1-ium (1,4-dimethylpyridin-1-ium; 1.85 g, 9.7 mmol) were dissolved in a mixed solution of MeOH:CH2Cl2 (1:1, 30 mL) and stirred under reflux overnight. Afterward, the reaction mixture was slowly cooled to room temperature, and the dark red solid was filtered. After drying, the crude product was filtered at high temperature using methanol (4 times) to obtain Py2 (2 g) as a dark red solid.

[0059] Yield: 86%. 1 H-NMR (600 MHz, DMSO-d6) δ 9.10 (d, J = 16.5 Hz, 1H), 9.03 (d, J = 9.6 Hz, 1H), 8.91 (d, J = 6.9 Hz, 2H), 8.69 (d, J = 8.2 Hz, 1H), 8.63 (d, J) = 6.9 Hz, 1H), 8.49 (d, J = 5.5 Hz, 2H), 8.40 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 15.1 Hz, 1H); HRMS (ESI + ):m / zfoundfor[C 32 H 26 N2] 2 + :cald. 437.2085. found437.1998.

[0061] (5) Synthesis of Py3

[0062] Compound 2 and 2-aminobenzenethiol (0.73 g, 4.67 mmol) were mixed in DMF (20 mL) in which PTSA (p-Toluenesulfonic acid; 40 mg) was dissolved and stirred overnight at 80 °C. Afterward, the reaction mixture was slowly cooled to room temperature and the solid was filtered. After drying, the crude product was hot-filtered with methanol (4 times) to obtain Py3 (0.7 g) as an orange solid.

[0063] Yield: 64%. 1 H-NMR (600 MHz, DMSO-d6) δ 9.38 (d, J = 13.1 Hz, 1H), 9.03-9.10 (m, 2H), 8.89 (d, J = 6.2 Hz, 3H), 8.68 (d, J = 7.6 Hz, 1H), 8.56 (d, J = 8.3 Hz, 1H), 8.51 (d, J = 7.6 Hz, 1H), 8.46-8.48 (m, 3H), 8.38-8.44 (m, 2H), 8.23 ​​(d, J = 9.0 Hz, 2H), 7.84 (d, J = 15.8 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.55 (d, J = 11.0 Hz, 1H), 4.30-4.24 (3H); 13 C-NMR (151 MHz, DMSO-D6) δ 167.88, 154.39, 152.66, 145.52, 136.82, 135.68, 134.15, 132.37, 132.27, 132.08, 130.69, 130.06, 129.86, 129.72, 129.57, 129.44, 128.89, 128.79, 127.88, 127.34, 126.73, 126.50, 126.39, 126.32, 125.30, 124.50, 124.43, 123.85, 122.71, 47.48; HRMS (ESI + ):m / zfoundfor[C 31 H 21 N2S] +:cald. 453.1420. found453.1404.

[0065] [Example 1] Spectroscopic Measurements

[0066] The photophysical properties of Py1, Py2, and Py3 of the above synthesis examples were confirmed. Absorption spectra and fluorescence spectra were recorded using a UV-Vis spectrophotometer (S-3100) and a fluorescence spectrophotometer (FS-2), respectively. Fluorescence quantum yield was measured using rhodamine-6G (Φ = 0.98 in MeOH) as a reference. 1 The 1H NMR spectrum was recorded using a 600 MHz NMR spectrometer (JNM-ECZR). Fluorescence images were obtained using a spectral confocal microscope (Leica TCS SP8).

[0067] As a result, according to Fig. 2, Py1, Py2, and Py3 exhibited maximum absorption at 399, 474, and 450 nm, respectively. Py1, Py2, and Py3 showed large Stokes shifts of 51, 127, and 140 nm, respectively, while emission peaks were 450, 601, and 590 nm, respectively. The weak fluorescence produced by commercially available PS inhibited their use as therapeutic diagnostic agents during imaging-assisted PDT. However, Py1, Py2, and Py3 each showed fluorescence quantum yields in DMF ( FL) values ​​of 89%, 34%, and 27% were shown. Most importantly, in the two-photon absorption cross-action study, along with the TPA (two-photon absorption) values ​​for Py2, the Py1, Py2, and Py3 probes with TPAs ​​of 720–1000 nm showed 714 GM at 750 nm, and Py3 showed 156 GM at 740 nm (Fig. 2c). Uniquely, regarding the fluorescence intensity of Py1, Py2, and Py3 with respect to solvent polarity, Py1 and Py2 exhibited strong fluorescence at low and high polarity, respectively, while Py3 exhibited fluorescence at both polarities, demonstrating asymmetric hybridization-induced fluorescence characteristics for both acceptors (Fig. 2d).

[0069] [Example 2] Singlet Oxygen Generation Measurement

[0070] Singlet oxygen generation by Py1, Py2, and Py3 under underwater conditions was measured using ABDA (9,10-anthracenediyl-bis(methylene)-dimalonic acid). In addition, Rose Bengal (RB), a well-known commercial PS, was used to compare singlet oxygen generation.

[0071] As a result, as shown in Fig. 3, ABDA degradation at 378 nm demonstrated that Py1 did not degrade ABDA, whereas the degradation rates of Py2 and Py3 were significantly faster than those of the commercially available PS, Rose Bengal (RB). At similar concentrations, the asymmetric A-π-A Py3 exhibited a slightly faster ABDA degradation rate than the symmetric Py2 (Fig. 3a). Next, Type I ROS generation was confirmed for the Py1, Py2, and Py3 probes using the formation of Rhodamine 123 through the oxidation of DHR 123 (Dihydrorhodamine 123). It was found that Py1 exhibited 5 times more ROS generation than Py2 and Rose Bengal (RB), and under similar conditions, the photo-induced oxidation of Py3 by DHR 123 was approximately 60 times higher than that of Py2 (Fig. 3b). This confirmed that installing asymmetric benzothiazole and pyridinium receptors in an A-π-A system with a pyrene π-bridge can yield a synergistic effect between the two receptors. It was found that the asymmetric pyrene A-π-A dye composed of a benzothiazole receptor for the promoted Type I PDT process, whereas pyridinium for the Type II PDT process, makes PS suitable for both normal oxygen and hypoxic conditions.

[0073] [Example 3] DFT (density functional theory) calculation

[0074] Theoretical calculations were performed to explain the ROS generation mechanisms (i.e., Type I and Type II) of Fig. 1b. The optimized structures, electronic energies, HOMOs, and LUMOs of the three photosensitizers (hereinafter referred to as PS) were obtained using density functional theory (DFT) calculations (M062X / 6-31g(d)). The intersystem crossover rate constants were calculated using the electronic energies of PS and the spin-orbit coupling constants between the singlet and triplet states.

[0075] In both Type I and Type II mechanisms, singlet excited states (S n Triplet excited states (T) in ) n It is important to efficiently generate the triplet state of PS through rapid intersystem crossing (ISC). Furthermore, electron transfer from the LUMO of PS to the oxygen molecule must occur efficiently for radical generation via a Type I mechanism, and efficient energy transfer from the PS in the T1 state to the oxygen molecule via a Type II mechanism for singlet oxygen ( 1 O2) must occur for generation.

[0076] As shown in Figure 4a, it was confirmed that HOMO was mainly distributed in the central pyrene group in the three PS, whereas the LUMO distribution varied depending on the substituents of the pyrene group. While the LUMO of Py1 was mostly distributed in the pyrene group, in Py2 and Py3, the LUMO was significantly distributed in the pyridium group because the pyridium group exhibited relatively strong electron-extraction characteristics. Accordingly, compared to Py1 (-1.50 eV) and Py3 (-1.92 eV) in Figure 4a, Py2 (-2.06 eV), which has two pyridium groups, was found to have the lowest LUMO energy.

[0077] In Table 1, the energies of the electron excited states were calculated using the time-dependent (TD)-DFT method. The energy of the first single excited state (S1) increased in the order of Py2 (2.88 eV) < Py3 (3.07 eV) < Py1 (3.47 eV).

[0078]

[0079] All three PS were calculated to have strong oscillator intensity for S1. The calculated electron absorption spectra were found to be in good agreement with the experimentally measured spectra. Furthermore, Py2 and Py3 showed smaller ΔE due to relatively better spatial HOMO-LUMO separation resulting from the electron withdrawal characteristics of pyridinium. ST It had a value.

[0080] To investigate the efficiency of triplet state generation in PS, the ISC rate constant k for possible ISC channels ISC is the spin-orbit coupling (SOC) constant and ΔE between the corresponding singlet and triplet states ST It was calculated theoretically using the values. As summarized in Figure 4a and Table 2, Py3 has the largest k ISC I confirmed that it represents the value of Py3's k. ISC It was confirmed that the reason for the large value is that the state transition from ISC to T2 is kinetically favorable. In contrast, Py1 has a large SOC constant due to sulfur atoms, but Py1's k ISC The value is small, and it was found that this is because the triplet state energy is not properly aligned with the S1 energy for an efficient ISC.

[0081]

[0082] In the case of the Type I mechanism, efficient electron transfer from PS to the oxygen molecule must occur for the oxygen molecule to become a superoxide anion radical (O2 - It is reduced to ), and the LUMO energy of PS is O2+ e - → O2 - It was observed that it was higher than the reduction potential of the half-reaction. In Fig. 4a, the LUMO energy decreases in the order of Py1 (-1.50 eV) > Py3 (-1.92 eV) > Py2 (-2.06 eV), which indicates that among the three PS molecules, Py1 transfers electrons to O2 -It was observed that it exhibited a relatively larger tendency to generate. However, the experiment observed that Py3 showed higher ROS generation than Py1, which is possible because Py3 has a k that is 10 times larger than Py1. ISC It was possible to confirm that it was because it had a value.

[0083] In the Type II mechanism, the T1 energy of PS and the electron energy of the oxygen molecule must be similar to ensure efficient triplet energy transfer from PS to the oxygen molecule; in this regard, the T1 energies of Py2 and Py3 were found to be relatively close to the electron excitation energy of the oxygen molecule in Fig. 4b, indicating that singlet oxygen generation is more efficient by Py2 and Py3 than by Py1. The reason Py3 generates singlet oxygen more efficiently than Py2 is that Py3 is k compared to Py2 ISC It was confirmed that this was because the value was larger.

[0085] [Example 4] Cytotoxicity Study

[0086] High phototoxicity and low oncogenicity upon light irradiation are critical for minimizing side effects and enhancing therapeutic efficiency in phototherapy; therefore, the biocompatibility of Py1, Py2, and Py3 in light-free BF10 melanoma cell lines was investigated using an MTT assay. According to Figure 5a, Py1 and Py3 were found to be non-toxic up to a concentration of 100 μM when PS were cultured in BF10 cells at 37 °C for 24 hours in a dark incubator, while Py2 showed IC10 values ​​without irradiation. 50 It was 75 μM. It demonstrated impressive biocompatibility of these PS in melanoma cells. Under aqueous conditions (0.1% DMSO), the quantum yields of Py2 and Py3 probes were determined to be 5% and 1%, respectively, and the two-photon cross-actions were, respectively With δ 21 and 4.2 GM, these PSs were suitable for OP (one-proton) and TP cell imaging without the aid of organic solvents. PSs were cultured in BF10 cell lines at 37 °C for 30 minutes in a dark incubator, and the dyes were washed with fresh cell medium prior to cell imaging. Conglomerate scanning laser microscope (CSLM) images obtained at 488 nm of BF10 cells containing 2 μM Py1 showed that the dye nano-aggregated outside the cells, which could explain the weak absorption in aqueous media. Next, fluorescence of 2 μM Py2 was observed inside BF10 cells under similar conditions. Interestingly, according to Figure 6, when applied to BF10 imaging, Py3 was found to selectively stain the cell membrane. Additionally, the ability of Py3 to stain membranes was investigated in other cell lines, such as melanoma (A375 and BL6 cell lines), non-melanoma (A431 cell line), and HT-29 cell line (colorectal cancer). In all cases, as with BF10 cells, it was confirmed that the thin layer surrounding the cell brightened with strong fluorescence emission in the 500 to 650 nm region. Overlaying brightfield and CSLM images of these cell lines allowed for a clear definition of the external shape of the cells.

[0087] To investigate the preferential targeting of the cell membrane by Py3, the commercial membrane tracer CMDR (cellMask deep red) NIR dye was co-stained with Py3 in BL6 cells for 30 minutes. Upon excitation at 488 nm (for Py3) and 633 nm (for CMDR), it was observed that the co-localization images of Py3 at 498–580 nm and the Pearson correlation coefficient of the NIR dye CMDR at 650–750 nm were 0.90, indicating significant overlap. Additionally, Py3 cell membrane staining at various depths in BL6 cell lines using the CMDR dye was measured using a CSLM. BL6 images at a distance of 1 μm from the z-axis depth of 0–13 μm confirmed that Py3 was localized with the cell membrane tracer CMDR (Pearson correlation coefficients ranged from 0.84 to 0.90). These results demonstrated that Py3 possesses amphiphilic and cationic properties and is particularly helpful in staining cell membranes in various living cell lines.

[0088] Next, the photostability of Py3 on the cell membrane of BF10 was investigated using CSLM, and it was confirmed that similar fluorescence intensity was observed within 260 seconds by extending the scan time. This characteristic indicated that Py3 possesses high photostability and serves as an excellent imaging agent for real-time imaging of cell membranes over a long period.

[0089] In addition, radiance 0.16 J / cm 2 The photo-induced cytotoxicity of Py1, Py2, and Py3 was measured using white light irradiation. Py1 clearly exhibited very weak toxicity due to extracellular aggregation. On the other hand, Py2 had an IC50. 50 It exhibited phototoxicity of 0.92 μM (Fig. 5b). Under similar light irradiation, Py3 was more phototoxic (IC) than Py2, which corresponds to the photo-induced ROS generation ability. 50It was found that the level (0.12 μM) increased approximately eightfold. In addition, the ability of Py3 to generate ROS via Type I processes under light irradiation was confirmed under cellular hypoxic conditions to utilize Type I PDT. Under hypoxic conditions, phototoxicity decreased slightly, but Py3 at a slightly higher concentration (IC10) than Py3 50 It can still completely eliminate melanoma cells at 1.43 μM, confirming that Py3 is an efficient PDT (Photodynamic therapy) agent in both normal oxygen and hypoxic conditions.

[0090] To investigate the cause of white light-induced cytotoxicity of Py3 in melanoma cell lines, non-fluorescent DCHF-DA (2,7-dichlorohydrofluorescein diacetate) was used as a cellular ROS indicator. In the presence of esterase and ROS, DCHF-DA is converted to DCF (dichlorofluorescein) and emits light; however, cellular Py3 and DCHF-DA exhibited very weak fluorescence between 498 and 545 nm, indicating that Py3 cannot generate ROS in the absence of light. However, 290 × 290 μm 2 Excitation for 100 scans (2.1 seconds per scan) in the 488 nm region significantly increased green fluorescence, indicating that Py3 can generate a large amount of ROS under light irradiation. Additionally, the presence of only Py3 or DCHF-DA showed a slight increase in fluorescence intensity in the green region under similar white light irradiation, further confirming that ROS actually generated Py3 light irradiation.

[0091] The photoprotective and radiation-protective capacity of melanin in melanoma cancer was evaluated by endogenous stimulation of melanin in BF10 cell lines. As shown in Figure 7, BF10 cell lines were treated with 0.1 to 5 nM MSH to induce the melanin synthesis process and irradiated under both 1 and TP conditions. When BF10 was irradiated using a single-photon confocal microscope, increased MSH concentrations (0 to 1 nM) showed a clear decrease in the percentage of dead cells at 50 and 100 scans, respectively (Figure 7a and b). In contrast, two light exposures at 750 nm in similar MSH-treated BF10 cell lines demonstrated that photo-induced cytotoxicity was independent of the cell's melanin content. To accurately evaluate melanin content versus PDT, non-melanoma (A437) and melanoma cell viability were studied using a cck assay. We observed that phototoxicity decreased linearly under single-photon irradiation conditions from non-melanoma cell lines to melanin-induced melanoma cell lines (Fig. 7c and d). These results indicated that two-photon-induced PDT is a viable approach for the treatment of melanoma.

[0092] Finally, by Py3 under OP and TP light in 2D melanoma cell cultures in vitroThe PDT results prompted the evaluation of PDT efficiency in multicellular tumor spheroids (MCTS). Light-induced PDT was demonstrated on melanoma MCTS measuring 300 μm. Fibroblast cells were co-cultured at different ratios to enhance the spherical stability of BF10. During the PDT evaluation, it was observed that fibroblasts at a ratio of 4:1 with BF10 maintained their shape. Py3 was incubated in the melanoma spheroids for 30 minutes (Fig. 7). A spatial target area with an internal diameter of 15 ± 5 μm was selected, and incident light at 488 nm (OP, 0.002 mW, 2.6 s per scan) and 750 nm (TP, 100 fs, 80 MHz, 3.6 mW, 2.6 s per scan) was irradiated for 200 scans. Then, propidium iodide (PI) was added to the MCTS, and the spheroids were incubated for 8 hours. As a result, according to Fig. 8, CSLM images at various depths showed that the unirradiated spheroids had very low red signal of PI, whereas the OP and TP scan areas showed well-stained PI dye inside the nucleus. Laser irradiation with Py3 on BF10 spheroids generated ROS on the cell membrane, causing membrane blistering and leakage, which led to necrosis of the spheroids. Thus, it was confirmed that Py3 can act as a PDT in specific areas within a mimic tumor.

[0094] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0095] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 Asymmetric pyrene derivative compounds represented by the following Chemical Formula 1 or Chemical Formula 2: [Chemical Formula 1] [Chemical Formula 2] In the above chemical formula 1 or chemical formula 2, R is a (C1~C4)alkyl. Claim 2 An asymmetric pyrene derivative compound according to claim 1, wherein R is methyl. Claim 3 An asymmetric pyrene derivative compound according to claim 1, wherein the pyrene derivative compound is 4-(2-(6-(benzo[d]thiazol-2-yl)pyrene-1-yl)vinyl)-1-methylpyridin-1-ium. Claim 4 A two-photon fluorescent dye comprising the compound of claim 1 as an active ingredient. Claim 5 A two-photon fluorescent dye according to claim 4, characterized in that the two-photon fluorescent dye selectively generates reactive oxygen species (ROS) by two-photon excitation (TPE) to selectively kill cells or tissues. Claim 6 A two-photon fluorescent dye according to claim 4, characterized in that the two-photon fluorescent dye is photoactivated by light in the range of 700 nm to 950 nm. Claim 7 A pharmaceutical composition for the prevention or treatment of cancer comprising the compound of claim 1 as an active ingredient. Claim 8 A pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the cancer is one or more selected from the group consisting of melanoma, skin cancer, colorectal cancer, breast cancer, liver cancer, lung cancer, blood cancer, sarcoma, and brain cancer. Claim 9 A method for promoting cancer cell death in an organism other than a human, comprising the steps of applying the two-photon fluorescent dye of claim 4 to a cell or tissue and irradiating the cell with light in the range of 700 nm to 950 nm.

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