Organic two-photon fluorescent substance exhibiting red fluorescence and use thereof

Abstract

The present invention relates to an organic two-photon fluorescent substance exhibiting red fluorescence and a use thereof and, more specifically, to: an organic two-photon fluorescent substance emitting red fluorescence around 600 nm when excited by light having a wavelength of 700 nm or more; and a method for imaging cells, tissues, or animals using same.

Description

Organic two-photon phosphor exhibiting red fluorescence and use thereof

[0001] This application claims the benefit of Korean Patent Application No. 10-2023-0176300, filed December 7, 2023, and Korean Patent Application No. 10-2024-0177797, filed December 3, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to an organic two-photon phosphor exhibiting red fluorescence and its use, and more particularly, to an organic two-photon phosphor emitting red fluorescence near 600 nm by excitation light having a wavelength of 700 nm or more, and a method for imaging cells, tissues, or animals using the same.

[0003]

[0004] Fluorescence bioimaging is a simple, non-invasive, and objective analytical method with numerous potential applications in living samples. It allows for real-time, high-resolution, and rapid monitoring of targets, even at very low concentrations. However, conventional fluorescence bioimaging methods, such as one-photon microscopy (OPM), remain limited by autofluorescence, photodamage, and shallow penetration depth.

[0005]

[0006] Two-photon microscopy (TPM), which utilizes near-infrared (NIR > 700 nm) two-photon excitation sources, has demonstrated the ability to provide long-term, high-resolution images with deep penetration into living specimens while minimizing autofluorescence and photodamage. Therefore, the construction of a two-photon system is a promising method for monitoring the structure and activity of specific regions of living specimens, as well as small-molecule bioactive compounds and proteins. Two-photon microscopy has attracted significant attention as it allows for the real-time observation of biological phenomena and the development and progression of diseases, potentially making groundbreaking contributions to the advancement of biology and medicine / pharmaceutics.

[0007]

[0008] To utilize two-photon microscopy to study various biological phenomena, a large number of two-photon fluorophores suitable for the desired purpose are required. However, existing fluorescent materials have limitations due to low two-photon fluorescence efficiency, and the number of fluorophores that can be used in two-photon microscopy and are commercially available is very limited. Since various biological phenomena occur simultaneously in the body, the utilization of two-photon microscopy for research and applications in biology and medicine requires the development of fluorophores that emit fluorescence across multiple wavelength ranges, such as blue, green, red, and far-red. However, currently, the only fluorophores specialized for two-photon microscopy are commercially available for use in the far-red channel.

[0009]

[0010] Fluorophores applied to the red detection region (RFP channel) of conventional microscopes exhibit very low fluorescence efficiency in aqueous solutions, which are the biological environment. This leads to analysis errors due to increased signal-to-noise ratios in microscopic analysis, and to overcome this, large amounts of fluorophore must be injected into biological samples, which can lead to sample damage. Furthermore, most known two-photon fluorophores near 600 nm exhibit a broad fluorescence spectrum, resulting in significant interference in the green and far-red detection regions. While multi-channel simultaneous analysis is an essential tool for biological imaging, this interference hinders the application of existing red-based two-photon fluorophores. Furthermore, a simple and flexible design strategy is needed to link fluorophores to various proteins and receptors.

[0011]

[0012] Under these circumstances, the inventor of the present invention sought to develop an organic fluorescent substance that, when excited by light in the near-infrared region of 700 nm or more, emits strong red fluorescence around 600 nm, minimizes interference with the green (GFP) and deep red (Far-Red) detection regions, and can efficiently bind to proteins.

[0013]

[0014] Accordingly, an object of the present invention is to provide a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0015] [Chemical Formula 1]

[0016]

[0017] In the above formula,

[0018] X1 and X2 are each independently hydrogen, C1~6 alkyl, -CO2H, -SO3H, , and is selected from the group consisting of, and X2 is present in the meta or para position with respect to X1,

[0019] The above R is , , and is selected from the group consisting of,

[0020] The above R' is hydrogen, C1~6 alkyl, phenyl, -CO2H, -CO2Me, -N3, -CH2N 3, , and is selected from the group consisting of,

[0021] The above n is an integer from 0 to 20.

[0022]

[0023] Another object of the present invention is to provide a conjugate in which a targeting moiety is conjugated to the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0024]

[0025] Another object of the present invention is to provide a method for imaging cells, tissues or non-human animals, comprising the step of treating the compound or conjugate with the cell, tissue or non-human animal and observing it.

[0026]

[0027] In order to achieve the above-described object of the present invention, the present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0028] [Chemical Formula 1]

[0029]

[0030] In the above formula,

[0031] X1 and X2 are each independently hydrogen, C1~6 alkyl, -CO2H, -SO3H, , and is selected from the group consisting of, and X2 is present in the meta or para position with respect to X1,

[0032] The above R is , , and is selected from the group consisting of,

[0033]

[0034] The above R' is hydrogen, C1~6 alkyl, phenyl, -CO2H, -CO2Me, -N3, -CH2N 3, , and is selected from the group consisting of,

[0035] The above n is an integer from 0 to 20.

[0036]

[0037] In order to achieve another object of the present invention, the present invention provides a conjugate in which a targeting moiety is conjugated to the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0038]

[0039] In order to achieve another object of the present invention, the present invention provides a method for imaging cells, tissues or non-human animals, comprising the step of treating the compound or conjugate with the cell, tissue or non-human animal and observing it.

[0040]

[0041] Hereinafter, the present invention will be described in more detail.

[0042]

[0043] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0044] [Chemical Formula 1]

[0045]

[0046] In the above formula,

[0047] X1 and X2 are each independently hydrogen, C1~6 alkyl, -CO2H, -SO3H, , and is selected from the group consisting of, and X2 is present in the meta or para position with respect to X1,

[0048] The above R is , , and is selected from the group consisting of,

[0049] The above R' is hydrogen, C1~6 alkyl, phenyl, -CO2H, -CO2Me, -N3, -CH2N 3, , and is selected from the group consisting of,

[0050] The above n is an integer from 0 to 20.

[0051]

[0052] In one aspect of the present invention, X1 and X2 are each independently hydrogen, -CO2H, and , and X2 may be characterized by existing in a para position with respect to X1.

[0053]

[0054] In another aspect of the present invention, R' is hydrogen, phenyl, -CO2Me, -CH2N3 or It can be characterized by being.

[0055]

[0056] In another aspect of the present invention, n may be an integer from 0 to 10, and preferably an integer from 0 to 5.

[0057]

[0058] In another aspect of the present invention, the compound represented by the chemical formula 1 may be characterized by being any one of the following chemical formulas 2 to 6:

[0059] [Chemical Formula 2]

[0060]

[0061] [Chemical Formula 3]

[0062]

[0063] [Chemical Formula 4]

[0064]

[0065] [Chemical Formula 5]

[0066]

[0067] [Chemical Formula 6]

[0068]

[0069]

[0070] In one aspect of the present invention, the compound or a pharmaceutically acceptable salt thereof may be characterized as being a two-photon absorbing fluorescent substance.

[0071]

[0072] As used herein, "alkyl" means an aliphatic hydrocarbon group unless otherwise defined. The alkyl group may be a "saturated alkyl group" that does not contain any double bond or triple bond. The alkyl group may also be an "unsaturated alkyl group" that contains at least one double bond or triple bond. Regardless of whether saturated or unsaturated, the alkyl group may be branched, straight-chain, or cyclic. For example, a C1 to C4 alkyl group indicates that the alkyl chain has 1 to 4 carbon atoms, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. For example, the alkyl group refers to a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a t-butyl group, a pentyl group, a hexyl group, an ethenyl group, a propenyl group, a butenyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and the like.

[0073]

[0074] In the present invention, the “pharmaceutical acceptable salt” refers to any organic or inorganic addition salt of the compounds of the present invention at a concentration that is relatively non-toxic and harmless and has an effective effect, and the side effects caused by the salt do not reduce the beneficial effects of the compounds of the present invention. These salts can use inorganic acids and organic acids as free acids. Inorganic acids that can be used include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc., and organic acids that can be used include citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, malonic acid, etc. In addition, these salts can include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.).For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, Salts such as tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc may be included, but are not limited thereto as long as they do not affect the efficacy of the compounds of the present invention and can be added.

[0075]

[0076] In one aspect of the present invention, the compound or a pharmaceutically acceptable salt thereof may be characterized as being a two-photon absorbing fluorescent substance.

[0077]

[0078] The present invention also provides a conjugate in which a targeting moiety is conjugated to a compound or a pharmaceutically acceptable salt thereof.

[0079]

[0080] In the present invention, the “targeting moiety” refers to a substance that has an affinity for or can bind to a molecule in a living body.

[0081]

[0082] In the present invention, the “in vivo molecule” includes proteins, glycoproteins, receptors, hormones, enzymes, antigens, etc. contained in cells, tissues, organs, and body fluids existing in or derived from a living body.

[0083]

[0084] In one aspect of the present invention, the targeting moiety can target intracellularly, extracellularly or to the cell membrane.

[0085]

[0086] In one aspect of the present invention, the targeting moiety can bind to a target located in the cytoplasm, cell membrane, or extracellular matrix (ECM), and the target located in the cell membrane can be a membrane protein, a glycosylated phospholipid, or a portion thereof present in the cell membrane.

[0087]

[0088] In one aspect of the present invention, the targeting moiety may be a small molecule, a peptide, a protein or a phospholipid, and may be, but is not limited to, a cell adhesion molecule, an antibody, an antibody fragment, a targeting protein, a membrane fusion protein, an aptamer, a hormone, a cytokine, a chemokine, a ligand, a peptide that is a portion of a cytokine, a peptide that is a portion of a ligand, or a fusion protein thereof.

[0089]

[0090] In one embodiment, the targeting moiety can be a cell-penetrating peptide (CPP) or a fragment thereof, wherein the cell-penetrating peptide can be, but is not limited to, Penetratin, Tat (trans-activating transcriptional activator), Transportan, MAP, KALA, P1, MPG, PEP-1, hCT, Mastoparan, SV40 nuclear transport signal peptide derivative, Antp, Mph-1, VP22, HP4 or multi-Arg.

[0091]

[0092] The targeting moiety may be directly bound to the compound represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof (e.g., via an amide bond or an ester bond), or may be bound via a linker.

[0093]

[0094] The linker may be selected from the group consisting of, but is not limited to, dicyclohexyl carbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMC), diisopropyl carbodiimide (DIC), N-Hydroxysuccinimide (NHS), and N-Hydroxysulfosuccinimide sodium salt (NHSS).

[0095]

[0096] The present invention also provides a method for imaging a cell, tissue or non-human animal, comprising the step of treating the cell, tissue or non-human animal with the compound or the conjugate and observing the same.

[0097]

[0098] In one aspect of the present invention, the method may further comprise the step of treating the compound or the conjugate with a cell, tissue, or an animal other than a human and irradiating the compound with excitation light.

[0099]

[0100] In one aspect of the present invention, the excitation light may have a wavelength in the range of 700 to 1000 nm, and preferably may have a wavelength in the range of 800 to 1000 nm.

[0101]

[0102] In one aspect of the present invention, the observation may be characterized by using a two-photon fluorescence microscope.

[0103]

[0104] According to the above method of the present invention, it may be possible to observe physiological phenomena in real time by imaging biomolecules in living cells or tissues.

[0105]

[0106] The novel compound according to the present invention provides an organic fluorescent substance that emits strong red fluorescence around 600 nm upon excitation with excitation light having a wavelength ranging from 700 to 1000 nm. The present invention provides a fluorescent substance that exhibits a strong two-photon excitation efficiency of over 200 GM and a fluorescence efficiency close to 100% in an aqueous solution, which is a biological environment, and minimizes interference with other detection areas during simultaneous multi-channel analysis, thereby providing very high precision in microscopic analysis. Furthermore, the present invention provides a fluorescent substance that can be easily linked to a desired protein, thereby enabling its use in various analyses.

[0107]

[0108] Figures 1a and 1b show the absorption and fluorescence spectra of AR2, the basic fluorescent framework of novel compounds according to the present invention. Figure 1a shows the absorption spectrum in an EtOH and phosphate buffer saline (PBS, pH=7.4) solution, and Figure 1b shows the fluorescence spectrum in the above solution.

[0109]

[0110] Figure 2 shows the two-photon action cross-section spectra (GM: 1 GM = 1 × 10) of AR2 fluorophore in EtOH and PBS solutions. -50 cm 4 sphoton -1 molecule -1 ) is shown.

[0111]

[0112] Figure 3 shows the image results for signal intensity analysis by detection channel of two-photon microscopy of AR2. The two-photon microscope used was IVM-MS-C manufactured by Ibeam Technology Co., Ltd., with an excitation wavelength of 920 nm and a 20x magnification lens. The detection areas used GFP, RFP, and Cy5 channels, respectively, and the degree of mutual interference was displayed after overlapping them.

[0113]

[0114] Figure 4 shows the image results for signal intensity analysis by two-photon microscopy detection channel of AR2. The two-photon microscope used was IVM-CM3 manufactured by Ibeam Technology Co., Ltd., with an excitation wavelength of 800-1000 nm and a 20x magnification lens. The detection areas used GFP, RFP, and Cy5 channels, respectively, and the degree of mutual interference was indicated after overlapping these.

[0115]

[0116] Figure 5 shows the image results for signal intensity analysis by two-photon microscopy detection channel after conjugating anti-CD31 antibody to AR5-NHS. The two-photon microscope used was IVM-CMS3 manufactured by Ibeam Technology Co., Ltd., with an excitation wavelength of 920 nm and a 20x magnification lens. The detection areas used GFP, RFP, and Cy5 channels, respectively, and the degree of mutual interference was displayed after overlapping them (merged).

[0117]

[0118] Figures 6a and 6b are two-photon microscope images of the ear skin area after injection of the AR5-NHS-CD31 antibody conjugate into a living mouse. Figure 6a is a two-photon microscope image taken in GFP (500 nm channel), RFP (600 nm channel), and Cy5 (670 nm channel) after injection of the AR5-NHS-CD31 antibody conjugate (40 micrograms) into the tail vein of the mouse (IV injection). Figure 6b is a two-photon microscope image of the entire ear skin area by combining the images from each channel. The mouse used was B6 (female), and the images were obtained using a 920 nm femtosecond laser, IVM-CMS3, Ibeam Technology Co., Ltd.

[0119]

[0120] Figures 7a and 7b are two-photon microscope images of the liver region after injection of AR5-NHS-CD31 antibody conjugate into a living mouse. Figure 7a shows two-photon microscope images taken in GFP (500 nm channel), RFP (600 nm channel), and Cy5 (670 nm channel) after injection of AR5-NHS-CD31 antibody conjugate (40 micrograms) and GFP channel dye into a blood vessel in the tail region of the mouse (IV injection, tail vein). Figure 7b shows a two-photon microscope image of the entire liver region by combining the images from each channel. The mouse used was B6 (female), and the images were obtained using a 920 nm femtosecond laser, IVM-CMS3, Ibeam Technology Co., Ltd.

[0121]

[0122] Figures 8a and 8b are two-photon microscope images of the spleen region after injection of AR5-NHS-CD31 antibody conjugate into a living mouse. Figure 8a is a two-photon microscope image taken in the GFP, RFP, and Cy5 channels, respectively, after injection of AR5-NHS-CD31 antibody conjugate (40 micrograms) and GFP channel dye into the tail vein of the mouse (IV injection, tail vein). Figure 8b is a two-photon microscope image of the entire spleen region by combining the images of each channel. The mouse used was B6 (female), and the images were obtained using a 920 nm femtosecond laser, CMS3, Ibeam Technology Co., Ltd.

[0123]

[0124] Figure 9 is a two-photon microscope image of the kidney region after injection of AR5-NHS-CD31 antibody conjugate into a live mouse. AR5-NHS-CD31 antibody conjugate (40 micrograms) and GFP channel dye were injected into the blood vessel in the tail region of the mouse (IV injection, tail vein), and these are two-photon microscope images taken in the GFP, RFP, and Cy5 channels, respectively. (Merged) This is a complete two-photon microscope image of the kidney region by combining the images from each channel. The mouse used was a B6 (female), and the images were obtained using a 920 nm femtosecond laser, CMS3, Ibeam Technology Co., Ltd.

[0125]

[0126] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0127]

[0128] 1. Materials and experimental methods

[0129] All chemicals were purchased from Sigma-Aldrich, and reactions were performed under a nitrogen atmosphere unless otherwise specified. The reaction progress was monitored using thin layer chromatography (TLC) plates (TLC Silica gel 60 F254, 1.05715.0001, Merck). The products were purified using medium-pressure liquid chromatography (AI-580S, YAMAZEN) using general-purpose or C18 column cartridges. NMR spectra were acquired using a 600 MHz NMR spectrometer (JNM-ECZ 600R, JEOL). High-performance liquid chromatography (HPLC) was performed using a Waters 2695 module (Waters, Milford, MA, USA) using a Waters X-Bridge column (C18, 5 μm, 4.6 × 250 mm). Preparative high-performance liquid chromatography (preparative HPLC) was performed on a Waters 2525 Binary Gradient module using a Waters X-Bridge Prep column (C18, 5 μm, 19 × 250 mm), and mass data were obtained using a HRMS system (Accela UPLC / LTQ-Orbitrap XL, Thermo Fisher Scientific) and a Q-TOF system (Dionex UPLC / TripleTOF 5600+, AB Sciex) provided by the Gyeonggi Business and Science Accelerator (GBSA, Korea). Oleic acid, amiodarone, tetracycline, cyclosporine, tamoxifen, and chloroquine were purchased from Sigma-Aldrich (St. Louis, MO). Propranolol was obtained from Tokyo Chemical Industry (Tokyo, Japan).

[0130]

[0131] 2. Spectroscopic experiment

[0132] Absorption spectra were acquired on an S-3100 UV-Vis spectrophotometer (SCINCO, Korea), and emission spectra were recorded on a FluoroMate FS-2 spectrometer (SCINCO, Korea) using a 1.0 cm optical path Hellma quartz cell with a Teflon stopper (HE.111.650QG, Hellma Analytics). Relative fluorescence quantum yields were established using cresyl violet (Φ = 0.54 in methanol) as a reference compound.

[0133]

[0134] 3. Measurement of the cross-sectional area of ​​the two-photon beam

[0135] The two-photon absorption cross-section (δ) was determined by the following general method. Fluorophores (1.0–5.0 μM) were dissolved in various solvents, and the two-photon excited emission was integrated using Rhodamine 6G as a reference compound with well-established TP properties. The TPEF intensities of each sample and Rhodamine 6G were detected at the same excitation wavelength (720–960 nm). The two-photon cross-section is δ = δ r (S s Φ r φ r c r ) / (S r Φ s φ s c s ), where the subscripts r and s represent the reference compound and sample, respectively. δ ris the two-photon cross-section of rhodamine 6G, S is the TPEF signal collected using a CCD system (Monora 320i monochromator with DV401A-BV detector, DONGWOO OPTRON, Korea), Φ is the fluorescence quantum yield, φ is the overall fluorescence collection efficiency of the experimental system, and c is the concentration of each sample.

[0136]

[0137] 4. Two-photon fluorescence microscopy

[0138] Two-photon fluorescence microscopy images of the PBS solution of AR-induced fluorescence and the rat were acquired using a spectral confocal (488 nm) and multiphoton microscope (Leica TCS SP8 MP, IVM-CM3, IVM-CMS3, IVM-MS-C, Ibeam Technology Co., Ltd.) with ×10 dry lens, ×40 oil lens, and ×100 oil objectives, numerical apertures (NA) of 0.30, 1.30, and 1.30, respectively. Images were acquired using a multiphoton microscope (Leica TCS SP8 MP, IVM-CM3, IVM-CMS-3, IVM-MS-C, IBeam Technology Co., Ltd.) by exciting the probe with a mode-locked titanium-sapphire laser source (Mai Tai HP; Spectra Physics, 80 MHz pulse frequency, 100 fs pulse width) set to an 800–1000 nm wavelength and 2673 mW output power, corresponding to an average power of approximately 2.19 mW at the focal plane. Live-cell imaging was performed using a live-cell incubator (Chamlide IC system, Live Cell Instrument, Korea) to maintain a stable cell environment. Fluorescence signals were collected at 8-bit unsigned 512 × 512 and 1024 × 1024 pixels at scan rates of 400 and 200 Hz, respectively, using internal PMTs.

[0139]

[0140] Example 1. Synthesis of a novel red fluorescent AR derivative

[0141] Organic dyes exhibiting high two-photon action (TPA) cross sections include high intramolecular charge transfer (ICT) efficiency. Conventional rhodamine-based dyes have ICT properties due to the presence of push-pull type dipoles or pseudo-quadrupoles within the molecule, but they suffer from relatively low fluorescence quantum yields and poor photostability. The newly developed AR dyes introduce pyrrolidine-fused tetrahydroquinoxaline (p-THQ), a strong electron donor, and conjugate it with julidine, a double-cyclic amine, to design fluorescent materials exhibiting extremely high intramolecular charge transfer (ICT) efficiency. In addition, by cyclizing the entire dye skeleton, we expected to improve photostability, exhibit a narrow fluorescence spectrum, and provide high quantum yields and two-photon action (TPA) cross-sections. Furthermore, we designed the structure to facilitate the efficient introduction of various receptors, such as protein linkers, into the p-THQ moiety. The synthetic methods for representative skeletons and example substances among the novel fluorophores, along with structural analysis data obtained by 600 MHz 1H-NMR, are illustrated below.

[0142]

[0143]

[0144]

[0145] AR2: In a flame-dried, double-necked, 50-mL round-bottomed flask, compound 2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbonyl)benzoic acid (1 g, 2.96 mmol) and methyl 6-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)-6-oxohexanoate (0.98 g, 2.96 mmol) were added under inert conditions. TFA (15 mL) was added and refluxed at 95°C overnight. After confirming consumption of the starting material, the reactant was evaporated to dryness and volatile impurities were removed using a toluene strip. The residue was purified using flash chromatography using acidic silica gel. To obtain the desired AR2 as a dark blue solid, CHCl3:MeOH was changed from (9.5:0.5) to (8:2). Yield after purification: (0.8 g 1.26 mmol 42.6 %); 1H-NMR (600 MHz, CDCl3+METHANOL-D4 1:1) δ 7.85-7,79 (m, 1H), 7.27 (s, 1H), 7.24-7.23 (m,1H), 6.74-6.78 (m, 1H), 6.37 (s, 1H), 6.31 (s, 1H), 6.25 (s, 1H), 3.33 (t, J = 12 Hz, 1H), 3.22 (s, 3H), 3.16 (t, J= 6 Hz, 2H), 3.11 (m, 3H), 3.06 (t, J= 6 2H), 2.92 (s, 1H), 2.67 (t, J = 5.4 2H), 2.63 (t, J= 7.2 1H), 2.24 (m, 3H),1.86 (m, 2H), 1.68 (m, 3H) 1.58 (bs, 3H), 1.00-1.18 (m, 4H) 0.82 (s,2H)

[0146]

[0147]

[0148]

[0149] AR2-NHS: AR2 (0.1 g, 0.16 mmol) and lithium hydroxide monohydrate (66 mg, 1.58 mmol) were added to THF:H2O (1:1), and a few drops of MeOH were added. The mixture was stirred at room temperature overnight. THF and MeOH were removed from the reaction mixture, the aqueous layer was acidified, and the aqueous layer was concentrated to dryness. The mixture was adsorbed onto silica gel and flash chromatographed in CHCl3:MeOH (9.5:0.5) to (8:2) to obtain the target dye as a dark blue solid. Yield: 21%; 1H-NMR (600 MHz, METHANOL-D4) δ 8.13 (d, J = 5.5 Hz, 1H), 7.65-7.70 (m, 3H), 7.23 (s, 1H), 6.70 (s, 2H), 3.68-3.74 (m, 2H), 3.63 (m, 1H), 3.49 (m, 8H), 3.38 (m, 1H), 3.04 (m, 4H), 2.66 (m, 4H), 2.07 (m, 4H), 1.94 (m, 8H).

[0150] Hydrolyzed form (0.50 g, 0.08 mmol), DCC (33 mg, 0.16 mmol), and NHS (20 mg, 0.16 mmol) were added to DMF (4 mL), and the reaction was stirred at RT overnight. The reaction was loaded onto reverse-phase flash chromatography in ACN:H2O (2.5:7.5) to (7.5:2.5) to obtain the target AR2-NHS.

[0151]

[0152]

[0153]

[0154] AR-C-NHS. 11-((Benzyloxy)carbonyl)-9-(2-((3-carboxypropyl)(methyl)carbamoyl)phenyl)-1,2,3,5,6,7,11,12,12a,13,14,15-dodecahydropyrrolo[1,2-a]quinolizino[1',9':6,7,8]chromeno[3,2-g]quinoxalin-4-ium (0.06 g, 0.08 mmol, 1 eq) was added to a 50 mL round-bottom flask filled with nitrogen, followed by 3 mL of DMF. The starting material was stirred, and then triethylamine (0.048 g, 0.24 mmol, 6 eq) was added and stirred for 5 min. N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate, O-[N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (0.072 g, 0.24 mmol, 3 eq) was added last and stirred for 3 h. TLC was performed to confirm the completion of the starting material. The reaction mixture was concentrated in vacuo and partitioned between methanol / dichloromethane in water. The organic layer was collected, dried over Na2SO4, and concentrated in vacuo. The crude material was then purified using flash column chromatography (methanol / dichloromethane). The fractions were collected and concentrated in vacuo to give the desired product. (0.04 g, 0.048 mmol, 58.80%). 1 H NMR (600 MHz (CD3OD)): δ = 7.69-7.76 (m, 2H), 7.58-7.63 (m, 2H), 7.15-7.48 (m, 6H), 6.77-6.88 (m, 2H), 3.55-3.57 (m, 4H), 3.34 (s, 3H), 3.05 (br.s 4H), 2.80-2.89 (m, 4H), 2.64-2.73 (m, 5H), 2.23-2.28 (m, 2H), 2.07-2.13 (m, 4H), 1.92-2.03 (m, 5H), 1.52-1.85 (m, 5H).

[0155]

[0156]

[0157]

[0158] AR5-N3: A 100 mL round-bottomed flask equipped with a condenser was charged with 2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline-9-carbonyl)terephthalic acid A (0.3 g, 0.786 mmol, 1 eq) and 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-1-(8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propan-1-one B (0.4 g, 0.94 mmol, 1.2 eq), followed by 10 mL DMF. The reaction mixture was stirred, and then trimethylsilyl polyphosphate (0.4 mL) was added. The mixture was heated at 80°C for 6 h. The resulting solution was concentrated in vacuo to dryness. The crude oil was further treated with NaOH and stirred overnight at 0°C. The crude oil was neutralized with HCl and MeOH was partitioned into DCM / water (4 x 50 mL). The combined layers were dried over Na2SO4 and concentrated in vacuo. The product was purified by flash column chromatography (methanol / dichloromethane). The combined fractions were concentrated to give AR5-N3 (0.1 g, 0.13 mmol, 16.6%). 1 H NMR (600 MHz, (CD3OD)): δ=8.13-8.17(m, 2H), 8.03-8.06(m, 1H), 7.73 (d, J=6 Hz, 1H), 6.79(s, 1H), 6.72-6.75 (m, 1H), 3.40-3.68 (m, 20H), 3.26-3.27 (m, 2H), 2.96-3.00 (m, 3H), 2.60-2.65 (m, 3H), 2.19-2.2 (m, 2H), 2.00-2.04 (m, 4H), 1.84-1.86 (m, 3H).

[0159]

[0160]

[0161]

[0162] AR5-NHS: 9-(2,5-dicarboxyphenyl)-11-(2,5,8,11-tetraoxatetradecan-14-oyl)-1,2,3,5,6,7,11,12,12a,13,14,15-dodecahydropyrrolo[1,2-a]quinolizino[1',9':6,7,8]chromeno[3,2-g]quinoxalin-4-ium A (0.07 g, 0.092 mmol, 1 eq) and a DMF:DCM solvent mixture (3:1) were added sequentially to a 50 mL round-bottomed flask filled with nitrogen. The starting material was stirred in a solvent mixture, then bis(2,5-dioxopyrrolidin-1-yl) carbonate B (0.106 g, 0.42 mmol, 4.5 eq) and triethylamine (0.028 mg, 0.278 mmol, 3 eq) were added. The reaction mixture was stirred at RT for 6 h, and the completion of the starting material was observed by TLC. The crude material was concentrated and taken directly for purification using flash column chromatography (methanol / dichloromethane). The fractions were collected and concentrated in vacuo to give the desired product (0.025 g, 0.29 mmol, 31.64%). 1 H NMR (600MHz (CD3OD)) δ = 8.36-8.39 (m, 1H), 8.18-8.22 (m, 1H), 8.05 (s, 1H), 7.33 (br.s 1H), 6.83-6.87 (m, 1H), 3.42-3.78 (m, 21H), 3.08 (br.s 1H), 2.89 (s, 4H), 2.67-2.71 (m, 5H), 2.36-2.57 (m, 2H), 2.24 (br.s, 1H), 2.09 (br.s, 4H), 1.93-1.94 (m, 3H), 1.48-1.60 (m, 2H).

[0163]

[0164] Example 2. Confirmation of the photophysical properties of AR dyes

[0165] The photophysical properties of AR2 dye, which has a representative fluorescent backbone, were evaluated in EtOH and PBS buffer solutions similar to physiological environments. AR2 exhibited an absorption spectrum near 570 nm and a narrow and strong fluorescence spectrum near 600 nm in these solutions (Figs. 1a and 1b, Table 1). In particular, the molar extinction coefficient (ε) in the PBS buffer solution was 6.4 × 10 4 M -1 cm -1 It exhibited 100% fluorescence quantum yield (Table 1). As expected, the two-photon action (TPA) cross section value (Φδmax) of AR2 showed a high value exceeding 220 GM when excited at 900 nm in an aqueous environment (Fig. 2, Table 1). Such 100% fluorescence quantum yield and two-photon action cross section value are the highest values ​​among existing rhodamine series dyes and can be used as a precise analysis tool together with bright two-photon microscopy images (Figs. 6a to 9).

[0166]

[0167] [Table 1]

[0168]

[0169]

[0170] Example 3. Two-photon microscopy imaging

[0171] Multichannel microscopy imaging is an essential element in biological image analysis. The main detection regions of currently widespread two-photon microscopy include GFP (500 nm), RFP (600 nm), and Cy5 (570 nm) channels. Among these regions, the only dye specifically designed for two-photon excitation is the Cy5 channel. To verify whether AR2 and AR5 fluorophores are suitable for the RFP detection region, we analyzed the signal intensity for each detection channel using two-photon microscopy. The two-photon microscope used was the IVM-CMS3 and IVM-MS-C manufactured by Ibeam Technology Co., Ltd., and a femtosecond pulse laser with a wavelength of 800-1000 nm was used for fluorophore excitation.

[0172]

[0173] As shown in Figures 3 to 5, the AR2 fluorophore selectively exhibited a high-intensity signal in the RFP channel and a weak signal in the remaining channels. In particular, the AR5-NHS and anti-CD31 antibody conjugate exhibited a significant fluorescence signal only in the RFP channel without interference in the GFP and Cy5 channels (Figure 5).

[0174]

[0175] Next, AR5-NHS-CD31 antibody conjugate (40 μg) was injected intravenously into the tail vein of a live mouse (B6, female), and two-photon microscopy images were captured in the GFP, RFP, and Cy5 channels (Figs. 6a to 9). Two-photon microscopy images were captured and analyzed from the ear (ear skin), liver (liver), spleen (spleen), and kidney (kidney) regions of the B6 mouse. To verify the usefulness of multi-channel analysis, widely used dyes were co-injected into the GFP channel. As shown in Figs. 6a to 9, the AR5-NHS-CD31 antibody conjugate clearly showed the blood vessels of these organelles and was suitable for precise analysis with minimal signal interference with other channels.

[0176]

[0177] In summary, these results confirm that the AR material according to the present invention can be used as a material useful for the red channel in two-photon microscopy images.

[0178]

[0179] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0180]

[0181] The novel compound according to the present invention provides an organic fluorescent substance that emits strong red fluorescence around 600 nm when excited with excitation light having a wavelength in the range of 700 to 1000 nm. It exhibits a strong two-photon excitation efficiency of 200 GM or more and a fluorescence efficiency close to 100% in an aqueous solution, which is a biological environment, and provides a fluorescent substance that minimizes interference with other detection areas during simultaneous multi-channel analysis, thereby providing very high precision in microscopic analysis. In addition, by providing a fluorescent substance that can be easily linked to a desired protein, it can be utilized for various purposes of analysis, and thus has very high industrial applicability.

Claims

1. A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above formula, X 1 and X 2 are each independently hydrogen, C1~6 alkyl, -CO 2 H, -SO 3 H, , and is selected from the group consisting of , and X 2 is X 1 It exists in the meta or para position, The above R is , , and is selected from the group consisting of, The above R' is hydrogen, C1~6 alkyl, phenyl, -CO 2 H, -CO 2 Me,-N 3 , -CH 2 N 3, , and is selected from the group consisting of, The above n is an integer from 0 to 20.

2. In paragraph 1, the X 1 and X 2 are each independently hydrogen, -CO 2 H, and is selected from the group consisting of X 2 is X 1 A compound characterized by being present in the para position, or a pharmaceutically acceptable salt thereof.

3. In paragraph 1, R' is hydrogen, phenyl, -CO 2 Me, -CH 2 N 3 or A compound characterized by: or a pharmaceutically acceptable salt thereof.

4. In the first paragraph, the compound represented by the chemical formula 1 is a compound characterized by being any one of the following chemical formulas 2 to 6 or a pharmaceutically acceptable salt thereof: [Chemical formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical formula 6] 5. A compound or a pharmaceutically acceptable salt thereof, characterized in that the pharmaceutically acceptable salt in paragraph 1 is an inorganic acid or an organic acid which is a free acid.

6. A compound or a pharmaceutically acceptable salt thereof, characterized in that in the fifth paragraph, the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid.

7. A compound or a pharmaceutically acceptable salt thereof, characterized in that in paragraph 5, the organic acid is selected from the group consisting of citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, and malonic acid.

8. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that the compound or a pharmaceutically acceptable salt thereof is a two-photon absorbing fluorescent substance.

9. A conjugate comprising a targeting moiety conjugated to a compound according to paragraph 1 or a pharmaceutically acceptable salt thereof.

10. A conjugate according to claim 9, wherein the targeting moiety has an affinity for or is capable of binding to a biological molecule.

11. A conjugate according to claim 9, characterized in that the targeting moiety is selected from the group consisting of a cell adhesion molecule, an antibody, an antibody fragment, a targeting protein, a membrane fusion protein, an aptamer, a hormone, an enzyme, an enzyme substrate, a cytokine, a chemokine, a ligand, a peptide which is a portion of a cytokine, a peptide which is a portion of a ligand, and a fusion protein thereof.

12. A method for imaging a cell, tissue or non-human animal, comprising the step of treating the cell, tissue or non-human animal with the compound according to paragraph 1 or the conjugate according to paragraph 6 and observing the same.

13. An imaging method according to claim 12, characterized by further performing a step of irradiating the compound or conjugate with excitation light after treating the compound or conjugate with a cell, tissue, or animal other than a human.

14. An imaging method according to claim 13, characterized in that the excitation light has a wavelength of 700 to 1000 nm.

15. An imaging method according to claim 12, characterized in that the observation uses a two-photon fluorescence microscope.

Images