Novel near-infrared phosphor compound and uses thereof

Abstract

The present invention relates to a novel phosphor compound and, more specifically, to a novel compound, which emits fluorescence according to the viscosity change, thereby enabling selective detection of ferroptosis and acting as a photosensitizer, leading to its application in photodynamic therapy.

Description

Novel near-infrared phosphor compound and its uses

[0001] The present invention relates to a novel near-infrared phosphor compound and its uses.

[0002]

[0003] The development of novel phosphor compounds that operate in the near-infrared region and possess excellent light transmittance is in high demand across various in vivo experiments and bioimaging fields, and their potential for application is also significant. However, currently known near-infrared phosphors generally have limitations, such as low photostability and signal instability caused by chemical degradation or oxidation reactions induced by light.

[0004] In particular, although commercially available cyanine-based phosphors are the most widely used, they have disadvantages such as easy photobleaching, low quantum yield, and short fluorescence duration. Furthermore, it has been reported that cyanine-based far-red phosphors can generate blue-shifted derivatives through photoconversion. Therefore, to utilize them in in vivo imaging and photodynamic therapy (PDT), there is a need to develop new photosensitizers and phosphors that operate in the near-infrared region.

[0005]

[0006] Against this background, the inventors completed the present invention by developing a fluorescent compound based on the small molecular weight organic compound 2,3-dihydro-1H-inden-1-one or 1H-indene-1,3(2H)-dione backbone while researching novel fluorescent compounds.

[0007] Accordingly, the objective of the present invention is to provide a novel compound.

[0008] Another objective of the present invention is to provide a fluorescent probe composition comprising a novel compound.

[0009] Another objective of the present invention is to provide a composition for photodynamic therapy comprising a novel compound.

[0010]

[0011] To solve the above-mentioned problem, the present invention provides a compound represented by the following chemical formula 1:

[0012] [Chemical Formula 1]

[0013]

[0014] In the formula, R1 is NR5R6 or OR7, and R5, R6, and R7 are each independently C 1-8 It is an alkyl or benzene, or R5 and R6 can be connected to form a 5- to 7-membered heterocycle substituted or unsubstituted with one or more substituents, L is a 5- to 14-membered arylene or heteroarylene substituted or unsubstituted with one or more substituents, and R2 is H, O, or C substituted or unsubstituted with one or more substituents. 1-6 It is a fused ring of two rings in which an alkyl, or a cycloalkyl of 5 to 7 groups substituted or unsubstituted with one or more substituents, is fused with an aryl group, and R3 and R4 are each independently H or F.

[0015] In the above compound, the above L is It could be.

[0016] In the above compound, R1 is NR5R6, and R5 and R6 are each independently C 1-3 It is alkyl or benzene, or R5 and R6 are connected to each other, piperazine or It can form.

[0017] In the above compound, R1 is OR7, and R7 is C 1-8 It can be an alkyl.

[0018] In the above compound, R2 is H, O, C(CN)2 or It could be.

[0019] In the above compound, the compound may be any one selected from the group consisting of the following compounds:

[0020]

[0021]

[0022]

[0023] In addition, the present invention provides a fluorescent probe composition comprising one or more of the above compounds.

[0024] In the above fluorescent probe composition, the fluorescent probe may exhibit near-infrared fluorescence or red fluorescence in response to an increase in viscosity.

[0025] In the above fluorescent probe composition, the fluorescent probe may be used for detecting lipid peroxidation in living cells or tissues.

[0026] In the above fluorescent probe composition, the fluorescent probe may be for detecting ferroptosis.

[0027] In addition, the present invention provides a composition for photodynamic therapy comprising one or more of the above compounds.

[0028] In the above photodynamic therapy composition, the composition may exhibit photosensitizing activity for light with a wavelength of 300 to 700 nm.

[0029]

[0030] Since the compounds of the present invention generate fluorescence upon changes in viscosity, they can selectively emit fluorescence in cells inducing ferroptosis. Therefore, the compounds of the present invention can function as biosensors for ferroptosis and can be utilized for the diagnosis of related diseases.

[0031] Furthermore, the compounds of the present invention exhibit excellent cell permeability and stably emit red fluorescence within cells, thereby enabling real-time tracking of ferrotopsis. They are suitable for in vivo imaging and are particularly advantageous for the detection of ferrotopsis in liver diseases.

[0032] In addition, the compounds of the present invention can be used as photosensitizers by reacting to light and can be used in photodynamic therapy.

[0033]

[0034] Figure 1 is a diagram showing the structure of the compound of the present invention in a schematic manner.

[0035] Figure 2 is a diagram showing the structure of the compound of the present invention in a schematic manner.

[0036] Figure 3 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0037] Figure 4 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0038] Figure 5 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0039] Figure 6 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0040] Figure 7 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0041] FIG. 8 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0042] FIG. 9 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0043] FIG. 10 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0044] FIG. 12 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0045] FIG. 13 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0046] FIG. 14 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0047] FIG. 15 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0048] FIG. 16 is a diagram showing the synthesis procedure of a specific compound of the present invention.

[0049] Figure 17 shows the absorption spectrum of 10 μM of TTM or TTB compounds measured in various solvents.

[0050] Figures 18a, 18b, and 18c show the absorption spectra of 10 μM of TTM or TTB compounds measured in various solvents.

[0051] Figure 19 shows the emission spectrum of 2 μM of the TTB compound measured in various solvents.

[0052] Figures 20a, 20b, 20c, 20d, 20e, and 20f show the emission spectra of 2 μM of TTM compound measured in various solvents.

[0053] Figures 21a, 21b, 21c, and 21d show the normalized emission spectra of 2 μM of TTM or TTB compounds measured in various solvents and photographs thereof.

[0054] Figures 22a, 22b, and 22c show the normalized emission spectra of 2 μM of TTM or TTB compounds measured in various solvents and photographs thereof.

[0055] Figures 23a and 23b show photographs of 10 μM MTdT-2 and Pz0T-2 excited with ultraviolet light (excitation wavelength = 365 nm) in glycerol / H2O mixed solutions (0-95%) at various concentrations, as well as fluorescence spectra and integrated intensity plots.

[0056] Figures 24a, 24b, 24c, 24d, 24e, and 24f show the fluorescence spectra (top) and integrated intensity plots (bottom) of 10 μM TTM-04, TTM-09, TTM-10, TTM-12, TTM-13, and MTdT-2 excited with UV light (excitation wavelength = 365 nm) in glycerol / H2O mixed solutions (0-95%) at various concentrations. For all compounds, the fluorescence intensity was stronger with increasing glycerol concentration, and the strongest fluorescence intensity was observed in the 95% glycerol / H2O mixed solution.

[0057] Figures 25a, 25b, 25c, and 25d show the fluorescence spectra (top) and integrated intensity plots (bottom) of MTdT-CN, PzdT-2, Pz0T-2, and Oxy8-0T-2 excited by UV light (excitation wavelength = 365 nm) in glycerol / H2O mixed solutions (0-95%) at various concentrations. For all compounds, the fluorescence intensity was stronger as the concentration of glycerol increased, and the strongest fluorescence intensity was observed in the 95% glycerol / H2O mixed solution.

[0058] Figure 26 is a graph confirming the photostability of TTM-04 and MTdT-2.

[0059] FIG. 27 is a graph showing the normalized emission spectra of (a) TTB-07 and (b) TTB-09 in the solid state (Inlet: solid powder of TTB / TTM compounds under visible light (left) and ultraviolet light (right).

[0060] Figure 28a is the result of confirming the absolute quantum yield in the solid powder state of TTB-07, Figure 28b is TTB-09, Figure 28c is TTM-07, Figure 28d is TTM-09, and Figure 28e is TTM-13, showing the excitation spectrum (left) and emission spectrum (right) of the measured compounds and blanks, respectively.

[0061] Figures 29a, 29b, and 29c are time-lapse profile graphs showing the emission spectra of H2DCF-DA (4 μM) upon white light irradiation in the presence of TTBs (1 μM). Measurements were taken at 5-second intervals from 0 to 60 seconds, and in all graphs, the intensity increased as time progressed.

[0062] Figures 30a, 30b, and 30c are time-lapse profile graphs showing the emission spectrum of H2DCF-DA (4 μM) upon white light irradiation in the presence of TTMs (1 μM). Measurements were taken at 5-second intervals from 0 to 60 seconds, and in each graph, the intensity increased as time progressed.

[0063] Figures 31a and 31b are time-lapse profile graphs showing the emission spectrum of H2DCF-DA (4 μM) upon white light irradiation in the presence of TTMs (1 μM). Measurements were taken at 5-second intervals from 0 to 60 seconds, and in each graph, the intensity increased as time progressed.

[0064] Figures 32a, 32b, and 32c are time-course profile graphs showing the changes in the emission spectrum of dihydrorhodamine 123 (DHR123, 10 μM) upon white light irradiation in the presence of TTBs (2 μM). Measurements were taken at 5-second intervals from 0 to 60 seconds, and in each graph, the intensity increased as time progressed.

[0065] Figures 33a, 33b, 33c, and 33d are time-course profile graphs showing the changes in the emission spectrum of dihydrorhodamine 123 (DHR123, 10 μM) upon white light irradiation in the presence of TTMs (2 μM). Measurements were taken at 5-second intervals from 0 to 60 seconds, and in each graph, the intensity increased as time progressed.

[0066] Fig. 34 shows a white LED (luminous intensity 30,000 lm / m² 2A graph (top) showing the relative luminescence intensity of H2DCF-DA (4 μM) as a function of irradiation time (5-second intervals) in the presence of TTM / B compound (1 μM), using a ) as a light source, and a white LED (luminance 30,000 lm / m² 2 This is a graph (below) showing the relative luminescence intensity of DHR123 (10 μM) in the presence of TTM / B compound (2 μM) as a function of irradiation time (5-second intervals) using ) as a light source.

[0067] Figures 35a, 35b, and 35c are graphs showing PDT activity under white LED irradiation. Cell viability was measured over a period of 0 to 20 minutes, and cell viability decreased as time progressed.

[0068] Figure 36 is a confocal laser fluorescence microscopy image (λ) of HepG2 cells treated with 10 mM H2O2 and 1 μM TTM-4. ex = 561 nm, λ em = 635-700 nm), and the scale bar represents 10 μm. The intracellular fluorescence intensity of TTM-4 is shown in the graph. Significance was confirmed by Student's t-test with ****p < 0.0001. Data are expressed as mean ± standard deviation (SD), and n = 3.

[0069] Fig. 37 is a confocal laser fluorescence microscopy image (λ) of HepG2 cells treated with 10 mM H2O2 and 1 μM TTM-7. ex = 488 nm, λ em = 560-635 nm), and the scale bar represents 10 μm. The intracellular fluorescence intensity of TTM-7 was graphed. Significance was confirmed by Student's t-test with **p < 0.01. Data were expressed as mean ± standard deviation (SD), and n = 3.

[0070] Figure 38 is an image confirming the fluorescence emission of TTM-4 in HepG2 cells cultured at different temperatures, and is a live cell fluorescence image obtained by treating HepG2 cells cultured at 37 °C, 0 °C, or 4 °C for 1 hour with 1 μM TTM-4 (λ ex = 561 nm, λ em = 635-700 nm). The scale bar indicates 50 μm.

[0071] Figures 39a, 39b, and 39c are images confirming that TTM-4 can detect intracellular lipid peroxidation during ferroptosis in cancer cells, and are live cell fluorescence imaging results of lipid peroxidation using 1 μM TTM-4 in HepG2 cells treated with 10 mM H2O2 (Figure 39a) and 10 μM erastin and 5 μM RSL3 (Figure 39b) in the presence or absence of ferroptosis inhibitors including Liproxstatin-1 (Lip-1), β-mercaptoethanol (β-ME), and N-acetyl-L-cysteine ​​(NAC). Figure 39c shows the results of co-culturing HepG2 and NIH-3T3 cells, inducing ferroptosis with 10 μM elastin, and detecting intracellular lipid peroxidation with 1 μM TTM-4 (TTM-4 observation condition: λ ex = 561 nm, λ em = 635-700 nm). The white arrow indicates NIH-3T3 cells, and the scale bar indicates 50 μm. Figure 39b D is a graph showing the results of quantifying the intracellular fluorescence intensity of TTM-4 (statistical significance was evaluated using Student's t-test, p<0.0001). Data are expressed as mean ± SD, and n = 15.

[0072] Figures 40a, 40b, and 40c show the PDT activity (left), absorption spectrum (middle), and emission spectrum (right) of InTz, InTz-F, and InTzCN under white LED irradiation.

[0073] Figures 41a, 41b, and 41c show the results of verifying ROS generation according to the presence of H2DCF-DA and white LED using InTz, InTz-F, and InTzCN.

[0074] Figures 42a, 42b, and 42c show the results of confirming ROS generation and relative luminescence intensity according to the presence of H2DCF-DA and white LED using InTz, InTz-F, and InTzCN.

[0075] Figures 43a, 43b, and 43c show the results of verifying ROS generation according to the presence of ABDA and white LED using InTz, InTz-F, and InTzCN.

[0076] Figures 44a, 44b, and 44c show the results of confirming ROS generation and relative luminescence intensity according to the presence of ABDA and white LED using InTz, InTz-F, and InTzCN.

[0077] Figures 45a, 45b, and 45c show the results of verifying ROS generation according to the presence of DHR123 and white LED using InTz, InTz-F, and InTzCN.

[0078] Figures 46a, 46b, and 46c show the results of confirming ROS generation and relative luminescence intensity according to the presence of DHR123 and white LED using InTz, InTz-F, and InTzCN.

[0079] Figures 47a, 47b, and 47c are diagrams relating to the near-infrared (NIR) emission of TTM-4 with changes in viscosity, Figure 47a shows (A) the structures of TTM-4, TTO-4, TTM-6, and TTO-6 and (B) a schematic diagram of fluorescence expression with changes in viscosity, and Figure 47 shows (C) fluorescence enhancement and (D) λ ex (E) Quantification of 10 μM TTM-4 in a glycerol-water mixed solvent at 576 nm, and fluorescence intensity of TTM-4 (10 μM) in the presence of various analytes (200 μM). Fig. 47c is a diagram visualizing lipid peroxidation by TTM-4 using (F) in vitro and in vivo MASLD models.

[0080] Figures 48a and 48b illustrate the sensitivity and selectivity of intracellular lipid peroxidation detection using TTM-4. Lipid peroxidation was induced in HepG2 cells by treating them with (A1) 10 mM H₂O₂ or (B1) 10 μM erastin and 5 μM RSL3, after which live cell fluorescence imaging was performed using 1 μM TTM-4 in the presence or absence of ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), β-mercaptoethanol (β-ME), and N-acetyl-L-cysteine ​​(NAC). (A2) and (B2) graph the fluorescence intensity emitted from TTM-4 and include the results of statistical analysis (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test, and is indicated as ***p < 0.001.

[0081] Figures 49a and 49b show the results of detecting intracellular lipid peroxidation using various concentrations of TTM-4 in HepG2 cells in which ferroptosis was induced with (C) 5 μM RSL3 (λ ex = 561 nm; λ em= 635-700 nm), control images were obtained using a 5 μM BODIPY 581 / 591 C11 probe, and oxidized BODIPY C11 is λ ex = 488 nm and λ em = 493-550 nm, reduced BODIPY C11 is λ ex = 561 nm and λ em = Measured at 570–620 nm. The scale bar represents 10 μm. (D1) Fluorescence images of living cells after treatment with 10 μM erastin, 5 μM RSL3, 10 μg / mL LPS, or 10 μM staurosporine for different durations to induce different apoptotic pathways in HepG2 cells stained with 1 μM TTM-4. The scale bar represents 50 μm. (D2) Fluorescence intensity of TTM-4 was quantified under different conditions. Data are expressed as mean ± standard deviation (mean ± SD), and measurements were taken in the n = 4 range. Statistical analysis was performed using two-way ANOVA, and values ​​were indicated as **p < 0.01 and ***p < 0.001.

[0082] Figures 50a, 50b, and 50c show the results of specific detection of lipid peroxidation within cell organelles using TTM-4, (AC) 1 μM TTM-4 (λ ex = 561 nm; λ em Lipid peroxidation within organelles was detected using BODIPY 493 / 503 (λ ) = 635-700 nm. Cell organelles were co-stained as follows: (A) BODIPY 493 / 503 (λ ) ex = 488 nm; λ em = 493-550 nm) or (B) ER Tracker Blue-White (λ ex = 405 nm; λ em(= 410–490 nm). Colocalization analysis results were presented as a histogram. The scale bar represents 10 μm. (C) Nuclear lipid droplets are indicated by white arrows. (DE) Lipid peroxidation levels in specific organelles were evaluated during ferroptosis induced by 10 μM erastin. The white arrow represents the ER. The scale bars represent 2 μm or 0.5 μm, respectively.

[0083] Figure 51 is a diagram related to the visualization of lipid peroxidation in an in vitro metabolic dysfunction-associated steatotic hepatitis (MASH) model using TTM-4, wherein (A1) nuclear lipid droplets (LD) induced by treatment with 10 μM erastin for 6 hours were treated with 1 μM TTM-4 (λ ex = 561 nm; λ em Detection was performed using (= 635-700 nm). HepG2 cells were pretreated with 50 μM lalistat-2 for 24 hours prior to erastin treatment. Cell organelles were BODIPY 493 / 503 (λ ex = 488 nm; λ em = 493-550 nm) and Hoechst(λ ex = 405 nm; λ em Co-stained with (= 410-500 nm). (A2) represents the change in the ratio of oxidized LDs to total LDs and the ratio of oxidized nLDs to total nLDs under conditions (A1). Data were expressed as mean ± standard deviation (mean ± SD) and measured in n > 4 regions with > 100 cells per region. Statistical analysis was performed using Student's t-test, where ****p < 0.0001, ***p < 0.001, *p < 0.05, ns: indicates not significant.

[0084] Figure 52 is a diagram showing the visualization of lipid peroxidation in an in vitro metabolic dysfunction-associated steatotic hepatitis (MASH) model using TTM-4. (B1) Lipid peroxidation was visualized with 1 μM TTM-4 in the presence or absence of 30 μM eicosapentaenoic acid (EPA) in lipid droplets (LD) induced by treating HepG2 cells with 500 μM palmitic acid for 24 hours. The scale bar represents 10 μm. (B2) shows the changes in the ratio of oxidized LDs to total LDs and the ratio of oxidized nLDs to total nLDs under condition (B1). Data are expressed as mean ± standard deviation (mean ± SD) and were measured in regions with n > 4, with >100 cells per region. Statistical analysis was performed using Student's t-test, where ****p < 0.0001, ***p < 0.001, *p < 0.05, ns: indicates not significant.

[0085] Figure 53 shows (C1) 1) representative IncuCyte images taken with 1 μM TTM-4 in the presence or absence of 100 μM deferoxamine (DFO) in AML12 cells treated with 5 μM RSL3 for 24 hours and 2) a graph showing TTM-4 fluorescence intensity, (D) TTM-4 positive cell (population) data obtained through FACS analysis and (EF) the results of performing FACS analysis on AML12 cells treated with (E) 5 μM RSL3 or (F) palmitate (250 or 1000 μM) for 24 hours, stained with 1 μM BODIPY 581 / 591 C11 or 1 μM TTM-4 for 1 hour.

[0086] Figures 54a and 54b show the results regarding the detection of ferroptosis in MASH mouse and human liver tissues using TTM-4. (A) Fluorescence signals of DAPI (blue), α-SMA (green), and TTM-4 (red) were analyzed in liver tissue biopsy samples from MASLD patients. The combined image shows the combined result of α-SMA and TTM-4 fluorescence. (B) Representative images of liver tissue stained with BODIPY 493 / 503 (green), TTM-4 (red), and DAPI (gray) from C57BL / 6J mice fed CDAHFD for 12 weeks. The scale bar indicates 0.5 mm. (C) Results of statistical analysis and comparison of BODIPY 581 / 591 C11 and TTM-4 fluorescence using confocal microscopy in mouse liver tissue samples at weeks 1, 6, and 12 of CDAHFD feeding (n = 3). The scale bar represents 50 μm. (D) Confocal microscopy images of nuclear lipid droplets (nLDs) observed using TTM-4 in liver tissue samples at weeks 1, 6, and 12 of CDAHFD administration. The scale bar represents 10 μm. Data were expressed as mean ± standard deviation (mean ± SD), with n = 5 regions and measurements taken from >100 cells per region. Statistical analysis was performed using a two-sided Student t-test, indicated as **p < 0.01 and ***p < 0.001.

[0087] Figures 55a, 55b, and 55c are diagrams related to the detection of lipid toxicity by CIDEC-mediated lipid droplet fusion using TTM-4, (A1-A2) expression levels of CIDEA, CIDEB, and CIDEC at different stages of metabolic dysfunction-associated fatty liver disease (MASH) in patient samples, (A3-A5) gene analysis of con-early-moderate NAFLD using public data (GSE135251), (A3) Heatmap (false-discovery rate 0.05, fold-change 1.5), (A4) volcano plot of CIDEA and CIDEC, and (A5) violin plot of CIDEC mRNA levels.

[0088] Figures 56a, 56b, 56c, 56d, and 56e relate to the detection of lipid toxicity by CIDEC-mediated lipid droplet fusion using TTM-4, including (B) quantitative real-time PCR (qRT-PCR) analysis results, 1) liver tissues (weeks 1, 6, and 12) of a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) mouse model, 2) Cidec mRNA levels in mouse primary hepatocytes after treatment with palmitic acid (PA; 100 and 300 μM), and 3) Cidec mRNA levels in AML12 cells after PA treatment (250 and 1000 μM); (C) fluorescence comparison and statistical analysis of BODIPY 493 / 503 and TTM-4 in AML12 cells treated with PA (250 and 1000 μM) (n = 5); and (D) fluorescence comparison of BODIPY 493 / 503 and TTM-4 in human liver biopsy samples. and statistical analysis (n = 3) (scale bar represents 50 μm), (E) results of nuclear lipid droplet (nLDs) detection using confocal microscopy (n = 4 regions, >70 cells per region) (eMASH: early MASH; aMASH: advanced MASH). Data are expressed as mean ± standard deviation (mean ± SD), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance was evaluated via Tukey's post-hoc test following one-way ANOVA. (F) Detection of lipid toxicity induced by CIDEC and TTM-4 in human MASH samples. Colocalization analysis results are shown as a histogram. Scale bar represents 10 μm.

[0089] Figures 57a, 57b, and 57c are diagrams related to the measurement of ferroptosis in mice using TTM-4. (A1-A2) Ferroptosis was induced in C57BL / 6J mice by a single intraperitoneal CCl4 injection or by feeding a methionine-choline-deficient (MCD) diet for 3 weeks, and fluorescence expression in liver tissue was observed using TTM-4. (B) A single dose of TTM-4 (5 mg / kg) was administered to ICR mice via tail vein injection, and a toxicity assessment was performed 2 hours after injection. Hepatotoxicity was evaluated by measuring serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) levels, and renal function was evaluated by comparing blood urea nitrogen (BUN) and creatinine (CRE) levels. The results were presented in a comparative graph. (C1-F2) Ferroptosis was induced in ICR mice using two methods. In one experiment, a single intraperitoneal injection of 100 μL of 20% CCl4 per 20 g body weight (n = 4) was administered during 24 hours of fasting. In the other experiment, the mice were fed an MCD diet for 3 weeks. On the last day, after 24 hours of fasting (n = 5), TTM-4 (5 mg / kg) was injected via the tail vein, and fluorescence expression was evaluated using IVIS 2 hours later. (C1) Schematic diagram of the animal experiment and (C2-C3) photographs of mouse livers. Fluorescence images were captured using IVIS.

[0090] Figures 58a and 58b show (D1-D2) serum AST and ALT levels, (E) results of measuring TTM-4 fluorescence expression at each institution 24 hours after oral administration of the same concentration of CCl4, and (F1-F2) fluorescence intensity was quantified using ImageJ software based on IVIS fluorescence images, and the results are presented in a comparison graph. Data are expressed as mean ± standard deviation (mean ± SD). ns: not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical significance was evaluated using a two-sided Student t-test.

[0091]

[0092] The present invention provides a novel compound, a fluorescent probe composition containing the same, and a composition for photodynamic therapy containing the same.

[0093] The present invention provides a compound represented by the following chemical formula 1:

[0094] [Chemical Formula 1]

[0095]

[0096] In the above formula, R1 is NR5R6 or OR7;

[0097] The above R5, R6, and R7 are each independently C 1-8 It is an alkyl or benzene, or R5 and R6 can be connected to each other to form a 5- to 7-membered heterocycle substituted or unsubstituted with one or more substituents;

[0098] L is a 5- to 14-membered arylene or heteroarylene substituted or unsubstituted with one or more substituents;

[0099] R2 is C substituted or unsubstituted with H, O, or one or more substituents. 1-6 It is a fused ring of two rings formed by fusing an alkyl, or a cycloalkyl of five to seven members substituted or unsubstituted with one or more substituents, with an aryl group;

[0100] R3 and R4 are each independently H or F.

[0101] In cases where a substituent is required at a position in a structural formula but no substituent is listed, the hydrogen substituent has been omitted, and this applies equally to all structural formulas of the present invention.

[0102] In a compound according to one embodiment of the present invention, L may be a π-spacer. For example, it may be thiophene, phenylene, etc.

[0103] In a compound according to one embodiment of the present invention, the L is C 1-3 It may be a 5 to 14-membered arylene or heteroarylene substituted or unsubstituted with an alkenyl group. For example, C 1-3 It may be thiophene, benzene, or benzothiadiazole substituted or unsubstituted with an alkenyl group.

[0104] In a compound according to one embodiment of the present invention, the L is It could be.

[0105] In a compound according to one embodiment of the present invention, R1 is NR5R6, and R5 and R6 are each independently C 1-3 It is alkyl or benzene, or R5 and R6 are connected to each other, piperazine or It may form.

[0106] In a compound according to one embodiment of the present invention, R1 is NR5R6, and R5 and R6 may be the same substituent.

[0107] In a compound according to one embodiment of the present invention, R1 is NR5R6, and R5 and R6 are C 1-3 or benzene, or connected to each other piperazine or It may form.

[0108] In a compound according to one embodiment of the present invention, the R1 is OR7, and the R7 is C 1-8 It can be an alkyl.

[0109] In a compound according to one embodiment of the present invention, the R1 is OR7, and the R7 is C 1-8 It is alkyl and can be O or C(CN)2.

[0110] In a compound according to one embodiment of the present invention, the R2 is H, O, C(CN)2 or It could be.

[0111] In a compound according to one embodiment of the present invention, R3 and R4 may simultaneously be H or simultaneously be F.

[0112] In a compound according to one embodiment of the present invention, the compound may be any one selected from the group consisting of the following compounds.

[0113]

[0114]

[0115]

[0116] The above compounds may be prepared according to the manufacturing examples of the present invention, but are not limited thereto.

[0117] Since the compound of the present invention is a fluorescent organism, it is easy to visualize its location or accumulation within the body using bioimaging by detecting fluorescence or luminescence images through light irradiation.

[0118] The compound of the present invention may exhibit fluorescence in a wavelength range of 600 to 1000 nm. For example, it may exhibit fluorescence in a wavelength range of 600 to 1000 nm, 600 to 900 nm, 600 to 800 nm, or 600 to 700 nm.

[0119] The compound of the present invention may express near-infrared fluorescence or red fluorescence in response to an increase in viscosity. More specifically, it may express near-infrared fluorescence.

[0120] The compound of the present invention may detect lipid peroxidation in living cells or tissues and generate fluorescence. The compound of the present invention may detect lipid peroxidation in cells or tissues isolated from living organisms and generate fluorescence.

[0121] The compound of the present invention may selectively detect ferrotopsis and generate fluorescence.

[0122] A compound according to one embodiment of the present invention can be used to diagnose lipid peroxidation metabolic disorder and fatty liver disease.

[0123] The present invention provides a diagnostic use of the compound of the present invention for metabolic disorder fatty liver disease (MASLD).

[0124] The present invention provides a method for diagnosing metabolic dyslipidemia (MASLD), comprising the step of detecting fluorescence in a biological sample using a compound of the present invention.

[0125] The present invention provides a fluorescent probe composition comprising a compound of the present invention. A fluorescent probe composition according to one embodiment of the present invention may be for bioimaging. For example, it may be for in vivo bioimaging.

[0126] A fluorescent probe composition according to one embodiment of the present invention may express near-infrared fluorescence or red fluorescence in response to an increase in viscosity. More specifically, it may express near-infrared fluorescence.

[0127] A fluorescent probe composition according to one embodiment of the present invention may exhibit fluorescence in a wavelength range of 600 to 1000 nm. For example, it may exhibit fluorescence in a wavelength range of 600 to 1000 nm, 600 to 900 nm, 600 to 800 nm, or 600 to 700 nm.

[0128] A fluorescent probe composition according to one embodiment of the present invention may be used for detecting lipid peroxidation in living cells or tissues. For example, it may be used for detecting lipid peroxidation in living cells or tissues in vivo, or for detecting lipid peroxidation in cells or tissues isolated from living organisms.

[0129] A fluorescent probe composition according to one embodiment of the present invention may be used for detecting ferroptosis. A fluorescent probe composition according to one embodiment of the present invention may selectively detect ferroptosis.

[0130] The compound of the present invention can be used as a photosensitizer.

[0131] Photosensitizers are substances that are activated by light of a specific wavelength and can induce apoptosis through the generation of reactive oxygen species upon exposure to light; the therapeutic method of killing cells using light and photosensitizers is called photodynamic therapy.

[0132] The present invention provides a composition for photodynamic therapy comprising a compound of the present invention.

[0133] The target diseases for the above photodynamic therapy are not particularly limited as long as they can be prevented or treated through light irradiation and cell death, and may be, for example, skin diseases or cancer.

[0134] The method of administering the above composition to a target subject is not particularly limited and can be appropriately selected by a person skilled in the art to which the present invention pertains. Examples include, but are not limited to, methods of application to the skin or spraying, intravenous injection or ingestion mixed into a beverage, nasal inhalation, or direct administration to the bladder or uterus.

[0135] The above composition may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, which is a compound of the present invention, or may be administered to mammals including humans. The adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

[0136] In addition, the above composition can be preferably formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the pharmaceutically effective amount of active ingredient described above for administration.

[0137] The above “pharmaceuticalally effective amount” refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. Other pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art.

[0138] The subject of the photodynamic therapy composition according to one embodiment of the present invention may be a mammal, including humans, but is not limited thereto.

[0139] The compound of the present invention or a composition for photodynamic therapy containing the same may exhibit photosensitizing activity to light of a wavelength of 300 to 700 nm. For example, it may exhibit photosensitizing activity to light of a wavelength of 400 nm to 600 nm.

[0140] In the present invention, the light is, for example, a light intensity of 30,000 to 36,000 lm / m² 2 White light can be used. In one embodiment, a luminous intensity of 30,000 lm / m² 2 White light can be used.

[0141] The present invention provides a use for the prevention or treatment of cancer comprising a compound of the present invention or a composition for photodynamic therapy containing the same.

[0142] The present invention provides a composition for the prevention or treatment of cancer comprising a compound of the present invention or a composition for photodynamic therapy containing the same.

[0143] The present invention provides a method for preventing or treating cancer comprising a compound of the present invention or a composition for photodynamic therapy containing the same.

[0144] In the present invention, the cancer may be breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, penile cancer, genitourinary cancer, testicular tumor, esophageal cancer, laryngeal cancer, stomach cancer, gastrointestinal cancer, skin cancer, keratosporoma, follicular carcinoma, melanoma, lung cancer, small cell lung carcinoma, non-small cell lung carcinoma (NSCLC), lung adenocarcinoma, squamous cell carcinoma of the lung, colon cancer, pancreatic cancer, thyroid cancer, papillary cancer, bladder cancer, liver cancer, cholangiocarcinoma, kidney, bone cancer, bone marrow disorder, lymphatic disorder, hair cell carcinoma, pharyngeal cancer, lip cancer, tongue cancer, oral cancer, salivary gland cancer, pharyngeal cancer, small intestine cancer, colon cancer, rectal cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, colorectal cancer, endometrial cancer, uterine cancer, brain cancer, central nervous system cancer, peritoneal cancer, hepatocellular carcinoma, head cancer, cervical cancer, Hodgkin's cancer, or leukemia.

[0145]

[0146] Preparation Example

[0147] 2.1. Synthesis of TTB-01, TTM-01, TTB-06, and TTM-06

[0148] 2.1.1. Bindone

[0149] [1,2'-biindenylidene]-1',3,3'(2H)-trione

[0150] Triethanolamine (700 μL) was added to a solution of 1H-indene-1,3(2H)-dione (600 mg, 4.1057 mmol) dissolved in ethanol (10 mL). The reaction mixture was stirred at 100 °C for 30 minutes. After cooling to room temperature, the contents of the flask were poured into ice water and neutralized by adding a few drops of HCl. The precipitated solid was filtered and recovered, and recrystallized with dioxane to obtain Bindone (550.1 mg, 97.7%), a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 9.69 (d, J = 8.1 Hz, 1H), 8.06 - 8.01 (m, 1H), 8.00 - 7.94 (m, 2H), 7.90 - 7.85 (m, 1H), 7.85 - 7.81 (m, 2H), 7.76 (t, J = 7.4 Hz, 1H), 4.18 (s, 2H).

[0151]

[0152] 2.1.2. MNI

[0153] 2-(3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile

[0154] 1H-indene-1,3(2H)-dione (500 mg, 3.4214 mmol) and malononitrile (760 μL, 13.6855 mmol) were dissolved in anhydrous ethanol (3.5 mL). The reaction mixture was stirred at room temperature for 30 minutes. Sodium acetate (337 mg, 4.1057 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour. The mixture was poured into water and hydrochloric acid was added to acidify the pH to 1-2. The precipitate was filtered to obtain MNI (604.2 mg, 90.9%), a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.71 - 8.63 (m, 1H), 7.99 (dd, J = 7.4, 2.2 Hz, 1H), 7.93 - 7.84 (m, 2H), 3.74 (s, 2H).

[0155]

[0156] 2.1.3. TPATA

[0157] 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde

[0158] 4-bromo-N,N-diphenylaniline (500 mg, 1.5422 mmol), (5-formylthiophen-2-yl)boronic acid (288.6 mg, 1.8506 mmol), and potassium carbonate (1065.7 mg, 7.711 mmol) were dissolved in a methanol (4 mL) / toluene (4 mL) mixture, and Bis(triphenylphosphine)palladium(II) dichloride (108 mg, 0.1542 mmol) was added. The reaction mixture was stirred at 75 °C for 16 hours. The resulting crude was diluted with water and extracted with methylene chloride (3 x 100 mL). After drying the organic layer with anhydrous sodium sulfate and evaporating the solvent, the residue was subjected to silica gel chromatography under hexane / ethyl acetate gradient elution conditions to obtain an orange solid, TPATA (374 mg, 72%). 1 H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 7.71 (d, J = 4.0 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.32 - 7.28 (m, 5H), 7.16 - 7.11 (m, 5H), 7.09 - 7.05 (m, 3H).

[0159]

[0160] 2.1.4. DMATA

[0161] 5-(4-(dimethylamino)phenyl)thiophene-2-carbaldehyde

[0162] 4-bromo-N,N-dimethylaniline (600 mg, 2.9988 mmol), (5-formylthiophen-2-yl)boronic acid (561 mg, 1.8506 mmol), and potassium carbonate (1657 mg, 11.9952 mmol) were dissolved in a methanol (6 mL) / toluene (6 mL) mixture, and Bis(triphenylphosphine)palladium(II) dichloride (210.5 mg, 0.2999 mmol) was added. The reaction mixture was stirred at 75 °C for 16 hours. The obtained crude was diluted with water and extracted with DCM (3 x 100 mL). The organic layer was dried using anhydrous sodium sulfate, the solvent was evaporated, and the residue was subjected to silica gel chromatography under hexane / ethyl acetate gradient elution conditions to obtain an orange solid, DMATA (305.3 mg, 44%). 1 H NMR (400 MHz, CDCl3) δ 9.82 (s, 1H), 7.68 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.24 (d, J = 4.0 Hz, 1H), 6.72 (d, J = 9.0 Hz, 2H), 3.03 (s, 6H).

[0163]

[0164] 2.1.5. General Procedure for Synthesis of TTB-01, TTM-01, TTB-06, and TTM-06

[0165] An aldehyde intermediate (1.0 equiv.) was added to a solution of Bindone or MNI (1.0 equiv.) dissolved in pyridine anhydrous. The reaction mixture was stirred at room temperature for 1 hour. Then, methanol was added. After stirring the reaction mixture for 30 minutes, the obtained crude was washed with methanol. The obtained residue was concentrated under reduced pressure to obtain TTB and TTM.

[0166]

[0167] 2.1.5.1. TTB-01

[0168] (Z)-2-((5-(4-(diphenylamino)phenyl)thiophen-2-yl)methylene)-[1,2'-biindenylidene]-1',3,3'(2H)-trione

[0169] A solution of Bindone (30 mg, 0.1094 mmol) dissolved in anhydrous pyridine (1 mL) was reacted with TPATA (38.9 mg, 0.1094 mmol) according to a general procedure to obtain a blue solid TTB-01 (20.9 mg, 31.2%). 1 H NMR (400 MHz, CDCl3) δ 8.97 (d, J = 7.8 Hz, 1H), 8.40 (s, 1H), 8.00 - 7.98 (m, 1H), 7.95 (d, J = 5.7 Hz, 1H), 7.87 (d, J = 7.1 Hz, 1H), 7.83 (s, 1H), 7.79 - 7.76 (m, 2H), 7.65 (dd, J = 16.4, 7.5 Hz, 4H), 7.39 (s, 1H), 7.30 (d, J = 7.4 Hz, 4H), 7.16 (d, J = 7.7 Hz, 4H), 7.11 (s, 2H), 7.06 (d, J = 8.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 190.83, 190.48, 189.39, 161.98, 159.81, 149.54, 147.01, 145.55, 143.44, 141.84, 140.99, 138.53, 136.41, 134.82, 134.75, 134.53, 133.42, 130.40, 129.66, 127.70, 126.64, 125.48, 124.58, 124.17, 123.77, 123.14, 123.09, 122.84, 122.24; HR-MS (FAB+): m / z calcd for C 41 H 26 NO3S [M+H]+: 612.1633, found: 612.1634.

[0170]

[0171] 2.1.5.2. TTM-01

[0172] (Z)-2-((5-(4-(dimethylamino)phenyl)thiophen-2-yl)methylene)-[1,2'-biindenylidene]-1',3,3'(2H)-trione

[0173] A solution of Bindone (15.4 mg, 0.0563 mmol) dissolved in anhydrous pyridine (0.6 mL) was reacted with DMTA (20 mg, 0.0563 mmol) according to a general procedure to obtain a blue solid TTM-01 (16.4 mg, 59.6%). 1H NMR (400 MHz, CDCl3) δ 8.91 (d, J = 6.3 Hz, 1H), 8.38 (s, 1H), 7.98 (dd, J = 5.6, 2.1 Hz, 1H), 7.93 (d, J = 6.4 Hz, 1H), 7.85 (d, J = 7.1 Hz, 1H), 7.81 (d, J = 3.6 Hz, 1H), 7.78 - 7.76 (m, 1H), 7.75 (dd, J = 3.8, 2.1 Hz, 2H), 7.63 (dt, J = 22.2, 7.4 Hz, 3H), 7.36 (d, J = 3.7 Hz, 1H), 6.73 (d, J = 8.3 Hz, 2H), 3.07 (s, 6H). ; HR-MS (FAB+): m / z calcd for C 31 H 22 NO3S [M+H]+: 488.1320, found: 488.1338.

[0174]

[0175] 2.1.5.2. TTB-06

[0176] 2-((Z)-2-((5-((E)-4-(diphenylamino)styryl)thiophen-2-yl)methylene)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile

[0177] A solution of MNI (10.1 mg, 0.0524 mmol) dissolved in anhydrous pyridine (0.5 mL) was reacted with VTPATA (20 mg, 0.0524 mmol) according to a general procedure to obtain a blue solid TTB-06 (20 mg, 68.4%). 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.67 (d, J = 6.6 Hz, 1H), 7.91 (d, J = 6.8 Hz, 1H), 7.77 - 7.71 (m, 3H), 7.38 (d, J = 8.7 Hz, 2H), 7.34 - 7.27 (m, 5H), 7.17 (d, J = 4.1 Hz, 1H), 7.13 (d, J = 7.6 Hz, 5H), 7.10 (d, J = 7.2 Hz, 2H), 7.02 (d, J = 8.6 Hz, 2H). 13 C NMR (100 MHz, CDCl3) δ 188.52, 160.77, 160.30, 149.26, 147.11, 146.56, 140.16, 137.89, 137.05, 135.80, 135.22, 134.93, 134.56, 129.28, 128.51, 127.12, 125.45, 124.09, 123.85, 122.29, 122.06, 118.89, 114.87, 114.76, 69.29; HR-MS (FAB+): m / z calcd for C 37 H 24 N3OS [M+H]+: 558.1640, found: 558.1645.

[0178]

[0179] 2.1.5.2. TTM-06

[0180] 2-((Z)-2-((5-((E)-4-(dimethylamino)styryl)thiophen-2-yl)methylene)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile

[0181] A solution of MNI (15.1 mg, 0.0777 mmol) dissolved in anhydrous pyridine (0.5 mL) was reacted with VDMATA (20 mg, 0.0777 mmol) according to a standard procedure to obtain a blue solid TTM-06 (17.8 mg, 52.8%). 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.67 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 6.7 Hz, 1H), 7.76 - 7.72 (m, 3H), 7.44 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 4.2 Hz, 1H), 7.09 (s, 1H), 6.70 (d, J = 8.5 Hz, 2H), 3.05 (s, 6H); HR-MS (FAB+): m / z calcd for C 27 H 20 N3OS [M+H]+: 434.1327, found: 434.1342.

[0182]

[0183] 2.2. Synthesis of TTB-03, TTM-03, TTB-04, and TTM-04

[0184] 2.2.1. VTPA

[0185] N,N-diphenyl-4-vinylaniline

[0186] Tetrakis(triphenylphosphine)palladium (0) (178 mg, 0.154 mmol) and tributylvinyl tin (0.54 mL, 1.85 mmol) were added to a solution of 4-bromo-N,N-diphenylaniline (500 mg, 1.542 mmol) dissolved in anhydrous toluene (7 mL). The reaction mixture was stirred at 70 °C for 16 hours. After concentration under reduced pressure, the resulting residue was filtered using silica gel chromatography under hexane / methylene chloride gradient elution conditions to obtain a white solid VTPA (143.5 mg, 34%). 1H NMR (400 MHz, CDCl3) δ 7.31 - 7.26 (m, 3H), 7.25 - 7.22 (m, 3H), 7.11 - 7.08 (m, 4H), 7.04 - 7.00 (m, 4H), 6.66 (dd, J = 17.6, 10.9 Hz, 1H), 5.64 (d, J = 17.6 Hz, 1H), 5.16 (d, J = 10.9 Hz, 1H).

[0187]

[0188] 2.2.2. VTPAPA

[0189] (E)-4-(4-(diphenylamino)styryl)benzaldehyde

[0190] N,N-Diphenyl-4-vinylaniline (60 mg, 0.2211 mmol), 4-bromobenzaldehyde (45 mg, 0.2432 mmol), tetrabutylammonium bromide (142.6 mg, 0.4422 mmol), and palladium(II) acetate (5 mg, 0.0221 mmol) were dissolved in an N,N-dimethylformamide (1 mL) / toluene (1 mL) mixture. Triethylamine (62 μL, 0.4422 mmol) was added. The reaction mixture was stirred at 95 °C for 16 hours. The resulting crude was diluted with water and extracted with methylene chloride (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel chromatography under hexane / methylene chloride gradient elution conditions to obtain an orange solid VTPAPA (51.6 mg, 62.2%). 1H NMR (400 MHz, CDCl3) δ 9.98 (s, 1H), 7.85 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 7.4 Hz, 3H), 7.21 (d, J = 16.3 Hz, 1H), 7.13 (d, J = 8.7 Hz, 4H), 7.10 - 6.97 (m, 6H).

[0191]

[0192] 2.2.3. VTPATA

[0193] (E)-5-(4-(diphenylamino)styryl)thiophene-2-carbaldehyde

[0194] N,N-diphenyl-4-vinylaniline (400 mg, 1.4741 mmol), 5-bromothiophene-2-carbaldehyde (310 mg, 1.6215 mmol), tetrabutylammonium bromide (950 mg, 2.9481 mmol), and palladium(II) acetate (36 mg, 0.1474 mmol) were dissolved in an N,N-dimethylformamide (7 mL) / toluene (1 mL) mixture, and triethylamine (411 μL, 0.1474 mmol) was added. The reaction mixture was stirred at 95 °C for 16 hours. The obtained crude was diluted with water and extracted with DCM (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel chromatography under hexane / ethyl acetate gradient elution conditions to obtain an orange solid VTPAPA (195.3 mg, 34.7%). 1H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 7.65 (d, J = 4.0 Hz, 1H), 7.36 (d, J = 8.6 Hz, 3H), 7.28 (s, 4H), 7.11 (s, 5H), 7.09 (s, 2H), 7.06 (d, J = 10.1 Hz, 5H).

[0195]

[0196] 2.2.4. VDMATA

[0197] (E)-5-(4-(dimethylamino)styryl)thiophene-2-carbaldehyde

[0198] Tetrakis(triphenylphosphine)palladium (0) (289 mg, 0.2499 mmol) and tributyl(vinyl)tin (951 mg, 2.9988 mmol) were added to a solution in which 4-bromo-N,N-dimethylaniline (500 mg, 2.499 mmol) was dissolved in anhydrous toluene (7 mL). The reaction mixture was stirred at 70 °C for 16 hours. After concentration under reduced pressure, the mixture was poured into water and hydrochloric acid was added to acidify the pH to 1-2, and extracted with DCM (3 x 100 mL). The mixture was poured into DCM (3 x 100 mL), and sodium hydroxide was added to the solution to alkalize it to pH 8-10, thereby obtaining colorless, oily crude N,N-dimethyl-4-vinylaniline (VDMA). In the crude state, N,N-dimethyl-4-vinylaniline (350 mg, 2.3774 mmol), 5-bromothiophene-2-carbaldehyde (500 mg, 2.6151 mmol), tetrabutylammonium bromide (1533 mg, 4.7548 mmol), and palladium(II) acetate (53 mg, 0.2377 mmol) were dissolved in N,N-dimethylformamide (4 mL). Triethylamine (662 μL, 0.47548 mmol) was added. The reaction mixture was stirred at 95 °C for 16 hours. The resulting crude was diluted with water and extracted with DCM (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel chromatography with a hexane / ethyl acetate gradient elution to obtain an orange solid VDMATA (413 mg, 67.5%). 1H NMR (400 MHz, CDCl3) δ 9.81 (d, J = 7.7 Hz, 1H), 7.60 (dd, J = 26.9, 3.9 Hz, 1H), 7.41 (s, 1H), 7.13 - 7.05 (m, 2H), 7.00 (d, J = 16.0 Hz, 1H), 6.69 (d, J = 8.9 Hz, 2H), 6.66 (d, J = 8.9 Hz, 1H), 3.01 (d, J = 6.6 Hz, 6H).

[0199]

[0200] 2.2.5. General process of TTB-03, TTM-03, TTB-04, and TTM-04 synthesis

[0201] An aldehyde intermediate was added to a solution of 1H-indene-1,3(2H)-dione dissolved in a methanol / toluene mixture. After adding one drop of piperidine, the reaction mixture was stirred at 60 °C for 4 hours. After concentration under reduced pressure, the residue was filtered by silica gel chromatography with a hexane / ethyl acetate gradient elution to obtain TTB and TTM compounds.

[0202]

[0203] 2.2.5.1. TTB-03

[0204] (E)-2-(4-(4-(diphenylamino)styryl)benzylidene)-1H-indene-1,3(2H)-dione

[0205] A solution of 1H-indene-1,3(2H)-dione (14 mg, 0.0959 mmol) dissolved in a methanol (1 mL) / toluene (1 mL) mixture was reacted with VTPAPA (30 mg, 0.0799 mmol) according to a general procedure to obtain a dark red solid TTB-03 (38 mg, 94.4%). 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 8.4 Hz, 2H), 8.00 (td, J = 5.8, 5.1, 3.1 Hz, 2H), 7.87 (s, 1H), 7.82 - 7.79 (m, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.4 Hz, 4H), 7.22 (s, 1H), 7.12 (d, J = 7.5 Hz, 4H), 7.08 - 7.05 (m, 3H), 7.02 (d, J = 11.2 Hz, 2H). 13 C NMR (100MHz, CDCl3) δ 190.71, 189.39, 148.37, 147.45, 146.52, 143.03, 142.70, 140.22, 135.38, 135.20, 132.24, 131.83, 130.59, 129.51, 128.40, 128.05, 126.64, 125.86, 125.00, 123.57, 123.34, 123.08; HR-MS (FAB+): m / z calcd for C 36 H 26 NO2[M+H]+: 503.1885, found: 503.1892.

[0206]

[0207] 2.2.5.2. TTM-03

[0208] (E)-2-(4-(4-(dimethylamino)styryl)benzylidene)-1H-indene-1,3(2H)-dione

[0209] A solution of 1H-indene-1,3(2H)-dione (11.7 mg, 0.079 mmol) dissolved in a methanol (1 mL) / toluene (1 mL) mixture was reacted with VDMAPA (20 mg, 0.079 mmol) according to a general procedure to obtain a dark red solid TTB-03 (19.1 mg, 63.7%). 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.5 Hz, 2H), 8.03 - 7.98 (m, 2H), 7.87 (s, 1H), 7.83 - 7.78 (m, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 16.2 Hz, 1H), 6.96 (d, J = 16.2 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 3.02 (s, 6H). 13 C NMR (151MHz, CDCl3) δ 190.84, 189.47, 146.75, 143.85, 142.71, 140.23, 135.32, 135.08, 132.73, 131.77, 128.49, 128.00, 126.35, 123.32, 123.29, 112.46, 40.45.; HR-MS (FAB+): m / z calcd for C 26 H 22 NO2[M+H]+: 380.1651, found: 380.1642

[0210]

[0211] 2.2.5.3. TTB-04

[0212] (E)-2-((5-(4-(diphenylamino)styryl)thiophen-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0213] A solution of 1H-indene-1,3(2H)-dione (15 mg, 0.1049 mmol) dissolved in a methanol (1 mL) / toluene (1 mL) mixture was reacted with VTPATA (40 mg, 0.1049 mmol) according to a general procedure to obtain a burgundy solid TTB-04 (46.8 mg, 87.4%). 1H NMR (400 MHz, CDCl3) δ 7.96 (dt, J = 6.2, 3.4 Hz, 2H), 7.93 (s, 1H), 7.86 (d, J = 4.0 Hz, 1H), 7.80 - 7.75 (m, 2H), 7.39 (d, J = 8.6 Hz, 2H), 7.32 - 7.27 (m, 4H), 7.17 - 7.12 (m, 6H), 7.12 - 7.01 (m, 5H). 13 C NMR (100 MHz, CDCl3) δ 190.65, 189.93, 156.92, 148.82, 147.26, 143.84, 142.17, 140.63, 136.17, 135.90, 135.05, 134.84, 133.49, 129.69, 129.57, 128.20, 127.08, 125.25, 123.84, 123.49, 123.04, 122.87, 122.64, 119.13; HR-MS (FAB+): m / z calcd for C 34 H 24 NO2S [M+H]+: 510.1528, found: 510.1522.

[0214]

[0215] 2.2.5.4. TTM-04

[0216] (E)-2-((5-(4-(dimethylamino)styryl)thiophen-2-yl)methylene)-1H-indene-1,3,(2H)-dione

[0217] A solution of 1H-indene-1,3(2H)-dione (140 mg, 0.958 mmol) dissolved in a methanol (10 mL) / toluene (10 mL) mixture was reacted with VDMATA (300 mg, 1.17 mmol) according to a standard procedure to obtain a burgundy solid TTM-04 (310.4 mg, 84.1%). 1H NMR (401 MHz, Chloroform-d) δ 7.97 - 7.93 (m, 2H), 7.92 (s, 1H), 7.86 (d, J = 3.9 Hz, 1H), 7.78 - 7.74 (m, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 16.3 Hz, 1H), 7.12 (d, J = 4.1 Hz, 1H), 7.07 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 8.9 Hz, 2H), 3.03 (s, 6H). 13 C NMR (101 MHz, CHLOROFORM-D) δ 190.78, 189.90, 144.12, 142.07, 140.53, 136.18, 135.20, 134.84, 134.61, 128.70, 126.35, 122.69, 112.34, 40.38 ; HR-MS (FAB+): m / z calcd for C 24 H 20 NO2S [M+H]+: 386.1215, found: 386.1220.

[0218]

[0219] 2.3. TTB-07, TTM-07, TTB-09, TTM-09 및 TTM-13의 합성

[0220] 2.3.1. DPABr

[0221] 4-bromo-N,N-dipropylaniline

[0222] N,N-Dipropylaniline (2000 mg, 11.28 mmol) was dissolved in acetonitrile anhydride (10 mL). N-bromosuccinimide (2007.8 mg, 11.28 mmol) was dissolved separately in acetonitrile anhydride (10 mL). The diluted N-bromosuccinimide solution was dropwise added to the diluted N,N-dipropylaniline solution while stirring at 0 °C for 30 minutes. The reaction mixture was stirred at 0 °C for 1 hour. The residue was purified using silica gel chromatography with a hexane / methylene chloride gradient elution, yielding a colorless oil-like DPABr (2885 mg, 99.9%). 1 H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 9.2 Hz, 2H), 6.49 (d, J = 9.1 Hz, 2H), 3.21 - 3.17 (m, 4H), 1.61 - 1.56 (m, 4H), 0.91 (t, J = 7.4 Hz, 6H). 1 H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 9.2 Hz, 2H), 6.49 (d, J = 9.1 Hz, 2H), 3.21 - 3.17 (m, 4H), 1.61 - 1.56 (m, 4H), 0.91 (t, J = 7.4 Hz, 6H).

[0223]

[0224] 2.3.2. DPATA

[0225] 5-(4-(dipropylamino)phenyl)thiophene-2-carbaldehyde

[0226] 4-Bromo-N,N-dipropylaniline (600 mg, 2.342 mmol), (5-formylthiophen-2-yl)boronic acid (438 mg, 2.8104 mmol), and potassium carbonate (1295 mg, 9.368 mmol) were dissolved in a methanol (4 mL) / toluene (4 mL) mixture. Bis(triphenylphosphine)palladium(II) dichloride (164.4 mg, 0.2342 mmol) was added. The reaction mixture was stirred at 75 °C for 16 hours. The resulting crude was diluted with water and extracted with DCM (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified using silica gel chromatography with a hexane / ethyl acetate gradient elution to obtain an orange oil DPATA (294.9 mg, 43.8%). 1 H NMR (401 MHz, CDCl3) δ 9.81 (s, 1H), 7.67 (d, J = 4.0 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.21 (d, J = 4.0 Hz, 1H), 6.64 (d, J = 8.7 Hz, 2H), 3.31 - 3.26 (m, 4H), 1.64 (q, J = 7.5 Hz, 4H), 0.95 (t, J = 7.4 Hz, 6H).

[0227]

[0228] 2.3.3. General Procedure for Synthesis of TTB-07, TTM-07, TTB-09, TTM-09, and TTM-13

[0229] An aldehyde intermediate (1.0 equiv.) and NaOH (2.0 equiv.) were added to a solution in which 2,3-dihydro-1H-inden-1-one (1.0 equiv.) was dissolved in an ethanol / toluene mixture. The reaction mixture was stirred at 0 °C for 4 hours. After concentration under reduced pressure, the residue was purified using silica gel chromatography with a hexane / ethyl acetate gradient elution, and TTB and TTM compounds were obtained.

[0230]

[0231] 2.3.3.1. TTB-07

[0232] (Z)-2-((5-((E)-4-(diphenylamino)styryl)thiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one

[0233] A solution of 2,3-dihydro-1H-inden-1-one (7 mg, 0.0524 mmol) dissolved in an ethanol (0.5 mL) / toluene (0.1 mL) mixture was reacted with VTPATA (20 mg, 0.0524 mmol) and NaOH (5.2 mg, 0.1048 mmol) according to general procedures, and an orange solid TTB-07 (19 mg, 73%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 7.7 Hz, 1H), 7.83 (t, J = 2.0 Hz, 1H), 7.64 - 7.57 (m, 2H), 7.43 (t, J = 7.3 Hz, 1H), 7.37 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 3.8 Hz, 1H), 7.30 - 7.28 (m, 3H), 7.15 - 7.10 (m, 6H), 7.08 - 7.04 (m, 5H), 7.00 (s, 1H), 3.99 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 193.76, 149.10, 149.05, 148.16, 147.47, 138.92, 138.26, 134.58, 134.52, 132.50, 130.36, 130.22, 129.52, 127.78, 127.66, 126.91, 126.88, 126.31, 124.98, 124.40, 123.55, 123.15, 119.63, 32.61; HR-MS (FAB+): m / z calcd for C 34 H 26 NOS [M+H]+: 496.1735, found: 496.1727.

[0234]

[0235] 2.3.3.2. TTM-07

[0236] (Z)-2-((5-((E)-4-(dimethylamino)styryl)thiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one

[0237] A solution of 2,3-dihydro-1H-inden-1-one (10.3 mg, 0.0777 mmol) dissolved in an ethanol (0.5 mL) / toluene (0.1 mL) mixture was reacted with VDMATA (20 mg, 0.0777 mmol) and NaOH (3.1 mg, 0.1554 mmol) according to a standard procedure, and an orange solid TTM-07 (14.5 mg, 50%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 7.6 Hz, 1H), 7.82 (s, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.59 - 7.56 (m, 1H), 7.41 (dd, J = 12.2, 8.1 Hz, 3H), 7.31 (d, J = 3.7 Hz, 1H), 7.02 (d, J = 5.9 Hz, 3H), 6.70 (d, J = 8.7 Hz, 2H), 3.96 (s, 2H), 3.01 (s, 6H).13 C NMR (100 MHz, CDCl3) δ 193.76, 150.61, 150.07, 149.13, 139.00, 137.43, 134.73, 134.39, 131.96, 131.18, 128.05, 127.70, 127.11, 126.29, 125.96, 124.74, 124.32, 117.10, 112.43, 40.46, 32.60; HR-MS (FAB+): m / z calcd for C 24 H 22 NOS [M+H]+: 372.1422, found: 372.1419.

[0238]

[0239] 2.3.3.3. TTB-09

[0240] (Z)-2-((5-(4-(diphenylamino)phenyl)thiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one

[0241] A solution of 2,3-dihydro-1H-inden-1-one (7.4 mg, 0.0563 mmol) dissolved in an ethanol (0.5 mL) / toluene (0.1 mL) mixture was reacted with VTPATA (20 mg, 0.0563 mmol) and NaOH (4.5 mg, 0.1125 mmol) according to general procedures, and a yellow solid TTB-09 (24 mg, 90.8%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 7.7 Hz, 1H), 7.84 (t, J = 1.9 Hz, 1H), 7.62 - 7.56 (m, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.42 (t, J = 7.0 Hz, 1H), 7.38 (d, J = 3.9 Hz, 1H), 7.30 - 7.26 (m, 5H), 7.13 (d, J = 8.7 Hz, 4H), 7.08 - 7.04 (m, 4H), 3.96 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 193.78, 150.01, 149.07, 148.38, 147.33, 138.88, 138.34, 134.89, 134.48, 132.13, 129.54, 127.73, 127.33, 127.03, 126.88, 126.30, 125.00, 124.35, 123.67, 123.30, 123.15, 32.58; HR-MS (FAB+): m / z calcd for C 32 H 24 NOS [M+H]+: 470.1579, found: 470.1567.

[0242]

[0243] 2.3.3.4. TTM-09

[0244] (Z)-2-((5-(4-(dimethylamino)phenyl)thiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one

[0245] A solution of 2,3-dihydro-1H-inden-1-one (11.4 mg, 0.0865 mmol) dissolved in an ethanol (1.0 mL) / toluene (0.2 mL) mixture was reacted with DMTA (20 mg, 0.0865 mmol) and NaOH (7 mg, 0.1750 mmol) according to a standard procedure, and an orange solid TTM-09 (23.9 mg, 80%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 7.6 Hz, 1H), 7.85 (s, 1H), 7.60 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.43 (t, J = 6.8 Hz, 1H), 7.38 (d, J = 3.8 Hz, 1H), 7.23 (d, J = 3.9 Hz, 1H), 6.74 (d, J = 8.1 Hz, 2H), 3.98 (s, 2H), 3.03 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 193.81, 151.44, 150.59, 149.12, 139.04, 137.12, 135.15, 134.31, 131.36, 127.65, 127.36, 127.10, 126.27, 124.27, 121.99, 112.52, 40.51, 32.58; HR-MS (FAB+): m / z calcd for C 22 H 20 NOS [M+H]+: 346.1266, found: 346.1269.

[0246]

[0247] 2.3.3.5. TTM-13

[0248] (Z)-2-((5-(4-(dipropylamino)phenyl)thiophen-2-yl)methylene)-2,3-dihydro-1H-inden-1-one

[0249] A solution of 2,3-dihydro-1H-inden-1-one (10.3 mg, 0.0777 mmol) dissolved in an ethanol (0.5 mL) / toluene (0.1 mL) mixture was reacted with DPATA (20 mg, 0.0777 mmol) and NaOH (3.1 mg, 0.1554 mmol) according to general procedure, and an orange solid TTM-13 (14.5 mg, 50%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 7.6 Hz, 1H), 7.84 (s, 1H), 7.59 (d, J = 6.9 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.41 (t, J = 7.1 Hz, 1H), 7.35 (d, J = 3.6 Hz, 1H), 7.18 (s, 1H), 6.70 - 6.56 (m, 2H), 3.95 (s, 2H), 3.28 (s, 4H), 1.64 (q, J = 7.4 Hz, 4H), 0.95 (t, J = 7.4 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 193.77, 151.77, 149.07, 148.50, 139.03, 136.69, 135.22, 134.21, 131.05, 127.58, 127.24, 126.22, 124.20, 121.54, 120.66, 111.70, 52.91, 32.54, 20.56, 11.53; HR-MS (FAB+): m / z calcd for C 26 H 28 NOS [M+H]+: 402.1892, found: 402.1898.

[0250]

[0251] 2.4. Synthesis of TTM-10 and TTM-12

[0252] 2.4.1. General Procedure for the Synthesis of TTM-10 and TTM-12

[0253] An aldehyde intermediate was added to a solution of 1H-indene-1,3(2H)-dione dissolved in a methanol / toluene mixture. After adding one drop of piperidine dropwise, the reaction mixture was stirred at 60 °C for 4 hours. After concentration under reduced pressure, the residue was filtered by silica gel chromatography with a hexane / ethyl acetate gradient elution to obtain TTB and TTM compounds.

[0254]

[0255] 2.4.1.1. TTM-10

[0256] 2-((5-(4-(dimethylamino)phenyl)thiophen-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0257] A solution of 1H-indene-1,3(2H)-dione (12.6 mg, 0.0865 mmol) dissolved in a methanol (0.6 mL) / toluene (0.6 mL) mixture was reacted with DMTA (20 mg, 0.0865 mmol) according to a standard procedure, and a dark red solid TTM-10 (26 mg, 83.6%) was obtained. 1 H NMR (400 MHz, CDCl3) δ 7.94 (s, 4H), 7.77 - 7.73 (m, 2H), 7.69 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 4.1 Hz, 1H), 6.73 (d, J = 8.7 Hz, 2H), 3.06 (s, 6H). 13 C NMR (100 MHz, CDCl3) δ 190.92, 190.03, 144.67, 142.12, 140.58, 136.57, 134.82, 134.58, 128.02, 122.86, 122.69, 122.33, 112.39, 40.50; HR-MS (FAB+): m / z calcd for C 22 H 18 NO2S [M+H]+: 359.0980, found: 359.0978.

[0258]

[0259] 2.4.1.2. TTM-12

[0260] 2-((5-(4-(dipropylamino)phenyl)thiophen-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0261] A solution of 1H-indene-1,3(2H)-dione (15.2 mg, 0.1043 mmol) dissolved in a methanol (1 mL) / toluene (1 mL) mixture was reacted with DPATA (30 mg, 0.1043 mmol) according to a standard procedure, and purple solid TTM-12 (42.5 mg, 98%) was obtained. 1H NMR (400 MHz, CDCl3) δ 7.94 - 7.90 (m, 4H), 7.74 - 7.71 (m, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.29 (d, J = 4.0 Hz, 1H), 6.63 (d, J = 8.1 Hz, 2H), 3.32 - 3.27 (m, 4H), 1.67 - 1.61 (m, 4H), 0.96 (t, J = 7.4 Hz, 6H). 13 C NMR (100 MHz, CDCl3) δ 190.97, 190.00, 144.80, 142.10, 140.54, 136.53, 134.71, 134.47, 128.20, 122.77, 122.60, 111.71, 77.48, 52.94, 20.62, 11.54; HR-MS (FAB+): m / z calcd for C 26 H 26 NO2S [M+H]+: 461.1684, found: 416.1693.

[0262]

[0263] 2.5. MTdT-2 및 MTdT-CN의 합성

[0264] 2.5.1. VPOMTA

[0265] (E)-5-(4-methoxystyryl)thiophene-2-carbaldehyde

[0266] 5-bromothiophene-2-carbaldehyde (640 mg, 3.35 mmol), tetrabutylammonium bromide (1438 mg, 4.46 mmol), and palladium(II) acetate (51 mg, 0.223 mmol) were dissolved in a solution of 4-methoxystyrene (300 mg, 2.23 mmol) dissolved in an N,N-dimethylformamide (4 mL) mixture. Triethylamine (623 μL, 4.46 mmol) was added. The reaction mixture was stirred at 90 °C for 16 hours. The resulting crude was diluted with water and extracted with DCM (3 × 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was purified by silica gel chromatography with a hexane / acetone gradient elution to obtain an orange solid, VPOMTA (175.1 mg, 32.1%). 1 H NMR (400 MHz, Chloroform-d) δ 9.84 (s, 1H), 7.65 (d, J = 3.9 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.10 (t, J = 4.3 Hz, 3H), 6.91 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H).

[0267]

[0268] 2.5.2. MTdT-2

[0269] (E)-2-((5-(4-methoxystyryl)thiophen-2-yl)methylene)-1H-indene-1,3,(2H)-dione

[0270] A solution of 1H-indene-1,3(2H)-dione (11.9 mg, 0.0818 mmol) dissolved in a methanol (1 mL) / toluene (1 mL) mixture was reacted with VPOMTA (20 mg, 0.0818 mmol) according to a standard procedure to obtain burgundy solid MTdT-2 (25.8 mg, 84.6%). 1 H NMR (400 MHz, CDCl3) δ 7.96 (dt, J = 6.35, 3.4 Hz, 2H), 7.93 (s, 1H), 7.87 (d, J = 3.46, 1H) 7.78 (t, J = 8.61 Hz, 2H), 7.50 (d, J = 8.61, x2H), 7.27 (d, J = 16.15 Hz, 1H), 7.16 (s, 1H), 7.15 (d, J = 13.57 Hz, 1H), 6.94 (d, J = 8.76 Hz, 2H), 3.11 (s, 3H). 13 C NMR (101 MHz, CHLOROFORM-D) δ 190.56, 189.81, 160.47, 156.70, 143.69, 142.13, 140.57, 136.12, 135.88, 135.01, 134.80, 133.40, 128.99, 128.61, 127.02, 123.55, 123.00, 122.84, 119.08, 114.53, 55.49; HR-MS (FAB+): m / z calcd for [M+H]+:, found:

[0271]

[0272] 2.5.3. MTdT-CN

[0273] 2-((Z)-2-((5-((E)-4-methoxystyryl)thiophen-2-yl)methylene)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile

[0274] A solution of MNI (15.89 mg, 0.0818 mmol) dissolved in anhydrous pyridine (0.5 mL) was reacted with VPOMTA (20 mg, 0.0818 mmol) according to a general procedure to obtain a blue solid MTdT-CN (19.3 mg, 56.1%). 1 H NMR (400 MHz, Chloroform-d) δ 8.83 (s, 1H), 8.68 (d, J = 6.5 Hz, 1H), 7.95 - 7.92 (m, 1H), 7.80 - 7.72 (m, 3H), 7.49 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 16.1 Hz, 1H), 7.20 (d, J = 4.1 Hz, 1H), 7.14 (d, J = 16.1 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H).; HR-MS (FAB+): m / z calcd for [M+H]+:, found:.

[0275]

[0276] 2.6. Synthesis of PzdT-2 and Pz0T-2

[0277] 2.6.1. Boc-PzA

[0278] Tert-butyl 4-phenylpiperazine-1-carboxylate

[0279] 1-Phenylpiperazine (1000 mg, 6.16 mmol) and tert-butoxycarbonyl protecting group (1614.4 mg, 7.392 mmol) were dissolved in anhydrous acetonitrile (10 mL) and deionized water (10 mL). The mixture was stirred at room temperature for 3 hours. Subsequently, the reaction mixture was concentrated under reduced pressure. The residue was diluted in deionized water and extracted with dichloromethane. Pale yellow solid Boc-PzA (1491.7 mg, 92.3%) was obtained. 1H NMR (401 MHz, Chloroform-d) δ 7.29 (d, J = 7.5 Hz, 2H), 6.94 (s, 3H), 3.59 (s, 4H), 3.14 (s, 4H), 1.48 (s, 9H). Chemical Formula: C 15 H 22 N2O2

[0280]

[0281] 2.6.2. Boc-PzABr

[0282] Tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate

[0283] Tert-butyl 4-phenylpiperazine-1-carboxylate (500 mg, 2.073 mmol) was dissolved in anhydrous acetonitrile (3 mL). N-bromosuccinimide (406 mg, 2.279 mmol) was dissolved in anhydrous acetonitrile (3 mL). Subsequently, while stirring the tert-butyl 4-phenylpiperazine-1-carboxylate solution at 0°C, the pre-dissolved N-bromosuccinimide was added dropwise. The mixture was stirred at 0°C for 1 hour, followed by stirring at room temperature for 16 hours. The mixture was concentrated under reduced pressure, and the product was filtered to obtain Boc-PzABr (291 mg, 41.2%) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 7.35 (d, J = 8.9 Hz, 2H), 6.79 (d, J = 8.3 Hz, 2H), 3.57 (s, 4H), 3.09 (s, 4H), 1.48 (s, 9H). Chemical Formula: C 15 H 21 BrN2O2

[0284]

[0285] 2.6.3. Boc-VPzA

[0286] Tert-butyl 4-(4-vinylphenyl)piperazine-1-carboxylate

[0287] Tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate (600 mg, 1.76 mmol) was dissolved in anhydrous acetonitrile (3.5 mL). Tributyl(vinyl)tin (670 mg, 2.11 mmol) and tetrakis(triphenylphosphine)palladium (0) (203.4 mg, 0.176 mmol) were dissolved in the same solvent. The mixture was stirred at 90°C for 16 hours. The reaction mixture was extracted with saturated sodium bicarbonate solution (100 mL) and dichloromethane (50 mL x 3). The bound organic layer was washed with brine, dried in Na2SO₄, filtered, and concentrated under reduced pressure. The obtained product was purified by flash chromatography on silica gel (hexane: ethyl acetate = 20:1) to obtain Boc-VPzA (214.5 mg, 42.3%) in the form of a white solid. 1 H NMR (401 MHz, Chloroform-d) δ 7.33 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.64 (dd, J = 17.6, 10.9 Hz, 1H), 5.60 (d, J = 18.6 Hz, 1H), 5.10 (d, J = 11.8 Hz, 1H), 3.58 (s, 4H), 3.15 (s, 4H), 1.48 (s, 9H). Chemical Formula: C 17 H 24 N2O2

[0288]

[0289] 2.6.4. Boc-VPzATA

[0290] Tert-butyl (E)-4-(4-(2-(5-formylthiophen-2-yl)vinyl)phenyl)piperazine-1-carboxylate

[0291] Tert-butyl 4-(4-vinylphenyl)piperazine-1-carboxylate (200 mg, 0.6935 mmol), 5-bromothiophene-2-carbaldehyde (145.74 mg, 0.763 mmol), tetrabutylammonium bromide (447.2 mg, 1.39 mmol), and palladium(II) acetate (15.72 mg, 0.07 mmol) were combined in acetonitrile (2 mL). Triethylamine (194 μL, 1.39 mmol) was added to the mixture and stirred at 95°C for 16 hours. The reaction mixture was extracted with water (100 mL) and dichloromethane (50 mL x 3). The combined organic layer was washed with brine, dried in Na2SO₄, filtered, and concentrated under reduced pressure. The obtained product was purified by flash chromatography on silica gel (hexane: ethyl acetate = 20:1) to obtain Boc-VPzATA (70.3 mg, 25.4%) in the form of an orange solid. 1 H NMR (400 MHz, Chloroform-d) δ 9.83 (s, 1H), 7.64 (d, J = 3.9 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.09 (s, 1H), 7.08 (s, 1H), 7.07 (s, 1H), 6.90 (d, J = 8.8 Hz, 2H), 3.59 (s, 4H), 3.22 (s, 4H), 1.49 (s, 9H). Chemical Formula: C 22 H 26 N2O3S

[0292]

[0293] 2.6.5. Boc-PzATA

[0294] Tert-butyl 4-(4-(5-formylthiophen-2-yl)phenyl)piperazine-1-carboxylate

[0295] Tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate (250 mg, 0.735 mmol), (5-formylthiophen-2-yl)boronic acid (137.59 mg, 0.88 mmol), and potassium carbonate (406.4 mg, 2.94 mmol) were combined in methanol (1.5 mL) and toluene (1.5 mL). Bis(triphenylphosphine)palladium(II) dichloride (51.6 mg, 0.0735 mmol) was added to the mixture. The solution was refluxed at 75°C for 16 hours. The reaction mixture was extracted with water (100 mL) and dichloromethane (50 mL x 3). The combined organic layer was washed with brine, dried in Na2SO₄, filtered, and concentrated under reduced pressure. The obtained product was purified by flash chromatography on silica gel (hexane: ethyl acetate = 30:1) to obtain Boc-PzATA (155 mg, 56.6%) in the form of a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 9.85 (s, 1H), 7.70 (d, J = 4.0 Hz, 1H), 7.59 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 4.0 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 3.61 - 3.58 (m, 4H), 3.26 - 3.22 (m, 4H), 1.49 (s, 9H).Chemical Formula: C 20 H 24 N2O3S

[0296]

[0297] 2.6.6. General Procedure for Synthesis of PzdT-2 and Pz0T-2

[0298] An aldehyde intermediate was added to a solution of 1H-indene-1,3(2H)-dione dissolved in a methanol / toluene mixture. One drop of piperidine was added, and the reaction mixture was stirred at 60 °C for 4 hours. After concentration under reduced pressure, the residue was purified by silica gel chromatography eluted under hexane / ethyl acetate gradient elution conditions to obtain PzdT-2 and Pz0T-2.

[0299]

[0300] 2.6.6.1. PzdT-2

[0301] (E)-2-((5-(4-(piperazine-1-yl)styryl)thiophen-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0302] 1H-indene-1,3(2H)-dione (11.0012 mg, 0.0753 mmol) and Boc-VPzATA (30 mg, 0.0753 mmol) were dissolved in acetonitrile anhydride (2 mL), and then one drop of piperidine was added. The mixture was stirred at 60°C for 4 hours and then concentrated under reduced pressure. The product was purified by flash chromatography on silica gel (hexane: ethyl acetate = 10:1). The purified compound (22 mg, 0.0417 mmol) was dissolved in dichloromethane (2 mL), and 0.4 mL of trifluoroacetic acid was added. The mixture was concentrated under reduced pressure and dried under vacuum to obtain PzdT-2 (17.1 mg, 95.9%) in the form of a purple solid.1 H NMR (400 MHz, Methanol-d4) δ 8.02 (d, J = 4.1 Hz, 1H), 7.94 (s, 3H), 7.87 (s, 2H), 7.56 (d, J = 8.9 Hz, 2H), 7.34 - 7.29 (m, 3H), 7.04 (d, J = 8.9 Hz, 2H), 3.50 (s, 4H), 3.38 (s, 4H), 1.29 (s, 1H). Chemical Formula: C 25 H 22 N2O2S

[0303]

[0304] 2.6.6.2. Pz0T-2

[0305] 2-((5-(4-(piperazine-1-yl)phenyl)thiophen-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0306] 1H-indene-1,3(2H)-dione (11.0012 mg, 0.0537 mmol) and Boc-PzATA (20 mg, 0.0537 mmol) were dissolved in acetonitrile anhydride (1.5 mL). One drop of piperidine was added, and the mixture was stirred at 60°C for 4 hours. The reaction mixture was concentrated under reduced pressure, and the product was filtered via flash chromatography on silica gel (hexane: ethyl acetate = 8:1) to obtain a purple solid. The purified compound (10 mg, 0.01997 mmol) was dissolved in dichloromethane (1 mL), and 0.2 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure, dried under vacuum, and obtained Pz0T-2 (7.2 mg, 90%) in the form of a reddish-brown solid. 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 4.2 Hz, 1H), 8.04 (s, 1H), 7.93 (s, 4H), 7.78 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 4.1 Hz, 1H), 7.12 (d, J = 8.9 Hz, 2H), 3.51 (s, 4H), 3.26 (s, 4H), 1.23 (s, 1H). Chemical Formula: C 24 H 20 N2O2S

[0307]

[0308] 2.7. Synthesis of Oxy8-dT-2 and Oxy8-0T-2

[0309] 2.7.1. Oxy8-PBr

[0310] 1-bromo-4-(octyloxy)benzene

[0311] 4-bromophenol (200 mg, 1.156 mmol), 1-bromooctane (335 mg, 1.734 mmol), and potassium carbonate (798.7 mg, 5.78 mmol) were combined in N,N-dimethylformamide (3 mL). The mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with water (100 mL) and dichloromethane (50 mL x 3). The combined organic layer was washed with brine, dried in Na2SO₄, filtered, and concentrated under reduced pressure to obtain Oxy8-PBr (245.8 mg, 74.5%) in the form of a colorless oil. 1H NMR (400 MHz, Chloroform-d) δ 7.37 (s, 1H), 7.34 (s, 1H), 6.78 (s, 1H), 6.76 (s, 1H), 3.91 (t, J = 6.6 Hz, 2H), 1.80 - 1.73 (m, 2H), 1.44 (p, J = 6.9 Hz, 2H), 1.31 (dd, J = 12.3, 6.9 Hz, 8H), 0.90 - 0.86 (m, 3H). Chemical Formula: C 14 H 21 BrO

[0312]

[0313] 2.7.2. Oxy8-VP

[0314] 1-(octyloxy)-4-vinylbenzene

[0315] 1-bromo-4-(octyloxy)benzene (490.1 mg, 1.718 mmol) was dissolved in N,N-dimethylformamide (9 mL). Tributyl(vinyl)tin (653.86 mg, 2.062 mmol) and tetrakis(triphenylphosphine)palladium (0) (198.56 mg, 0.1718 mmol) were added. The mixture was stirred at 90°C for 16 hours. After the reaction, the mixture was filtered through Celite and extracted with saturated sodium bicarbonate solution (100 mL) and dichloromethane (50 mL x 3). The bound organic layer was washed with brine, dried in Na2SO₄, filtered, concentrated under reduced pressure, and purified by flash chromatography on silica gel (hexane: dichloromethane = 30:1) to obtain Oxy8-VP (390.3 mg, 97.8%) in the form of a colorless oil. 1H NMR (400 MHz, Chloroform-d) δ 7.33 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.66 (dd, J = 17.6, 10.9 Hz, 1H), 5.60 (d, J = 18.4 Hz, 1H), 5.11 (d, J = 11.6 Hz, 1H), 3.95 (t, J = 6.6 Hz, 2H), 1.62 (d, J = 7.4 Hz, 2H), 1.47 - 1.41 (m, 2H), 1.31 (d, J = 18.9 Hz, 8H), 0.89 (d, J = 7.6 Hz, 3H). Chemical Formula: C 16 H 24 O

[0316]

[0317] 2.7.3. Oxy8-VPTA

[0318] (E)-5-(4-octyloxy)styryl)thiophene-2-carbaldehyde

[0319] In N,N-dimethylformamide (8 mL), 1-(octyloxy)-4-vinylbenzene (390.3 mg, 1.68 mmol), 5-bromothiophene-2-carbaldehyde (417.16 mg, 2.183 mmol), tetrabutylammonium bromide (1083 mg, 3.36 mmol), and palladium(II) acetate (37.7 mg, 0.168 mmol) were combined. Triethylamine (469 μL, 3.36 mmol) was added to the mixture. The solution was stirred at 95°C for 16 hours. After the reaction, the mixture was filtered through Celite and extracted with water (100 mL) and dichloromethane (50 mL x 3). The bound organic layer was washed with brine, dried in Na2SO₄, filtered, concentrated under reduced pressure, and purified by flash chromatography on silica gel (hexane: ethyl acetate = 20:1) to obtain Oxy8-VPTA (163.3 mg, 28.5%) in the form of a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 9.84 (s, 1H), 7.65 (d, J = 3.9 Hz, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.10 (t, J = 4.6 Hz, 3H), 6.90 (d, J = 8.8 Hz, 2H), 3.98 (t, J = 6.6 Hz, 2H), 1.83 - 1.75 (m, 2H), 1.49 - 1.42 (m, 2H), 1.31 (dt, J = 16.6, 5.1 Hz, 8H), 0.91 - 0.87 (m, 3H). Chemical Formula: C 21 H 26 O2S

[0320]

[0321] 2.7.4. Oxy8-PTA

[0322] 5-(4-octyloxy)phenyl)thiophene-2-carbaldehyde (Oxy8-PTA)

[0323] 1-bromo-4-(octyloxy)benzene (245.8 mg, 0.8618 mmol), (5-formylthiophen-2-yl)boronic acid (161.3 mg, 1.034 mmol), and potassium carbonate (476.4 mg, 3.4472 mmol) were combined in methanol (1.5 mL) and toluene (1.5 mL). Bis(triphenylphosphine)palladium(II) dichloride (60.5 mg, 0.08618 mmol) was added. The mixture was refluxed at 75°C for 16 hours. After the reaction, the mixture was extracted with water (100 mL) and dichloromethane (50 mL x 3). The bound organic layer was washed with brine, dried in Na2SO₄, filtered, concentrated under reduced pressure, and purified by flash chromatography on silica gel (hexane: ethyl acetate = 40:1) to obtain Oxy8-PTA (127.7 mg, 46.8%) in the form of a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 9.86 (s, 1H), 7.71 (d, J = 4.0 Hz, 1H), 7.60 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 3.9 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 1.84 - 1.76 (m, 2H), 1.50 - 1.43 (m, 2H), 1.38 - 1.25 (m, 8H), 0.92 - 0.86 (m, 3H). Chemical Formula: C 19 H 24 O2S

[0324]

[0325] 2.7.5. General Procedure for the Synthesis of Oxy8-dT-2 and Oxy8-0T-2

[0326] An aldehyde intermediate was added to a solution of 1H-indene-1,3(2H)-dione dissolved in a methanol / toluene mixture. Subsequently, one drop of piperidine was added. The reaction mixture was stirred at 60 °C for 4 hours. After concentration under reduced pressure, the residue was purified by silica gel chromatography under hexane / ethyl acetate gradient elution conditions to obtain Oxy8-dT-2 and Oxy8-0T-2.

[0327]

[0328] 2.7.5.1 Oxy8-dT-2

[0329] (E)-2-((5-4-(octyloxy)styryl)thiophene-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0330] 1H-indene-1,3(2H)-dione (12.8 mg, 0.087 mmol) and (E)-5-(4-octyloxy)styryl)thiophene-2-carbaldehyde (30 mg, 0.087 mmol) were dissolved in methanol (1 mL) and toluene (1 mL). One drop of piperidine was added to the solution, and the mixture was stirred at 60°C for 4 hours. The reaction mixture was concentrated under reduced pressure. The product was filtered via flash chromatography on silica gel (hexane: ethyl acetate = 60:1) to obtain Oxy8-dT-2 (5.3 mg, 12.8%) in the form of a purple solid. 1H NMR (400 MHz, Chloroform-d) δ 7.98 - 7.94 (m, 2H), 7.93 (s, 1H), 7.87 (d, J = 4.1 Hz, 1H), 7.79 - 7.76 (m, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.30 (s, 1H), 7.16 (d, J = 2.8 Hz, 1H), 7.13 (d, J = 14.9 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 3.99 (t, J = 6.6 Hz, 2H), 1.84 - 1.76 (m, 2H), 1.47 (dt, J = 15.2, 6.6 Hz, 2H), 1.36 - 1.28 (m, 8H), 0.88 (d, J = 7.1 Hz, 3H). Chemical Formula: C 30 H 30 O3S

[0331]

[0332] 2.7.5.2 Oxy8-0T-2

[0333] 2-((5-(4-octyloxy)phenyl)thiophene-2-yl)methylene)-1H-indene-1,3(2H)-dione

[0334] 1H-indene-1,3(2H)-dione (13.8538 mg, 0.0948 mmol) and 5-(4-octyloxy)phenyl)thiophene-2-carbaldehyde (30 mg, 0.0948 mmol) were dissolved in methanol (1 mL) and toluene (1 mL). After adding one drop of piperidine, the mixture was stirred at 60°C for 4 hours. Subsequently, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (hexane: ethyl acetate = 40:1), resulting in Oxy8-0T-2 (30 mg, 71.2%) in the form of an orange solid. 1H NMR (400 MHz, Chloroform-d) δ 7.98 − 7.95 (m, 4H), 7.79 − 7.76 (m, 2H), 7.72 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 1 = 1.1 Hz), J ,6. Hz 2H), 4.01 (t, J = 6.6 Hz, 2H), 1.85 - 1.77 (m, 2H), 1.48 (p, J = 7.2 Hz, 2H), 1.38 - 1.27 (m, 8H), 0.92 - m 0.87). Chemical Formula: C 28 H 28 O3S

[0335]

[0336] 2.8. InTz, InTzCN 및 InTz-F의 합성

[0337] 2.8.1. PrpN_Bpin

[0338] N,N-dipropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline

[0339] DPABr (1000 mg, 3.903 mmol), Bis(pinacolato)diboron (1486.84 mg, 5.955 mmol), and potassium acetate (459.7 mg, 4.684 mmol) were dissolved in a 1,4-dioxane (15.61 ml) mixture. [1,1'-Bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane (159.38 mg, 0.195 mmol) was added. The reaction mixture was stirred at 95 °C for 15 hours. The resulting crude was diluted with water and extracted with DCM (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was filtered by silica gel chromatography under a hexane / EtOAc gradient to obtain PrPN_Bpin (783.3 mg, 66.2%) as a white solid. 1 H NMR (400 MHz, Acetone-d6) δ 7.54 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.9 Hz, 2H), 3.34 - 3.29 (m, 4H), 1.65 - 1.56 (m, 4H), 1.28 (s, 8H), 0.92 (t, J = 7.4 Hz, 6H).

[0340]

[0341] 2.8.2. PrpN_Tz_Ald

[0342] 7-(4-(dipropylamino)phenyl)benzo[c][1,2,5]thiadiazole-4-carbaldehyde

[0343] PrpN_Bpin (500 mg, 1.649 mmol), 7-bromobenzo[c][1,2,5]thiadiazole-4-carbaldehyde (440.87 mg, 1.814 mmol), and potassium carbonate (683.64 mg, 4.946 mmol) were dissolved in a 1,4-dioxane (12 mL) / water (3 mL) mixture. Tetrakis(triphenylphosphine)palladium (0) (95.26 mg, 0.082 mmol) was added. The mixture was stirred at 90 °C for 16 hours. The resulting crude was diluted with water and extracted with EtOAc (3 x 100 mL). The organic layer was dried with anhydrous sodium sulfate, the solvent was evaporated, and the residue was filtered by silica gel chromatography under hexane / EtOAc gradient elution conditions to obtain a deep red solid PrPN_Tz_Ald (521.8 mg, 93.3%). 1 H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.07 (d, J = 9.0 Hz, 2H), 7.98 (d, J = 7.5 Hz, 1H), 6.83 (d, J = 9.1 Hz, 2H), 3.32 (d, J = 5.6 Hz, 5H), 1.59 (h, J = 7.4 Hz, 5H), 0.92 (t, J = 7.4 Hz, 6H).

[0344]

[0345] 2.8.3. InTz

[0346] 2-((7-(4-(dipropylamino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)methylene)-1H-indene-1,3(2H)-dione

[0347] PrpN_Tz_Ald (20 mg, 0.059 mmol) and 1H-indene-1,3(2H)-dione (8.61 mg, 0.059 mmol) were dissolved in a toluene (1.5 ml) mixture. Piperidine (10 µL, 0.118 mmol) was added. The reaction mixture was stirred at 60 °C for 6 hours. After concentration under reduced pressure, the residue was filtered by silica gel chromatography under hexane / EtOAc gradient elution conditions, the crude was filtered with cooling MeOH, and washed with hexane to obtain a red-orange-brown solid InTz (12.5 mg, 41.2%). 1 H NMR (400 MHz, Chloroform-d) δ 9.76 (d, J = 7.9 Hz, 1H), 9.01 (s, 1H), 8.09 (d, J = 8.9 Hz, 2H), 8.04 (dt, J = 5.5, 2.9 Hz, 2H), 7.87 (d, J = 7.9 Hz, 1H), 7.82 (dd, J = 5.6, 3.0 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 3.37 - 3.33 (m, 4H), 1.74 - 1.65 (m, 5H), 0.99 (d, J = 7.4 Hz, 6H)

[0348]

[0349] 2.8.3. InTzCN

[0350] (Z)-2-(2-((7-(4-(dipropylamino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)methylene)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile

[0351] PrpN_Tz_Ald (70 mg, 0.206 mmol) and MNI (97.3 mg, 0.423 mmol) were dissolved in a chloroform (33.6 ml) mixture. Pyridine (0.22 mL, 0.271 mmol) was added. The reaction mixture was stirred at 75 °C for 16 hours. The reaction mixture was cooled to 50 °C, and MeOH (110 mL) was added. The resulting mixture was filtered with MeOH, and the residue was further purified by dry column chromatography using a DCM / EtOAc gradient to obtain InTzCN (76.5 mg, 72.0%) in the form of a deep purple solid. 1 H NMR (401 MHz, Chloroform-d) δ 9.62 (s, 1H), 9.30 (d, J = 7.9 Hz, 1H), 8.73 (d, J = 7.7 Hz, 1H), 8.13 (d, J = 9.0 Hz, 2H), 7.96 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.78 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 3.39 - 3.34 (m, 4H), 1.70 (h, J = 7.5 Hz, 4H), 0.98 (t, J = 7.4 Hz, 6H).

[0352]

[0353] 2.8.4. InTz-F

[0354] 2-((7-(4-(dipropylamino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)methylene)-5,6-difluoro-1H-indene-1,3(2H)-dione

[0355] PrpN_Tz_Ald (50 mg, 0.147 mmol) and 5,6-difluoro-1H-indene-1,3(2H)-dione (54.24 mg, 0.236 mmol) were dissolved in a THF (3.5 ml) mixture. Piperidine (8.78 µL, 0.103 mmol) was added. The reaction mixture was stirred at 35 °C for 6 hours. The reaction mixture was cooled to room temperature, and MeOH (80 mL) was added. The reaction mixture was filtered with MeOH, and the residue was further purified by dry column chromatography under a hexane / EtOAc gradient to obtain a dark green solid, InTz-F (44.4 mg, 60.0%). 1 H NMR (400 MHz, Chloroform-d) δ 9.70 (d, J = 7.9 Hz, 1H), 8.98 (s, 1H), 8.10 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 7.9 Hz, 1H), 7.81 - 7.77 (m, 2H), 6.79 (d, J = 7.1 Hz, 2H), 3.35 (t, J = 7.6 Hz, 4H), 1.68 (d, J = 6.0 Hz, 5H), 0.98 (d, J = 7.4 Hz, 7H).

[0356]

[0357] Experimental Example

[0358] 1. Materials and Methods

[0359] 1-1. Optical Characterization of Spherical Compounds

[0360] The UV-Vis absorption spectrum and fluorescence emission spectrum of the compounds synthesized in the above preparation example were measured at room temperature using an Orion AquaMate 8100 UV-VIS spectrophotometer (Thermo Scientific, WI, USA) and an FP-8350 fluorescence spectrometer (JASCO, Tokyo, Japan), respectively. The photostability of the synthesized compounds was monitored for 90 minutes by irradiating the samples every 25 seconds under conditions of glycerol, methanol (MeOH), and distilled water (DW) using a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, California, USA).

[0361]

[0362] 1-2. Human liver tissue samples and mouse experiments

[0363] This study was conducted in accordance with the principles regarding human participant research specified in the Declaration of Helsinki. It was approved by the Institutional Review Board (IRB) of Boramae Hospital (IRB No. 16-2013-45), and written informed consent was obtained from all participants in the study cohort. Human liver specimens were collected using a 16-gauge disposable needle, immediately immersed in liquid nitrogen, embedded in an optimal cutting temperature (OCT) solution, and frozen sections were prepared. The sections were subsequently fixed in 4% paraformaldehyde. Suitable specimens were selected for confocal microscopy to visualize BODIPY 581 / 591 C11 and TTM-4 fluorescence. For all animal experiments, 7–9 week old male C57BL / 6J and ICR mice (Naravio, Seoul, South Korea) were housed in a pathogen-free facility at the Laboratory Animal Research Institute, Seoul National University (Seoul, South Korea). All animal experiments were conducted in compliance with appropriate welfare measures throughout the study period. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-241021-1-1, SNU-250122-2) and were conducted in accordance with the guidelines for the care and use of laboratory animals.

[0364]

[0365] 1-3. Statistical Analysis

[0366] The significance of the difference between the two groups was analyzed using a two-sided Student's test. Values ​​are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

[0367]

[0368] 2. Results

[0369] 2-1. Optical Properties of Each Compound and Fluorescence Response to Viscosity

[0370] The absorption spectra of 10 μM of TTM or TTB compounds in various solvents (Figs. 17 and 18), the emission spectra of 2 μM of TTB compounds (Fig. 19), and the emission spectra of 2 μM of TTM compounds (Fig. 20) were examined. Additionally, the normalized emission spectra of 2 μM of TTM or TTB compounds in various solvents were examined (Figs. 21 and 22). Table 2 below shows the maximum absorption of the sphere compounds in each solvent, and Table 3 below shows the maximum emission of the sphere compounds in each solvent.

[0371] Compoundλ abs a (nm)ε max b (M -1 cm -1 )TolueneEt 2OTHFCHCl3EtOHDMSOH2OTris 7.5TTB-0160359359763050061760061222400TTB-0348447348148748448748148721800TTB-0453052052853753654251952537100TTB-066146016056306016205936215300TTB-0745944845846546746945944838300TTB-0944143143844144844844444737900TTM-0163062964965847665759359218800TTM-0350148849950650151641841821800TTM-0455053855356256257345650832900TTM-0664162963665364066459460429700TTM-0746745747346847948643443445100TTM-0945043945045246046742342324700TTM-1053151853354154255244044441700TTM-1254853755056156056852762543300TTM-1346145046346647247846247522700MTdT-249448549350349650340841358400MTdT-CN56555855957255556752755428800PzdT-252251552651952752743744439800Pz0T-250049550549850650241847735300Oxy8-dT-249848149550849650337940512400Oxy8-0T-245845245946946547039340242700

[0372]

[0373] Compoundλ abs a (nm)λ em b(nm)TolueneEt₂OTHFCHCl₃EtOHDMSOH₂OTris 7.5TTB-01616888898889.5896886874.5882889TTB-03488624.5668758.5744896.5849736.5736.5TTB-04540651682740743797833776770.5TTB-06617740892.5826824827828846832.5TTB-07470568583643650.5766737.5709.5709.5TTB-09449530545.5596608.5718.5675.5630.5631TTM-01663889890890878883.5883.5892.5673TTM-03518661714792756.5888.5844851.5856.5TTM-04573677.5711.5766737.5816.5843866.5866.5TTM-06664775.5832.5853.5847.5868.5893.5875898.5TTM-07487595616686.5674794.5781735735.5TTM-09466544.5565.5621613.5726698893.5867TTM-10553614.5625673.5652.5713745.5836837TTM-12569616.5630.5670.5662714737.5861.5863TTM-13479547565619618.5724698670.5861MTdT-2504578.5583.5606609.5636.5646761.5760MTdT-CN561642637699662.5690.5751849846PzdT-2543686716763.5740.5781.5805.5865817Pz0T-2509618.5637684.5655.5690.5712768.5893Oxy8-dT-2500582.5583609.5607.5639.5663847840Oxy8-0T-2469534.5531.5542.5548.5559573702707

[0374]

[0375] In addition, regarding viscosity, fluorescence spectra and integrated intensity plots were examined for each 10 μM spherical compound excited with ultraviolet light (excitation wavelength = 365 nm) in glycerol / H2O mixed solutions (0-95%) of various concentrations (Figs. 23 to 25). As a result, it was confirmed that for all measured compounds, the fluorescence intensity increased as the glycerol concentration increased, that is, as the viscosity increased. From this, it can be confirmed that fluorescence occurs with changes in viscosity.

[0376] The photostability of TTM-04 and MTdT-2 was confirmed, and their stability was verified as the fluorescence remained constant over time (Fig. 26). The normalized emission spectra of TTB-07 and TTB-09 in the solid state were examined (Inlet: Solid powder of TTB / TTM compounds under visible light (left) and ultraviolet light (right)) (Fig. 27 and Table 4).

[0377] CompoundSolidλ abs a (nm)λ em b (nm)Φ f c TTB-046147180.02TTB-075776560.23TTB-094965990.47TTM-076127000.04 TTM-095436430.01TTM-106497280.00TTM-127107910.01TTM-135506340.06

[0378]

[0379] The absolute quantum yields of (a) TTB-07, (b) TTB-09, (c) TTM-07, (d) TTM-09, and (e) TTM-13 in the solid powder state were confirmed, and the excitation and emission spectra of the measured compounds and blanks were confirmed (Fig. 28).

[0380] The absorption and emission spectra of compounds InTz, InTz-F, and InTzCN were also confirmed (Fig. 40).

[0381]

[0382] 2-2. ROS Measurement Using H2DCF-DA

[0383] Sodium hydroxide solution (10 mM, 0.9 mL) was added to a DMSO solution (10 mM, 100 μL) of H2DCF-DA to hydrolyze it, and the mixture was stirred at room temperature under dark conditions for 30 minutes. Subsequently, the solution was diluted with 1 mL of phosphate buffer (PBS) to obtain a 0.5 mM DCFH solution. After passing air through the solution, the test compound (1 μM) and DCFH (4 μM) were added. Then, the solution was subjected to a white LED (30,000 lm / m²). 2 After irradiating with ), the fluorescence spectrum was measured at a wavelength of 480 nm, and the fluorescence signal at 525 nm was monitored.

[0384] As a result, it was confirmed that the luminescence intensity of most compounds increased over time when irradiated with white light (Figs. 41 and 42). From this, it can be seen that the specific compounds of the present invention can function as photosensitizers as they are activated by light and continuously generate ROS.

[0385]

[0386] 2-3. O2 Measurement Using DHR123

[0387] 988 μL of air-saturated 1X PBS solution was prepared, to which 2 μM of the test compound (2 μL from a 1 mM DMSO stock solution) and 10 μM of DHR123 (10 μL from a 1 mM DMSO stock solution) were added. The resulting mixture was irradiated with a white LED light source (30,000 lm / m²), and the fluorescence spectrum was measured at 5-second intervals at an excitation wavelength of 490 nm. The superoxide generation rate of the compound was evaluated by monitoring the fluorescence intensity at an emission wavelength of 530 nm.

[0388] As a result, it was confirmed that the luminescence intensity increased over time during all measured white light irradiation (Figs. 32 and 33, 46 and 47). From this, it can be seen that the specific compounds of the present invention can be activated by light and function as photosensitizers.

[0389]

[0390] 2-4. Relative luminescence intensity in the presence of TTB / TTM compounds

[0391] White LED (luminous intensity 30,000 lm / m² 2 Using a light source, the relative luminescence intensity of H2DCF-DA (4 μM) in the presence of TTM / B compound (1 μM) was determined according to irradiation time (5-second intervals), and a white LED (luminance 30,000 lm / m²) was used as a light source. 2 Using ) as a light source, the relative luminescence intensity of DHR123 (10 μM) in the presence of TTM / B compound (2 μM) was determined according to irradiation time (5-second intervals).

[0392]

[0393] 2-5. PDT Activation by LED Irradiation

[0394] In addition, to investigate PDT activity, cell viability over time was examined at various concentrations of each specific compound (Fig. 35). HeLa cells were used. As a result, it was confirmed that cell viability decreased as the concentration increased and as time progressed. In particular, for TTM-04, TTM-07, TTM-09, TTM-13, MTdT-2, PzdT-2, and Pz0T-2, a significant decrease in HeLa cell viability was observed as the concentration increased. For compounds InTz, InTz-F, and InTzCN, a significant decrease in HeLa cell viability was also observed as the concentration increased and time progressed (Fig. 40). Table 5 shows IC in HeLa cells 50 Indicated the value.

[0395] CompoundIC50 irradiated by white LED (μM)5 min10 min20 minTTM-01No phototoxicityTTM-03No phototoxicityTTM-0445.66 ± 0.7782.827 ± 0.0952.122 ± 0.076TTM-06No phototoxicityTTM-0718.34 ± 0.0735.615 ± 0.0203.341 ± 0.033TTM-0914.37 ± 0.0394.561 ± 0.0132.944 ± 0.017TTM-10--24.86 ± 0.101TTM-121.627 ± 0.0090.975 ± 0.012~0.6TTM-134.504 ± 0.0172.512 ± 0.0161.346 ± 0.019MTdT-215.69 ± 0.03912.58 ± 0.068.044 ± 0.024MTdT-CNNo phototoxicityPzdT-24.238 ± 0.0192.938 ± 0.0132.083 ± 0.007Pz0T-27.627 ± 0.0264.791 ± 0.0273.629 ± 0.015Boc-PzdT-2No phototoxicityBoc-Pz0T-2-8.446 ± 0.2124.616 ± 0.045Oxy8-dT-2No phototoxicityOxy8-0T-2No phototoxicity

[0396]

[0397] 2-6. Confirmation of Fluorescence Emission Based on Viscosity Changes of TTM-04 and TTM-07

[0398] Confocal laser fluorescence microscopy image of HepG2 cells treated with 10 mM H2O2 and 1 μM TTM-4 (λ ex = 561 nm, λ em (= 635–700 nm) was captured, and the intracellular fluorescence intensity of TTM-4 was graphed (Fig. 36). Similarly, confocal laser fluorescence microscopy images (λ) of HepG2 cells treated with 10 mM H2O2 and 1 μM TTM-7 were obtained. ex = 488 nm, λem = 560-635 nm) was captured, and the intracellular fluorescence intensity of TTM-7 was graphed.

[0399] As a result, it can be seen that strong fluorescence appears in cells treated with H2O2. Therefore, it can be confirmed that when treated with H2O2, which is a reactive oxygen species, perotopes occurs in the cells, and as a result, viscosity increases, and the specific compound of the present invention can emit fluorescence by detecting the change in intracellular viscosity caused by such perotopes.

[0400]

[0401] 2-7. Confirmation of Fluorescence Emission Based on Viscosity Change of TTM-04

[0402] Fluorescence emission of TTM-4 in HepG2 cells cultured at different temperatures was confirmed. Live cell fluorescence images were obtained by treating HepG2 cells cultured at 37 ℃, 20 ℃, or 4 ℃ for 1 hour with 1 μM TTM-4 (λ ex = 561 nm, λ em = 635-700 nm). As a result, it was confirmed that viscosity increases as temperature decreases, and that the specific compound of the present invention can detect this change in viscosity and emit fluorescence (Fig. 38).

[0403]

[0404] 2-8. Confirmation of Fluorescence Emission Based on Viscosity Change of TTM-04

[0405] We investigated whether TTM-4 could detect intracellular lipid peroxidation during ferroptosis in cancer cells. Live cell fluorescence imaging of lipid peroxidation using 1 μM TTM-4 was performed in HepG2 cells treated with 10 mM H2O2 or 10 μM elastin and 5 μM RSL3 in the presence or absence of ferroptosis inhibitors including Liproxstatin-1 (Lip-1), β-mercaptoethanol (β-ME), and N-acetyl-L-cysteine ​​(NAC) (Fig. 39 A, B). Additionally, HepG2 and NIH-3T3 cells were co-cultured, ferroptosis was induced with 10 μM elastin, and intracellular lipid peroxidation was detected with 1 μM TTM-4 (TTM-4 observation condition: λ ex = 561 nm, λ em = 635-700 nm) (Fig. 39 C). The results of the quantification of the intracellular fluorescence intensity of TTM-4 were graphed (Fig. 39 D).

[0406] As a result, it was confirmed that the fluorescence induced by TTM-04 decreased when treated with a ferroptosis inhibitor, and increased when treated with a substance that induces ferroptosis. From this, it was proven that the compound of the present invention detects intracellular ferroptosis and emits fluorescence.

[0407] In addition, to confirm the sensitivity of TTM-4 to the detection of intracellular lipid peroxidation and ferroptosis, ferroptosis was induced in HepG2 cells by treating them with 5 μM RSL3, and the luminescence levels were examined at various concentrations of TTM-4. As a result, it was found that clearer fluorescence was observed as the concentration of TTM-4 increased.

[0408]

[0409] 2-9. Detection of cell viscosity during ferroptosis using TTM-4

[0410] To confirm the viscosity sensitivity of TTM-4, HepG2 cells treated with 10 mM H2O2 were imaged using a confocal microscope. TTM-4 fluorescence was significantly increased in the treated group compared to the control group, resulting in a signal-to-noise ratio improvement of more than 80-fold (A1-A2 in EH 49). This response was attenuated upon co-administration with liproxstatin-1 (Lip-1), β-mercaptoethanol (β-ME), or N-acetyl-L-cysteine ​​(NAC), suggesting redox dependence. Since ferroptosis involves lipid peroxidation and redox imbalance, TTM-4 was evaluated in cells treated with the ferroptosis inducers Erastin and RSL3. A strong red signal was observed (B1-B2 in Fig. 48b), which was inhibited by ferroxstatin-1 (Fer-1). And Lip-1 indicated that TTM-4 is useful for monitoring ferroptosis through lipid-ROS.

[0411] Cells were treated with various concentrations of TTM-4 to localize initial lipid peroxidation. At 200 nM, fluorescence was concentrated at the LD, but at higher concentrations, it showed a pattern of diffusion to other organelles (Fig. 49a C). Compared to BODIPY 581 / 591 C11, TTM-4 exhibited a stronger red-shifted "activation" emission, was more suitable for in vivo use, and required lower concentrations (<1 μM vs. 5 μM). MTT assays confirmed that cytotoxicity was minimized, supporting the finding that 1 μM is the optimal dose. Additionally, the sensitivity of TTM-4 in ferroptotic cells was compared to that in apoptotic or pyroptotic cells. In cells treated with staurosporine (an apoptosis inducer) and lipopolysaccharide (LPS; a pyroptosis inducer), the fluorescence intensity of TTM-4 was significantly lower than in cells treated with RSL3. This suggests that the increase in organelle viscosity occurs more rapidly in ferroptosis cells than in apoptotic or pyroptosis cells (D1-D2 in Fig. 49b). Collectively, these data demonstrated that TTM-4 selectively detects lipid peroxidation during ferroptosis.

[0412]

[0413] 2-10. Real-time monitoring of cellular lipid peroxidation using TMM-4

[0414] To evaluate the detection of organelle-specific lipid peroxidation by TTM-4, ferroptotic HepG2 cells were co-stained with BODIPY 493 / 503 or endoplasmic reticulum (ER) tracers. The TTM-4 signal showed high colocalization with the LD and ER (Pearson r = 0.85 and 0.75; AB in Fig. 50a). This suggests that ferroptosis primarily induces lipid peroxidation in these compartments. In particular, nuclear lipid droplets (nLD) were detected using TTM-4 in RSL3 or erastin-treated cells (C in Fig. 50b). Nuclear localization of nLD was confirmed by 3D imaging using Hoechst stain (white arrows). Four-hour time-lapse imaging of erastin-treated cells showed the dynamic transmission of the TTM-4 signal from the cytoplasmic LD (cLD) through the ER to the nucleus (Fig. 50c). These results suggest that nLD formation may represent an adaptive response to ER stress, which could potentially aid in early MASLD detection.

[0415]

[0416] 2-11. Visualization of lipid peroxidation using an in vitro MASLD model

[0417] MASH is the most severe symptom of MASLD, which can progress to liver cirrhosis and hepatocellular carcinoma. First, we tested whether the inhibition of lysosomal acidic lipase (LAL) by lalistat-2 modulates TTM-4 signaling in HepG2 cells. LAL inhibition, which mimics lipid accumulation by blocking lipid clearance via autophagy, significantly reduced TTM-4 signaling in the ER surrounding cLD and decreased oxidized nLD (Fig. 51), while cLD oxidation remained unchanged. These results suggest that the ER-mediated transport of peroxidized lipids from cLD to nLD is disrupted by lalistat-2, implying that LD degradation is a key step in nLD formation during ferroptosis. Given that LD comes into contact with organelles via the membrane contact site (MCS), MASLD was further modeled in vitro using HepG2 cells treated with palmitic acid (PA), resulting in increased TTM-4 fluorescence and increased levels of oxidized LD and nLD (Fig. 52). This is consistent with the clinical findings of nLD accumulation in MASH patients.

[0418] As ferroptosis is increasingly recognized as a contributing factor to MASH, we investigated whether the inhibition of ACSL3, a key enzyme in ferroptosis and LD biosynthesis, affects lipid oxidation. Treatment with eicosapentaenoic acid (EPA) inhibited ACSL3 and significantly reduced oxidized LD and nLD in PA-treated cells, suggesting a protective role of EPA against lipotoxic ferroptosis. To validate these results in a more physiological environment, AML12 mouse hepatocytes were used, and ferroptosis was induced using RSL3. RSL3 induced potent TTM-4 fluorescence inhibited by DFO (Fig. 53), confirming ferroptosis specificity. Notably, TTM-4 demonstrated superior performance compared to BODIPY 581 / 591 C11 in the detection of oxidized lipids in both RSL3- and PA-treated cells (Figs. 53 E, F). These results demonstrate that TTM-4 is an excellent probe for monitoring ferroptosis and oxidative lipid remodeling in the MASH model, suggesting that nLD is a potential biomarker of ferroptotic stress in steatohepatitis.

[0419]

[0420] 2-12. Visualization of Lipid Peroxidation in Liver Tissue Samples

[0421] Considering the potential of TTM-4 for detecting ferroptosis, it was applied to liver tissue samples. In liver biopsy samples from MASLD patients (human liver fibrosis stage 3), ferroptosis was frequently detected using TTM-4 in regions where the fibrosis marker α-SMA was highly expressed (Fig. 54a A). To mimic the pathological stages observed in human MASLD samples, C57BL / 6J mice were fed a choline-deficient, L-amino acid-deficient, high-fat diet (CDAHFD) for 1, 6, or 12 weeks. The effect was evaluated by detecting ferroptosis using TTM-4 at each time point. At the 12-week mark, which corresponds to a stage similar to that indicated in Fig. 54a A, TTM-4 successfully detected ferroptosis in mouse liver tissue. In addition, ferroptosis was observed in the same region as neutral lipids detected using BODIPY 493 / 503, indicating that TTM-4 can detect oxidized lipids in damaged liver tissue (Fig. 54a B). Similar to the findings in cells, in experiments evaluating detection at different time points after CDAHFD administration, TTM-4 exhibited fluorescence earlier in the Week 1 samples than in BODIPY 581 / 591 C11, highlighting the higher sensitivity of TTM-4. Ferroptosis occurred more frequently in liver tissue samples than in BODIPY 581 / 591 C11 (Fig. 54b C). This is thought to be due to the characteristic detection of TTM-4 over a longer wavelength range. Similar to the in vitro results, detection using TTM-4 confirmed that oxidized LD, which was first observed in the cytoplasm at Week 1, was abundant in the nucleus at Week 6. This pattern continued until week 12 (D in Fig. 54b). These results indicate that TTM-4 can track the migration of oxidized lipids in liver tissue samples.

[0422]

[0423] 2-13. Verification of gene expression related to LD fusion in the liver during MASLD progression

[0424] cLD undergoes oxidation and aggregation before lipid peroxides are detected in the ER, and CIDE proteins are involved in this process. Under normal conditions, CIDEB is dominant; however, when metabolic disorders such as MASLD occur, CIDEA and CIDEC increase, promoting LD-LD fusion and lipid storage, a process that can reduce lipotoxicity. CIDE gene expression was analyzed in a cohort of MASLD patients at Seoul National University Boramae Hospital. Among the CIDE family genes, CIDEC expression consistently increased with disease progression and showed a correlation with the severity of fatty degeneration and fibrosis (A1, A2 in Fig. 55a). This trend was also confirmed in the open dataset GSE135251 (n=216), where CIDEC showed the most significant upregulation when healthy controls transitioned to progressive NAFLD (A3-A5 in Figs. 55b and 55c). This trend was replicated particularly after 12 weeks in the mouse CDAHFD model (B1 in Fig. 56a). To evaluate this in more detail, primary hepatocytes treated with 100 or 300 μM PA showed significant CIDEC induction at 300 μM (B2 in Fig. 56a). Similarly, AML12 cells treated with increasing PA concentrations showed a dose-dependent increase in CIDEC, unlike with CIDEA (B3 in Fig. 56a). In TTM-4 and BODIPY 493 / 503 imaging, 250 μM PA increased cLD, while 1000 μM PA decreased neutral lipids (BODIPY 493 / 503 signal) but increased lipid oxidative accumulation (TTM-4 signal), which was consistent with increased CIDEC expression (C in Fig. 56b). In human liver biopsies, BODIPY showed that as fatty degeneration progressed to MASH, the 493 / 503 signal decreased and the TTM-4 signal increased, suggesting progression from lipid accumulation to lipid peroxidation (Fig. 56cD). In advanced MASH (aMASH), oxidized nLD increased (Fig. 56dE).Finally, confocal colocalization of TTM-4 and CIDEC in aMASH liver tissue confirmed signal overlap, suggesting that the upregulation of CIDEC contributes to oxidized LD formation and ferroptosis stress (F in Fig. 56e).

[0425]

[0426] 2-14. Real-time detection of ferroptosis in live animals with chemically induced acute liver injury and diet-induced chronic liver disease models using TTM-4

[0427] Ferroptosis was induced in mice using carbon tetrachloride (CCl4) or a dietary model (methionine and choline deficiency; MCD). After a single intraperitoneal injection of 20% CCl4 and 24 hours of incubation, TTM-4 fluorescence increased in liver tissue, indicating increased ferroptosis (A1 in Fig. 57a). Similarly, mice fed the MCD diet for 3 weeks showed a stronger TTM-4 signal compared to the control group (A2 in Fig. 57a). This confirms diet-induced ferroptosis. TTM-4 was suitable for non-invasive IVIS imaging due to its long emission wavelength.

[0428] To ensure safety, 5 mg / kg of TTM-4 was administered via the tail vein, and serum liver function indicators (AST, ALT, GGT) and renal function indicators (BUN, CRE) were measured 2 hours later; no acute toxicity was observed (Fig. 57b B). For in vivo imaging, mice were fasted (24 hours for the CCl4 diet, or the last day for the MCD diet) to minimize autofluorescence; TTM-4 was then injected, and IVIS imaging was performed 2 hours later (Fig. 57c C1). Liver damage was confirmed by macroscopic morphology (Fig. 57c C2-C3), and serum AST and ALT levels were elevated (Fig. 58a D1, D2). Strong IVIS fluorescence was continuously detected in ferroptosis-induced mice. To accurately identify the signal source, five major organ sites were dissected, and the highest fluorescence was observed in the liver (Fig. 58b E). Overall, the CCl4MCD diet effectively induced ferroptosis in vivo, and TTM-4 enabled real-time non-invasive imaging of ferroptosis, as verified by quantitative IVIS analysis (F1, F2 in Fig. 58b).

[0429]

[0430] [National R&D projects that supported this invention]

[0431] [Project ID] 2710011831

[0432] [Project No.] 2021R1A2C2010647 (RS-2021-NR059390)

[0433] [Ministry Name] Ministry of Science and ICT

[0434] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0435] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0436] [Research Project Title] Development of Reactive Theranostics Based on Small Molecule Compounds

[0437] [Name of Project Performing Organization] Seoul National University

[0438] [Research Period] 2021.03.01 ~ 2026.02.28

[0439]

[0440] [Project ID] 2710068986

[0441] [Project No.] RS-2022-NR067269

[0442] [Ministry Name] Ministry of Science and ICT

[0443] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0444] [Research Project Name] National R&D Project

[0445] [Project Title] Development of Multi-omics-based Therapeutic Technology Utilizing Clinical and Animal Model Samples by Stage of Metabolic Fibrosis

[0446] [Name of Project Performing Organization] Seoul National University

[0447] [Research Period] 2025.01.01 ~ 2026.12.31

Claims

Compound represented by the following chemical formula 1: [Chemical Formula 1] In the above formula, R1 is NR5R6 or OR7, and The above R5, R6, and R7 are each independently C 1-8 It is an alkyl or benzene, or R5 and R6 can be connected to each other to form a 5- to 7-membered heterocycle substituted or unsubstituted with one or more substituents, and L is a 5- to 14-membered arylene or heteroarylene substituted or unsubstituted with one or more substituents, and R2 is C substituted or unsubstituted with H, O, or one or more substituents. 1-6 It is a fused ring of two rings in which an aryl group is fused with a cycloalkyl group of 5 to 7 members that is substituted or unsubstituted with an alkyl group or one or more substituents, and R3 and R4 are each independently H or F. In claim 1, The above L is Phosphorus, compound. In claim 1, The above R1 is NR5R6, and The above R5 and R6 are each independently C 1-3 It is alkyl or benzene, or R5 and R6 are connected to each other, piperazine or A compound that forms. In claim 1, The above R1 is OR7, and The above R7 is C 1-8 Alkyl compounds. In claim 1, The above R2 is H, O, C(CN)2 or Phosphorus, compound. The compound of claim 1, wherein the compound is any one selected from the group consisting of the following compounds: A fluorescent probe composition comprising a compound of any one of claims 1 to 6. A fluorescent probe composition according to claim 7, wherein the fluorescent probe expresses near-infrared fluorescence or red fluorescence in response to a fluorescent reaction to an increase in viscosity. A fluorescent probe composition according to claim 7, wherein the fluorescent probe is for detecting lipid peroxidation in living cells or tissues. A fluorescent probe composition according to claim 7, wherein the fluorescent probe is for detecting ferroptosis. A composition for photodynamic therapy comprising a compound of any one of claims 1 to 6. A composition for photodynamic therapy according to claim 11, wherein the composition exhibits photosensitizing activity to light of a wavelength of 300 to 700 nm.

Images