KR20170136177A - Photosensitizer composition and preparing method thereof - Google Patents

Abstract

The present invention relates to a dimer compound formed from a fluorescent emission compound and a phosphorescent compound, a photosensitizer composition containing the dimer compound, and a preparing method of the dimer compound. Using the advantages of the dimer compound of the present invention, the dimer compound can be used as a biomedical imaging reagent and can also be usefully used for cancer treatment.

Description

[0001] PHOTOSENSITIZER COMPOSITION AND PREPARING METHOD THEREOF [0002]

The present invention relates to a dyad compound formed from a fluorescence emitting compound and a phosphorescent emitting compound, a monomolecular molecular oxygen photosensitizer composition comprising the dimer compound, and a method for producing the compound.

Singlet molecular oxygen ( ¹ O ₂ ) can be used for direct oxygenation, and such properties do not appear in triplet dioxygen. Such strong oxygenation of ¹ O ₂ has been used in a variety of applications including waste water treatment and organic conversion. Photodynamic therapy (photodynamic therapy) also uses a ¹ O ₂ sensitizer, which is emerging as a promising approach to cancer therapy. It is understood that the ¹ O ₂ is functional through any mechanism in a living body would be essential for the successful implementation of such the photodynamic therapy, the problem of the moment ¹ O ₂ in vivo sensing (sensing) of ^one of the O ₂ There is a difficulty in clearly grasping the biological mechanism.

In this regard, Korean Patent Publication No. 10-2015-0037225 ("Chemical Sensor Compound for Simultaneously Chemically Detecting Mercury and Active Oxygen and Method for Producing the Same") and Korean Patent Publication No. 10-2014-0107854 species measured MWNTs for platinum nanoparticles, and this introduction of the electrodes and sensors "), such as research for detecting active oxygen species efficiently are wateuna been conducted in various fields, among the various active oxygen species, especially singlet molecular oxygen ⁽¹ O ₂ ) has not been known.

In order to solve the problems of the prior art, the present invention provides a dimeric compound formed from a fluorescent emission compound and a phosphorescent compound, a monomolecular molecular oxygen photosensitizer composition containing the compound, and a method for producing the compound.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

A first aspect of the invention provides a compound represented by formula 1:

[Chemical Formula 1]

.

.

The second aspect of the present invention provides a photosensitive composition comprising a compound represented by Formula 1 above.

A third aspect of the present invention provides a process for preparing a compound represented by Formula 1, which comprises a process represented by the following Reaction Scheme 1:

[Reaction Scheme 1]

.

.

According to embodiments of the present invention, the development of a dyad compound that can be easily prepared as a dimer of a fluorescent emission compound and a phosphorescent emission compound enables selective sensitization and visualization of singlet oxygen ( ¹ O ₂ ) Is advantageous. In particular, the dual photofunction of the dimer compound means that the dimer compound exhibits a ratiometric optical luminescence reaction to ¹ O ₂ and also possesses high photocell toxicity, It is possible to induce the death of cancer cells of ¹ O ₂ -induced mammals through the dual photoperiodicity of the dimeric compound, and to simultaneously perform the photometric luminescence real-time monitoring of the rate of cancer cell death have. By using the dimeric compound of the present invention as such, the dimeric compound can be used as a biomedical imaging reagent and can also be usefully used for cancer treatment.

1A to 1D show the changes in the optical luminescence spectra of a mixture of Irpbt-TEG and C314-TEG, which are reference compounds of the dimer compound (EJ1) of the present invention, under 365 nm light irradiation according to one embodiment of the present invention, FIGS. 1B and 1C show the results of adding 100 mM histidine or 100 mM NaN _3, which is a ¹ O ₂ scavenger, to the mixture of Irpbt-TEG and C314-TEG, .
Figure 2a shows the time-dependent change in ultraviolet-visible light absorption spectra of solutions containing Irpbt-TEG and C314-TEG, reference compounds of the dimer compound (EJ1) of the present disclosure, according to one embodiment of the present application, 2b is, ¹ O _2, according to one embodiment of the present application - is a result of measuring the rate constant for the bimolecular reaction medium.
FIG. 3A shows ultraviolet-visible light absorption spectra of a solution containing the dimeric compound (EJ1) of the present invention in the case of performing 365 nm light irradiation according to an embodiment of the present invention, and FIG. And shows the change in the Nesss spectrum.
Figure 4 shows the proposed mechanism based on ¹ O ₂ reactions with respect to phosphorescence and fluorescence behavior of Irpbt-TEG and C314-TEG, reference compounds of the dimeric compound (EJ1) of the present invention.
Figure 5a shows the change in the optical luminescence spectrum of EJ1 following exposure to 365 nm light in the absence (black) and presence (gray) of 100 mM histidine, which is a ¹ O ₂ scavenger according to ^one embodiment of the invention And Figure 5b shows the time course of the ratio of the optical luminescence intensity at 460 nm and 561 nm of EJ1 in the absence and presence of 10 mM and 100 mM histidine, according to one embodiment of the invention.
Figure 6 is a graph showing that the photoluminescence response of EJ1, according to one embodiment of the present application, is selective for ¹ O ₂ among various active oxygen species.
FIG. 7A is a clear sky image (top) and a ratio scale image (bottom) shown to visualize endogenously generated ¹ O ₂ in accordance with one embodiment of the invention, and FIG. Lt; RTI ID = 0.0 > lambda < / RTI > profiles of the micrographs of the cells shown below.
FIG. 8A is a graph showing MTT cell viability assessment results performed to quantify the phototoxicity of EJ1 according to one embodiment of the present invention, and FIG. 8B is a graph showing the results of MTT cell viability assay performed on EJ1-treated cells according to one embodiment of the present invention Is a photoluminescent micrograph showing the induction of apoptosis in the early stage and nectrosis in the later stage.
Figure 9 shows that the photocell toxicity of EJ1 in accordance with one embodiment of the present invention is related to triggering intracellular mitochondrial dysfunction by singlet oxygen ( ¹ O ₂ ) generated by light irradiation (colocalization) experiment results.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

Throughout this specification, when a member is "on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure.

The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination (s) thereof " included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to these embodiments and examples and drawings.

A first aspect of the invention provides a compound represented by formula 1:

[Chemical Formula 1]

.

.

A second aspect of the present invention provides a photosensitive composition comprising a compound represented by formula 1:

[Chemical Formula 1]

.

.

In one embodiment of the present invention, the compound represented by Formula 1 may be a dimer compound formed from a phosphorescent emitting compound represented by Formula 2 below and a phosphorescent emitting compound represented by Formula 3 below, Can:

(2)

,

,

(3)

.

.

Herein, the dimer compound represented by Formula 1 is referred to as "EJ1 ", and the compound that is one of the reference compounds of the dimer compound represented by Formula 1 as the fluorescence emitting compound represented by Formula 2 is" C314- TEG "and the compound which is one of the reference compounds of the dimer compound represented by Formula 1 as the phosphorescent compound represented by Formula 3 is referred to as" Irpbt-TEG " have.

In one embodiment of the present invention, the compound represented by Formula 1 may be, but is not limited to, a compound acting as a photosensitizer for singlet oxygen ( ¹ O ₂ ).

In one embodiment of the present invention, the compound represented by Formula 1 may exhibit a selective ratiometric photoluminescence reaction or a photocytotoxicity reaction to singlet oxygen ( ¹ O ₂ ) , Or singlet oxygen ( ¹ O ₂ ), both of which have a dual optical functionality indicating both a ratio scale optical luminescence response and a photo cytotoxic response.

In one embodiment of the present invention, the photo-cytotoxicity reaction may include, but is not limited to, a method wherein the compound represented by Formula 1 induces cell death under light irradiation.

In one embodiment of the present invention, the photocell toxicity may include inducing apoptosis by inducing intracellular mitochondrial dysfunction by singlet oxygen ( ¹ O ₂ ) generated by light irradiation, But may not be limited.

In one embodiment of the invention, the cell may comprise, but is not limited to, cancer cells.

In one embodiment of the present invention, the photosensitizer composition comprises a ratio scale of the compound represented by Formula 1 generated during the progress of cell death due to singlet oxygen ( ¹ O ₂ ) under the light irradiation, by recording the change Sons, it may be used for the content of ¹ O ₂ ⁽¹ O ₂ therapeutic dosage) for the treatment to monitor in real time, but may not be limited thereto.

A third aspect of the present invention provides a process for preparing a compound represented by the following formula (1), comprising a step represented by the following Reaction Scheme 1:

[Reaction Scheme 1]

,

,

[Chemical Formula 1]

.

.

The manufacturing method of the third aspect of the present invention can be more specifically explained by way of [Scheme 1] of the following [Embodiment], but it is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are given for the purpose of helping understanding of the present invention, but the present invention is not limited to the following Examples.

[ Example ]

1. Materials and Experimental Methods

In this example, commercially available chemicals were used as received. Anhydrous CH ₂ Cl ₂ was purchased from Aldrich and used without further drying. Phosphate buffer (PBS) was purchased from Sigma. The PBS buffer solution was prepared in (10 mM) Milli-Q water (18.2 MΩ · cm) and the pH was adjusted using standard KOH (45 wt%, Aldrich) and HCl (1 N, Fluka) solution. The EJ1 stock solution was dissolved in DMSO (Aldrich) at a concentration of 10 mM and stored frozen. The solution was thawed prior to spectrometric determination. Generally, 30 μM of EJ1 stock solution was provided in 3.0 mL of PBS buffer solution at pH 7.4 containing 10 vol% DMSO. Therefore, the EJ1 concentration in the mixture of PBS and DMSO was 10 [mu] M. DMSO was introduced to inhibit particulate form. A 1 cm x 1 cm fluorometer cell with a Teflon stopper (Hellma) was used for optical measurements. All steady-state photophysical measurements were performed at 37 ° C unless otherwise noted. O ₂ ^{· -,} OCl ^-, a solution of t -BuO · and · OH were prepared by employing a conventional method [EWMiller, AEAlbers, A.Pralle, EYlsacoff , CJChang, J.Am. Chem . Soc . 2005, 127, 16652; Y. Chen, W. Guo, Z. Ye, G. Wang, J. Yuan, Chem . Commun . 2011, 47, 6266]. t- BuOOH and H ₂ O ₂ were purchased from Aldrich and used as received. To generate the light radiation ¹ O ₂ of (1 pH 7.4 in PBS:: DMSO = volume ratio 9) bubbling the solution with-methylene blue (Sigma, 1 μM) or Irpbt-TEG O ₂ containing (10 μM) . ^{The 1} H and ¹³ C { ¹ H} NMR spectra were collected on a Bruker Ultrashield 500, 400, and 300 plus NMR spectrometer and a Jeol JNM-AL300 spectrometer. Chemical shifts were used based on TMS. Electrospray ionization mass spectra were obtained by introducing a Thermo Electrons Co., Ltd Finnigan LCQ Advantage Max spectrometer.

2. Synthesis of EJ1 and reference compounds

In this example, EJ1 and the reference compounds Irpbt-TEG and C314-TEG were synthesized through a multistep process of scheme 1 below:

[Scheme 1]

.

.

Scheme 1 above illustrates a base-promoted aromatic substitution of chlorine and triethylene glycol in 5-chloro-1,10-phenanthroline followed by coumarin 343 (i.e., the free-acid form of coumarin 314) (Mitsunobu) condensation of distant hydroxyl groups. The [mu] -chloride Ir (III) 2 molecular sieve precursor of the 2-phenylbenzothiazole cyclometalating ligand was prepared according to the standard Nonoyama protocol [M. Nonoyama, Bull. Chem . Soc . Jpn . , 1974, 47, 767-768]. As a result, continuous (subsequent) substituted by chelation and NH ₄ PF ₆ in the modified 1,10-phenanthroline ligands are intended to give a dimer (EJ1) in a yield of 56%. Structural analysis data obtained from polynuclear NMR [ ¹ H NMR and ¹³ C { ¹ H} NMR], high resolution mass spectrometry (MS), and elemental analysis were consistent with the proposed structure. The specific synthesis process of each intermediate substance shown in Scheme 1 above and the analysis result thereof are described in detail below.

(One) Scheme 1 of  Synthesis of Compound 1

KOH (3.66 g, 65.2 mmol) was added to a stirred DMSO solution (100 mL) of triethylene glycol (9.79 g, 65.2 mmol). After 1 hour at room temperature, 5-chloro-1,10-phenanthroline (2.80 g, 13.0 mmol) was introduced into the solution stirred at 60 ° C for an additional 40 hours . The reaction mixture was poured onto 200 mL EtOAc and washed thoroughly with water (300 mL x 5 times) to remove DMSO. The organic layer was collected, which was dried over anhydrous MgSO _4, was concentrated in vacuo. The silica gel column purification gave a yellow liquid in 67% yield by performing an increase of the polarity of the effluent from 100% CH ₂ Cl ₂ to CH ₂ Cl ₂ : CH ₃ OH = volume ratio 9: 1.

^{1 H NMR (400 MHz, CDCl} 3) δ: 3.47 (m, 2H), 3.64 (m, 2H), 3,75 (m, 4H), 3.83 (m, 2H), 4.07 (t, J = 4.8 Hz , 2H), 4.44 (t, J = 4.8 Hz, 2H), 6.98 (s, 1H), 7.56 (dd, J = 8.2, 4.0 Hz, 1H), 7.65 (dd, J = 8.4, 4.4 Hz, 1H) , 8.72 (d, J = 6.4 Hz, 1 H), 9.0 (m, 1 H). ¹³ C { ¹ H} NMR (100 MHz, CDCl ₃ ) δ: 61.76, 68.06, 69.64, 70.53, 71.10, 72.64, 101.88, 122.72, 123.31, 123.65, 129.20, 131.09, 134.75, 142.98, 146.79, 148.01, 150.73, 152.16. MS (positive mode) m / z 329.20 ([M + H] < ⁺ >). HR MS (FAB ⁺ , m -NBA): Calcd for C ₁₈ H ₂₁ N ₂ O ₄ ([M + H] ⁺ ), 329.1501; found, 329.1502.

(2) Scheme 1 of  Synthesis of Compound 2

(0.400 g, 1.21 mmol), coumarin 343 (0.379 g, 1.33 mmol), diisopropyl azodicarboxylate (0.368 g, 1.82 mmol) and triphenylphosphine (0.477 g, 1.82 mmol) were added to a 100 mL round bottom flask. Anhydrous CH ₂ Cl ₂ (70 mL) was transferred to a flask using a glass syringe under an Ar atmosphere. The reaction mixture was stirred at room temperature for 48 hours and then concentrated under reduced pressure. The silica gel column purification was carried out using a CH ₂ Cl ₂ : CH ₃ OH effluent in which the volume ratio of CH ₃ OH was increased from 2 vol% to 10 vol%. A brown solid was obtained in 17% yield.

^{1 H NMR (300 MHz, CDCl} 3) δ: 1.87 (m, 4H), 2.58 (t, J = 6.3 Hz, 2H), 2.70 (t, J = 6.3 Hz, 2H), 3.22 (m, 4H), 3.83 (m, 4H), 3.88 (t, J = 5.2 Hz, 2H), 4.09 (t, J = 5.3 Hz, 2H), 4.38 (t, J = 4.4 Hz, 2H), 4.49 (t, J = 4.6 Hz, 2H), 6.71 (s , 1H), 6.91 (s, 1H), 7.53 (dd, J = 8.1, 4.6 Hz, 1H), 7.61 (dd, J = 8.3, 4.4 Hz, 2H), 8.08 (dd J = 8.2, 1.7 Hz, 1H), 8.26 (s, 1H), 8.64 (dd, J = 8.3, 1.7 Hz, 1H), 9.00 (dd, J = 4.4, 1.9 Hz, 1H), 9.15 J = 4.3, 1.8 Hz, 1H). ¹³ C ( ¹ H) NMR (125 MHz, CDCl ₃ ) ?: 20.05, 21.02, 27.26, 29.71, 30.90, 41.02, 49.80, 50.21, 64.00, 68.22, 29.29, 29.64, 70.93, 101.76, 107.37, 119.16, 122.64, 123.17, 126.87, 128.52, 129.24, 131.06, 132.15, 134.74, 142.82, 146.57, 147.72, 148.57, 149.31, 150.64, 152.20, 153.35, 158.56, 164.40. HR MS (FAB ⁺ , m -NBA): Calcd for C ₃₄ H ₃₄ N ₃ O ₇ ([M + H] ⁺ ), 596.2347; found 596.2401

(3) Scheme 1 of  Synthesis of Compound EJ1

A Cl-bridged dinuclear iridium precursor of 2-phenylbenzothiazole ([(μ-Cl) Ir (pbt) ₂ ] ₂ , pbt = 2-phenylbenzothiazolate) was prepared according to the procedure reported previously [ H. Woo, S. Cho, Y. Han, W.-S. Chahe, Y. Yu, W. Nam, J. Am. Chem . Soc . 2013, 135, 4771]. A 50 mL 2-inlet round bottom flask was filled with 2 (47 mg, 80 μmol) and [(μ-Cl) Ir (pbt) ₂ ] ₂ (52 mg, 40 μmol). Anhydrous CH ₂ Cl ₂ (30 mL) was added into the flask and the reaction mixture was refluxed for 15 h under an Ar atmosphere. NH ₄ PF ₆ (130 mg, 800 μmol) was then slowly added to the solution at 0 ° C and the solution was stirred at room temperature for an additional 4 hours. The remaining NH ₄ PF ₆ was removed by passing the reaction mixture through a glass filter, after which the reaction mixture was concentrated in vacuo. Flash column chromatography on silica gel and further purification using preparative TLC technique provided 56% yield of orange powder.

^{1 H NMR (400 MHz, CD} 3 CN) δ: 1.78 (m, 4H), 2.53 (m, 4H), 3.25 (m, 4H), 3.64-3.71 (br m, 6H), 4.00 (m, 2H) , 4.21-4.31 (br m, 2H) , 4.38 (m, 2H), 5.82 (d, J = 8.4 Hz, 1H), 5.92 (d, J = 8.4 Hz, 2H), 6.48 (t, J = 7.6 Hz , 2H), 6.72 (s, 1H), 6.84 - 6.95 (br m, 4H), 7.15 (t, J = 7.2 Hz, 2H), 7.24 (td, J = 7.2, 0.8 Hz, 2H), 7.36 (s J = 8.0, 4.8 Hz, 1H), 7.88 (dd, J = 8.4, 5.2 Hz, 1H), 7.94 (m, 4H), 8.14 ( s, 1H), 8.25 (dd, J = 5.2, 1.2 Hz, 1H), 8.43 (dd, J = 8.4, 1.2 Hz, 1H), 8.46 (dd, J = 5.2, 1.2 Hz, 1 H), 8.85 (dd, J = 8.4, 1.6 Hz, 1 H). ¹³ C { ¹ H} NMR (100 MHz, CD ₃ CN) 隆: 20.61, 20.80, 21.79, 22.28, 27.98, 50.42, 50.91, 55.40, 64.77, 69.86, 69.90, 70.11, 70.67, 71.49, 71.61, 104.41, , 107.35, 108.06, 120.64, 126.97, 127.02, 127.10, 127.85, 128.01, 128.80, 129.12, 129.68, 129.80, 132.57, 132.63, 132.78, 132.95, 134.48, 135.06, 138.46, 141.69, 141.75, 144.83, 149.27, 149.78, 150.07 , 150.20, 150.29, 150.79, 150.92, 153.05, 154.46, 154.75, 158.83, 165.04, 182.70. MS (positive mode) m / z 1208.07 ([M-PF ₆ ] ^< ^{+ &} gt ^; ). HR MS (FAB ⁺ , m -NBA): Calcd for C ₆₀ H ₄₉ IrN ₅ O ₇ S ₂ ([M-PF ₆ ] ⁺ ), 1208.2703; found, 1208.2698.

(4) Scheme 1 of Irpbt - TEG  synthesis

Irpbt-TEG was prepared according to the same procedure as that for synthesizing EJ1, except that 1 was used instead of 2. Obtained as a brown powder in a yield of 41%.

^{1 H NMR (300 MHz, CDCl} 3) δ: 3.61 (m, 2H), 3.68-3.79 (br m, 7H), 4.06 (m, 2H), 4.58 (m, 2H), 5.83 (d, J = 2.7 Hz, 2H), 6.49 (t , J = 11 Hz, 2H), 6.93 (m, 5H), 7.14 (t, J = 10 Hz, 2H), 7.61 (s, 1H), 7.76-7.87 (br m, 7H), 8.17 (d, J = 5.1 Hz, 1H), 8.38 (dd, J = 7.1, 2.0 Hz, 1H), 8.72 (dd, J = 12, 2.0 Hz, 1H), 9.05 (dd, J = 11 , 2.2 Hz, 1H). MS (positive mode) m / z 999.13 ([M + NaCl] < ⁺ >).

(5) Scheme 1 of  C314- TEG  synthesis

2 was employed and only triethylene glycol was used instead of 1. As a white powder in 34% yield.

^{1 H NMR (400 MHz, CDCl} 3) δ: 1.98 (m, 4H), 2.76 (t, J = 6.5 Hz, 2H), 2.87 (t, J = 6.4 Hz, 2H), 3.33 (m, 3H), 2H), 3.64 (m, 2H), 3.64 (m, 2H), 3.64 (m, 2H) ). ¹³ C { ¹ H} NMR (100 MHz, CDCl ₃ ) δ: 15.39, 20.33, 21.43, 50.14, 50.15, 64.29, 66.86, 69.45, 70.07, 70.92, 106.76, 107.02, 107.76, 119.37, 127.24, 148.79, 149.52, 158.84, 164.41. MS (positive mode) m / z 440.16 ([M + Na] < ^{+ &} gt ^; ).

3. Methods for characterization of EJ1 and reference compounds

(1) Normal-state ultraviolet-visible light absorption measurement

Ultraviolet-visible light absorption spectra were collected using an Agilent Cary 300 spectrometer. Unless otherwise noted, 10 μM or 50 μM solutions were used for the measurement.

(2) normal-state light Luminesence  Measure

The optical luminescence spectrum was obtained at 37 DEG C using a Quanta Master 400 scanning spectrofluorimeter. The temperature was maintained by introducing water circulation. 10 μM solution was used for the measurement. The excitation wavelength of EJ1, C314-TEG and Irpbt-TEG was 300 nm. Optical luminescence quantum yield (PLQYs; photoluminescence quantum yields) was relatively measured according to the following standard _{equation: PLQY = PLQY ref x (I} / I ref) x (A ref / A) x (n / n ref) 2 , Where A is the absorbance at the excitation wavelength, I is the integrated optical luminescence intensity, and n is the refractive index of the solvent. 9,10-Diphenylanthracene in a toluene solvent was used as an external reference (PLQY _ref = 1.00).

(3) Optical Luminesence  Life measurement

A 50 μM solution in Ar-saturated CH ₃ CN was used to measure the optical luminescence lifetime. Photo-luminescence decay trace (decay trace), the time-linked single-photon-on the basis of the counting (time-correlated single-photon- counting) described using FluoTime 200 device (PicoQuant, Germany) Pico-(λ _obs = 484 nm) and nanoseconds (? _Obs = 600 nm) pulsed laser excitation. A 377 nm diode laser (PicoQuant, Germany) was used as the excitation source. The optical luminescence signals at 484 nm and 600 nm were obtained through a monochromator with an automated power plant. The optical luminescence attenuation profiles were analyzed using a single (λ _obs = 600 nm) or double (λ _obs = 484 nm) index attenuation model embedded in OriginLab, OriginPro 8.0 software.

(4) Cell culture

Modified Eagle's medium (-containing Dulbecco; (FBS fetal bovine serum) HeLa , A549, MCF7, and RAW 264.7 cells, 50 μg mL ^-1 penicillin and 10% fetal bovine serum under 5% CO ₂ and 37 ℃ Dulbecco ' s modified Eagles ' s medium; DMEM; 4.5 g L < ^-1 > glucose, L- glutamic acid, and sodium pyruvate;

(5) Endogenous in RAW 264.7 cells Generated ^One O ₂ of  Visualization

RAW 264.7 cells were treated with 100 ng mL- ¹ lipid polysaccharide (LPS, Sigma; 16 hours) and 400 U mL- ¹ interferon (IFN) -γ (Sigma; 2.5 hours) to stimulate ¹ O ₂ production. As a control, RAW 264.7 cells pretreated with LPS and IFN-y were incubated in 10 mM histidine for 15 minutes. The 10 mM EJ1 stock solution was prepared in DMSO (biotech grade, Sigma). A aliquot of the EJ1 stock solution was taken and diluted in fresh DMEM to a concentration of 5 μM prior to cell treatment. RAW 264.7 cells pretreated with LPS and IFN-y were washed twice with DPBS (Thermo Scientific). EJ1 solution in DMEM (2 mL) was added to the culture dish, and the cells were incubated for 15 minutes in a humidified incubator. As a result, RAW 264.7 cells were treated with 10 nM phorbol 12-myristate 13-acetate (PMA, Sigma; 30 min). The optical luminescence microscope images were obtained through two light emitting channels (λ _obs = blue channel of 410 nm to 499 nm, λ _obs = 507 nm to 691 nm red channel). Ratio scale images were constructed by dividing red channel images into blue channel images. Zeiss ZEN software was used for image processing. The lambda profile of the optical luminescence signals was recorded through 32 light emitting channels and was constructed using OriginLab, Origin 8.0 software.

(6) Light treatment

HeLa, MCF7, and A549 cells were sprayed on a poly-L-lysine coated microscope culture dish one day prior to treatment. Cells were treated with 5 μM EJ1 for 15 min in a humidified incubator at 37 ° C and 5% CO ₂ . After two washes with DMEM, fresh DMEM was supplemented. Light irradiation was performed inside the incubator using a stripe of commercial blue LED.

(7) MTT  evaluation

MTT assays were performed according to the manufacturer's protocol (Promega, CellTiter 96® non-radioactive cell proliferation assay). HeLa, A549, and MCF7 cells were plated on 96-well plates one day prior to the experiment. Cells were incubated with EJ1 or cisplatin for 1 hour (0.1 μM to 200 μM, 100 μL / well) and incubated for an additional 1 hour under blue light illumination or dark conditions. 15 [mu] L of MTT reagent was added to each well, and the cells were further incubated at 37 [deg.] C for 3 hours. Eventually, 100 μL of stop solution was injected into the wells. After incubation for an additional 1 hour, the absorbance at 570 nm was recorded using a VersaMax microplate reader.

(8) Cell death apotosis ) detection Of kit  Visualization

HeLa cells preincubated with EJ1 were treated with the Annexin V-FITC apoptosis detection kit (Sigma) according to the manufacturer's protocol. In summary, 5 μL of annexin V-FITC and 10 μL of propidium iodide solution were directly injected into the culture medium. Cells were incubated in a humid incubator for 30 minutes. Plasma membrane stains due to Annexin V-FITC and nuclear staining due to propidium iodine were visualized via lambda _obs = 498 nm to 560 nm and lambda _obs = 568 nm to 673 nm, respectively.

(9) Position correspondence ( colocalization ) Experiments

HeLa cells were treated with 2 μM of DRAQ5 for 30 min, treated with 100 nM of LysoTracker Deep Red (molecular probe) for 25 min, or treated with 1 μM ER-Tracker Red (BODIPY® TR Glibenclamide, ) For 25 min, or treated with 200 nM MitoTracker Deep Red FM (molecular probe) for 30 min. After two washes with DPBS, the cells were incubated with 5 μM EJ1 for 15 min. Cells were washed with fresh DMEM and DMEM supplemented with culture dishes. Fluorescence microscopy image visualization was performed through the following emission of a channel: DRAQ5, λ _obs = 630 nm to 650 nm; _{LysoTracker Deep Red, λ obs = 647} nm to 668 nm; ER-Tracker Red _{,? Obs} = 587 nm to 615 nm; MitoTracker Deep Red FM,? _Obs = 644 nm to 665 nm; EJ1, [lambda] _obs = 410 nm to 499 nm and 507 nm to 691 nm. Acquisition and image analysis were performed using Zeiss ZEN software.

(10) DFT / TD- DFT  Calculation

Quantum chemistry calculations based on density function theory (DFT) were performed using the Gaussian 09 program. Coumarin 314 in methyl ester form was employed as a model structure for the active site of EJ1. The iminium form of the model compound was constructed based on the MS results. The geometry optimization and single point calculation for the model structure was performed using Becke's 3-parameter B3LYP exchange-locality matching functions and 6-311 + G (d, p) based set . A polarizable continuum model (CPCM), expressed as a parameter for water, was applied during the geometric optimization step. Frequency calculations were performed continuously to evaluate the stability of the convergence. For TD-DFT computation, the functionals and base set used for geometry optimization are applied to the optimized geometry. A polarizable continuum model (CPCM) with parameters for water was applied to calculate the solvation effect. Thirty of the least significant and triplet states were calculated and analyzed. The simulation of ultraviolet-visible light absorption spectra was performed using the GaussSum program.

4. Characterization results of EJ1 and reference compounds

The molecular structure of EJ1 was constructed based on fluorescent coumarin 314 (C314) and phosphorescent Ir (III) complex (Irpbt). Such strategies for integrating fluorophore and phosphorescent metal complexes have been used for photocatalysis, electroluminescence, and hypoxia and RNA imaging, but for dimeric compounds such as EJ1 herein and for their use as photosensitizers, This is the first time I've tried.

In this example, continuous exposure (30 W) to 365 nm light (4 W) of an air-equilibrium aqueous solution containing 10 μM Irpbt-TEG and 10 μM C314-TEG (PBS buffer pH 7.4: DMSO = 9: 1 by volume) Min) cause rate scales to cause fluorescence changes. In this connection, in the present FIG. 1a to 1d is as shown a light luminescence spectrum changes under 10 μM Irpbt-TEG and 10 μM 365 nm light irradiation of a mixture of C314-TEG, Figure 1a O ₂ - equilibrium PBS: DMSO = volume ratio of 9: 1 solution and 37 ° C, and FIG. 1b shows the change of the optical luminescence spectra at a PBS: DMSO = volume ratio of 9: 1 solution containing 100 mM histidine 1c shows the change of the optical luminescence spectra under the condition of PBS: DMSO = volume ratio 9: 1 solution containing 100 mM NaN ₃ , and Fig. 1d shows the change of Ar-saturated PBS: DMSO = volume ratio 9: 1 solution and And the change in the optical luminescence spectrum at 37 ° C. As can be seen in FIG. 1 a, the photoluminescence spectrum of the mixture showed the generation of new bands in the blue emission region (λ _ems = 436 nm). This reaction was not observed in aqueous solutions containing either 10 μM C314-TEG or 10 μM Irpbt-TEG. As seen on Figure 1b and Figure 1c, adding to the 100 mM histidine or 100 mM NaN ₃ ¹ O ₂ scavenger (scavenger) in a mixture of Irpbt-TEG and C314-TEG, greatly weakening the fluorescence change . Also, as can be seen in FIG. 1d, de-aeration by Ar saturation caused the reaction to hardly occur. A faster rate of fluorescence reaction was observed in an O ₂ -saturated aqueous DMSO solution containing D ₂ O (DMSO: D ₂ O = 1: 9 by volume). These results strongly indicate that the fluorescence reaction is due to the photosensitive ¹ O ₂ .

Meanwhile, Causes within the molecular structure of the ¹ O ₂ reaction can be determined by electron spray ionization mass spectrometry (positive mode) measurement using either Irpbt-TEG or C ³ O ₁₄ irradiated in the presence of methylene blue used as a ¹ O ₂ photoresist on clinical treatments -TEG solution. ≪ / RTI > In both cases, it has been found that the julolidine moiety in C314-TEG is converted to the iminium form. In this regard, the conversion of the julolididine moiety to the iminium form at the C314-TEG moiety of the dimeric compound EJ1 of the present disclosure can be represented by the following reaction scheme 2:

[Reaction Scheme 2]

.

.

In this regard, the ultraviolet-visible light absorption spectrum showed the disappearance of the absorption band as a function of the transfer of charge transfer from the julolidine to the carbonyl group (λ _abs = 449 nm). This may determine through the Figures 2a and 2b, specifically, Figure 2a O ₂ containing 30 μM C314-TEG and 10 μM Irpbt-TEG under irradiation with light (λ _ex = 365 nm) at 37 ℃ - saturated (PBS: DMSO = volume ratio 9: 1) of the aqueous buffer solution (PBS: DMSO = volume ratio 9: 1) showing the corresponding decrease in absorbance at 449 nm, FIG. 2b shows the results of measuring the rate constants of the ¹ O ₂ -mediated biomolecule reaction. In Figures 2a and 2b, the rate of the 449 nm band of 10 μM C314-TEG photobleached to light was measured at 9.6 × 10 ^-4 s ^-1 in the presence of 10 μM Irpbt-TEG, The rate was linearly increased with the concentration of Irpbt-TEG. Similar absorption changes were observed with the addition of (NH ₄ ) ₂ Ce ^IV (NO ₃ ) ₆ , a powerful mono-electron oxidant. Similar to the fluorescence reactions, this result indicated the occurrence of α-dehydrogenation by ¹ O ₂ . Quantum chemical calculations based on time-dependent density function theory also supported this hypothesis. Previous studies have reported similar ¹ O ₂ -mediated oxidation of 7- (dialkylamino) coumarin compounds. The chemical processes underlying the reaction are the mechanism proposed by Baciocchi and co-workers [E. Bacciocchi, TDGiacco and A. Lapi , Org . Lett . , 2004, 6, 4791-4794. This can be accomplished by extracting an a-hydrogen atom from a tertiary amine by ¹ O ₂ , followed by the addition of a single electron to the corresponding perhydric acid group (HOO go to) is associated to those for obtaining H ₂ O _2. In fact, in this embodiment, H ₂ O ₂ could be detected through iodine titration.

3A and 3B, light irradiation for 10 μM EJ1 solution (pH 7.4: PBS buffer: DMSO = 9: 1 by volume, at 37 ° C.) at 365 nm resulted in an interaction with ¹ O ₂ Resulting in the disappearance of the charge-transfer absorption band of the coumarin moiety ([lambda] _abs = 445 nm; see the ultraviolet-visible light absorption spectrum change of Fig. 3a), the absorption change is accompanied by the generation of a new luminescent band at 441 nm (See the change of the optical luminescence spectrum in Fig. 3B). These spectral features were the same as those measured for C314-TEG.

On the other hand, FIG. 4 shows the proposed mechanism based on the ¹ O ₂ reaction, in which the phosphorescence intensity (λ _ems = 526 nm) of the Ir (III) complex was increased, indicating that the triplet- It is the result of inhibition of triplet energy transfer and the increase in the phosphorescence lifetime of the Ir (III) complex component (λ _obs = 600 nm) from 2.44 μs to 3.80 μs supports this mechanism.

In addition, the total photoluminescence quantum yield (phi _PL ) was increased from 0.015 to 0.10. The extent of the quantitatively evaluated rate scale luminescence change as a ratio of the optical luminescence intensity at 460 nm and 561 nm ( I ₄₆₀ / I ₅₆₁ ) was increased from 0.0048 to 2.16. The I ₄₆₀ / I ₅₆₁ value was reduced in proportion to the concentration of added histidine, which was as expected for the ¹ O ₂ reaction. In this regard, FIG. 5A shows the change in the optical luminescence spectrum of 10 μM EJ1 according to the absence of 100 mM histidine ( ¹ O ₂ scavenger) (black) and exposure to 365 nm light (30 min) And FIG. 5B shows the temporal change in the ratio of the optical luminescence intensity at 460 nm and 561 nm of EJ1 in the absence and presence of 10 mM and 100 mM histidine, wherein the experimental conditions were PBS: DMSO = And a volume ratio of 9: 1 and 37 [deg.] C.

To On the other hand, as identified in Table 1 in, EJ1 exhibited a high quantum yield in both the ¹ O ₂ dose (dosimetric) reaction in (0.98) and after (1.00) for the optical sensitization of ¹ O ₂ (φ _Δ ). This small difference in φ _Δ is beneficial for sustained ¹ O ₂ generation and is due to the slow rate of dose response of the coumarin moiety. The value of [ _{Delta] [Delta] [Delta} ] is kept high for a sustained exposure to the excitation beam, which is due to the excellent light stability of the Irpbt moiety. Specifically, the photophysical data of EJ1 and reference compound before and after it has ¹ O ₂ The reaction was listed for Table 1:

[Table 1]

In the above Table 1, superscript a is 10 μM (PBS: DMSO = 9: 1 by volume), b is the excitation wavelength 330 nm, c is 9,10-diphenylanthracene ; PhMe) standard material, d is the optical luminescence lifetime (parenthesized values represent the observed wavelength), e is the singlet oxygen generation quantum yield, methylene blue reference material (φΔ = 0.52) (1,3-diphenylisobenzofuran) ¹ O ₂ substrate.

On the other hand, the fact that the photoluminescence reaction of EJ1 is selective for ¹ O ₂ among various active oxygen species is due to the fact that the photoluminescence reaction of 10 μM EJ1 (EJ1 or methylene blue) FIG. 6 shows the result of the analysis. As it will be confirmed from FIG. ^{_{6, O 2 · - (1.0}} mM KO 2), ClO - (1.0 mM NaOCl), 1.0 mM t-BuOOH, 1.0 mM H 2 O 2, t-BuO · (1.0 mM FeSO 4 + the handle 100 μM t-BuOOH), and ^{· OH (1.0 mM FeSO 10 μM} EJ1 that compete with the active oxygen species comprising a _{4 + 100 μM H 2 O 2} ) was filled to make any change in the spectrum. In contrast, photoluminescence rate scale responses were measured by irradiating a mixture of EJ1 and methylene blue with a wavelength of 510 nm, which was not absorbed by EJ1. The results of these experiments clearly show that EJ1 is ¹ O ₂ -selective.

Such an EJ1 After confirming selective detection and detection capabilities for ¹ O ₂ , experiments were performed in this example to confirm the bioavailability of such properties for mammalian cells. A number of mammalian cells capable of penetrating EJ1 have been found, including HeLa, A549, MCF7, and RAW 264.7 macrophages. Fluorescent microscope images of RAW 264.7 cells treated with 5 μM EJ1 for 15 minutes in a humid incubator (5% CO ₂ , 37%) showed fluorescence microscopic images in the blue region (λ _obs = 507 nm to 691 nm) (&_Amp;_thetas; _obs = 410 nm to 499 nm). In contrast, 100 ng mL- ¹ lipopolysaccharide (LPS, 16 hours) and 100 UmL- ¹ interferon (IFN) -γ (2.5 hours) followed by 10 nM phorbol 12- myristate 13- PMA, 0.5 hr) stimulated blue light emission from RAW 264.7 cells. The rate scale images constructed using the blue and red images shown in Figure 7a helped visualize endogenously generated ¹ O ₂ . Specifically, FIG. 7a shows the effect of the endogenously generated RAW 264.7 cells on stimulation with 100 ng mL ^-1 LPS, 100 U mL ^-1 IFN- ?, and 100 ng mL ^-1 PMA (scale bar 50 μm) as a visualization of ¹ O _2, an image of the top of the specified field image (bright field) image, and the image at the bottom of the blue channel image of the red channel image (λ _obs = 507 nm to 691 nm) as a ratio scale image (λ _obs = 410 nm to 499 nm). Significant attenuation of the ratio scale responses in Figure 7a was observed by treating cells with 10 mM histidine, a ¹ O ₂ scavenger, confirming that the reactions were from ¹ O ₂ . In addition, the spectral changes in the lambda profiles of the micrographs of the cells shown in Figure 7b further supported what was observed in the microscopic images.

On the other hand, EJ1 was cytotoxic to cancer cells under light irradiation. The results of MTT cell viability assay performed for the quantification of photocell toxicity are shown in FIG. 8A. Specifically, ICs for HeLa cells treated with 0.2 to 200 [mu] M EJ1 for 1 hour and continuous light irradiation with blue LED for an additional hour in a humidified incubator (5% CO ₂ , 37 ° C) ₅₀ values [IC ₅₀ (persons); (Red graph of FIG. 8A), and IC ₅₀ values for HeLa cells treated with 0.2-200 μM EJ1 for 1 hour and dark for an additional hour in a humidified incubator (5% CO ₂ , 37 ° C.) [IC ₅₀ (cancer); Black graph of Fig. 8A] was measured. In addition, IC ₅₀ values (blue graph of FIG. 8A) derived from cisplatin under dark conditions and IC ₅₀ values (red graph of FIG. 8A) derived from the photoproduct of EJ1 under light irradiation are also shown in FIG. 8A . As a result of the MTT evaluation in FIG. 8A, an IC ₅₀ (person) value of 0.45 μM was obtained, while an IC ₅₀ (cancer) value of cells incubated in darkness as a control group was as high as 50 μM. The IC ₅₀ (persons) was smaller than the value obtained from cisplatin (IC ₅₀ = 2.8 μM), which demonstrated the cytotoxic potential of EJ1 under light irradiation. An IC ₅₀ (n) value of 0.48 μM indicates an assessment of the EJ1 photoproduct, indicating that the cytotoxicity remains high and even when the ¹ O ₂ -dose response is complete, it remains high. The photocell toxicity index (PI), defined as IC ₅₀ (cancer) / IC ₅₀ (persons), reached 110. Similar PI values were obtained for A549 (PI = 217) and MCF7 (PI = 51) cells. PI values were comparable to those obtained from an Ir (III) complex containing a pyrene-containing Ru (II) complex or a carbene ligand of a N-heterocycle or poly (ethylene glycol).

In addition, the application of annexin V-FITC (apoptosis marker) and propidium iodide (necrosis marker) to EJ1-treated HeLa cells shows the mechanism by which photo-induced cell death occurs Respectively. In this regard, FIG. 8b shows the results obtained by photoirradiation and apoptosis detection kits (Annexin V-FITC and propidium iodine (PI)) of HeLa cells incubated with 5 占 퐉 EJ1 as optical luminescent micrographs 8b is a ratio scale image and the red channel image of EJ1 (? _Obs = 507 nm to 691 nm) is divided into a blue channel image of EJ1 (? _Obs = 410 nm to 499 nm) ( _Obs = 498 nm to 560 nm) and PI (red, lambda _obs = 568 nm to 673 nm) luminescent images, while the bottom image is an overlay of anexin V- (bright field) image. In the experiment in which Figure 8b (scale bar = 50 μm) was derived, cells were exposed to the blue LED for the indicated time before imaging. As can be seen in Figure 8b, green staining of plasma membranes, representing annexin V-FITC, suggested apoptosis predominates in early stages of apoptosis. In addition, additional light irradiation on EJ1-treated cells ultimately led to necrotic cell death, which was labeled as nuclear iodine as propidium iodide. In addition, EJ1 was preferentially localized in mitochondria relative to other cellular organelles, such as nuclei, lysosomes, and endoplasmic reticulum, as can be seen in FIG. Specifically, FIG. 9 shows the colocalization of EJ1 and cell organelle-specific staining. From left to right, DRAQ5 (nuclear staining), ER-Tracker Red (rough endoplasmic reticulum staining), LysoTracker Deep Red (lysosome staining) , And MitoTracker Deep Red FM (mitochondria staining). In addition, the upper part of FIG. 9 shows the overlapping images of EJ1 (green) and cell organ-specific staining (red) optical luminescence microscopy, and the lower part shows corresponding loci scattering points. Referring to the results of FIG. 9, photo-induced cell death may be closely related to dysfunction of mitochondria triggered by photo-generated ¹ O ₂ . The progression of photo-induced apoptosis was also tracked by measuring the rate-scale optical luminescence responses of EJ1. As shown in FIG. 8B, induction of apoptosis and necrosis correlates with the accumulation level of ¹ O ₂ , which is as quantified by the ratio scale image. These results indicate that EJ1 has potential use in controlled photodynamic therapy applications. In this regard, it should be noted that Ir (III) complexes can perform ¹ O ₂ sensitization through a two-proton process without interference with the optical excitation wavelengths for imaging.

The foregoing description of the disclosure is exemplary, It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention .

Claims (10)

(1)
compound:
[Chemical Formula 1]

.
A compound represented by the following general formula (1)
Photosensitizer composition:
[Chemical Formula 1]

.
3. The method of claim 2,
Wherein the compound represented by Formula 1 is a dimer compound formed from a phosphorescent emitting compound represented by Formula 2 below and a phosphorescent emitting compound represented by Formula 3 below:
(2)

,
(3)

.
3. The method of claim 2,
Wherein the compound represented by Formula 1 serves as a photosensitizer for singlet oxygen ( ¹ O ₂ ).
3. The method of claim 2,
The compound represented by Formula 1 may exhibit a selective ratiometric photoluminescence reaction or a photocytotoxicity reaction to singlet oxygen ( ¹ O ₂ ), or a singlet oxygen ( ¹ O ₂ ) having dual photoprotective properties that exhibit both a ratio scale photoluminescence response and a photo cytotoxic response.
6. The method of claim 5,
Wherein the photocell toxic reaction comprises that the compound represented by Formula 1 induce apoptosis of cells under light irradiation.
6. The method of claim 5,
Wherein the photocell toxicity comprises inducing apoptosis by inducing intracellular mitochondrial dysfunction by singlet oxygen ( ¹ O ₂ ) generated by light irradiation.
8. The method according to claim 6 or 7,
Wherein said cells comprise cancer cells.
The method according to claim 6,
The ratio scale of the compound represented by the formula (1) generated during the progress of the cell death by the singlet oxygen ( ¹ O ₂ ) under the light irradiation was recorded so that the ¹ O ₂ content ( ¹ ) < / ^RTI > ( ₂ ).
1. A process for the preparation of a compound of formula < RTI ID = 0.0 >
A process for producing a compound represented by the following formula (1)
[Reaction Scheme 1]

,
[Chemical Formula 1]

.

Images