KR102572743B1 - Compound, composition including the same, photosensitizer and composition for diagnosis or therapy of tumors including the composition, and photodynamic therapy using the composition for diagnosis or therapy of tumors - Google Patents

Abstract

Disclosed are a compound, a composition containing the compound, a photosensitizer containing the composition, a composition for diagnosing or treating tumors, and a photodynamic treatment method using the composition for diagnosing or treating tumors. The compound may be represented by Formula 1 below:
<Formula 1>
In Formula 1, R _1a , R _{1b ,} R ₂ , and R ₃ are each as disclosed in the specification.

Description

A compound, a composition including the same, a photosensitizer and a composition for diagnosing or treating tumors including the composition, and a photodynamic treatment method using the composition for diagnosing or treating tumors {Compound, composition including the same, photosensitizer and composition for diagnosis or therapy of tumors including the composition, and photodynamic therapy using the composition for diagnosis or therapy of tumors}

It relates to a compound, a composition containing the same, a photosensitizer and a composition for diagnosing or treating tumors including the composition, and a photodynamic treatment method using the composition for diagnosing or treating tumors.

Photodynamic therapy (PDT) is a medical technology approved in many countries for the treatment of various types of cancer and other diseases. For photodynamic therapy (PDT), a photosensitizer (PS), light, and oxygen are required. That is, PS is activated under light irradiation, and the excited photosensitizer interacts with oxygen molecules to generate cytotoxic reactive oxygen species (ROS), and oxidizes biomolecules to kill cancer cells. .

Among photosensitizers (PS), boron dipyrromethene (BODIPY) PS has excellent stability and physical and chemical properties and is being developed. However, PS with heavy atoms introduced into BODIPY has concerns about toxicity, shortened triplet excitation lifetime, and cost. Therefore, it is necessary to develop PS without introducing heavy atoms.

From this point of view, there is a need for a compound having a novel structure, a composition containing the compound, a photosensitizer containing the composition, a composition for diagnosing or treating tumors, and a photodynamic treatment method using the composition for diagnosing or treating tumors. .

One aspect is to provide a novel compound that does not introduce heavy atoms.

Another aspect is to provide a composition comprising the compound or a pharmaceutically acceptable salt thereof.

Another aspect is to provide a photosensitizer comprising the composition.

Another aspect is to provide a composition for diagnosing or treating tumors comprising the composition.

Another aspect is to provide a photodynamic treatment method using the composition for diagnosing or treating tumors.

According to one aspect,

A compound represented by Formula 1 is provided:

<Formula 1>

In Formula 1,

R _1a and R _1b may be each independently a deuterium (-D), a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, or a combination thereof;

R ₂ and R ₃ are each independently hydrogen, deuterium (-D), a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, -N(Q ₁ )(Q ₂ ), -N ⁺ (Q ₁ )(Q ₂ )(Q ₃ ), or combinations thereof;

Q ₁ to Q ₃ may be each independently hydrogen, heavy hydrogen (-D), a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, or a combination thereof.

According to other aspects,

the aforementioned compound or a pharmaceutically acceptable salt thereof; and solvent, acid, base, and buffer solutions; A composition comprising at least one selected from is provided.

According to another aspect,

A photosensitizer comprising the aforementioned composition is provided.

According to another aspect,

A composition for diagnosing or treating tumors, including the aforementioned composition, is provided.

According to another aspect,

contacting the above-described composition for diagnosing or treating tumor with a subject of a non-human mammal;

allowing time for the composition for diagnosing or treating tumors to be distributed in target cells; and

There is provided a photodynamic therapy method comprising the step of irradiating light to a target cell region of the object.

A novel compound in which heavy atoms are not introduced into the compound represented by Formula 1 according to one aspect may be provided. A composition containing the compound or a pharmaceutically acceptable salt thereof and a photosensitizer containing the composition generate active oxygen by light irradiation and can be used for fluorescence imaging and photodynamic therapy. The composition can be used for diagnosing or treating a tumor. A photodynamic therapy method may be provided using a composition including the compound.

1a is a chemical structure and synthesis route of a compound according to one embodiment.
Figure 1b is a schematic diagram of a process for synthesizing a micellar nanoparticle composition comprising a compound or a pharmaceutically acceptable salt thereof according to one embodiment.
1c is a DLS measurement result and a TEM image of Example 1 of a composition (Example 1) and Comparative Example 1 according to one embodiment.
1d is a result of measuring UV-vis absorption spectrum and PL spectrum of Example 1 and Comparative Example 1.
1e is a result of measuring ROS generating ability of Example 1 and Comparative Example 1.
1f is a fluorescence intensity measurement result of Example 1 according to a two-photon excitation wavelength.
1g is a fluorescence intensity measurement result of Example 1 according to the irradiation time of the two-photon excitation wavelength.
Figure 2a is a fluorescence microscope image before and after treatment of Example 1 in cancer cells and normal cells, respectively.
Figure 2b is a fluorescence microscopy image before/after treatment of Example 1 in the ROS probe (DCFH-DA) and the apoptotic cell marker (PI), respectively.
Figure 2c is the result of analyzing the viability of HeLa cells treated with Example 1 at various concentrations.
3 is a result of measuring the ROS generating ability of HeLa cells treated with DCFH-DA and/or Example 1.
Figure 4a is an overlay image and a brightfield image of HeLa cells treated with Example 1 and Hoechst 33342.
Figure 4b is a result of measuring the viability of cells irradiated at different laser powers after treatment with Example 1.
4C is a correlation between Example 1 and laser output.
Figure 4d is a result of measuring cell viability after light excitation irradiation at 780 nm.
Figure 5a is a 3D image of ROS generation of HeLa spheroids treated with DHR123 (dihydrorhodamine123).
Figure 5b is a 3D image of ROS generation of HeLa spheroids treated with Example 1.
5c is a fluorescence image of HeLa spheroids treated with DHR123.
5d is a fluorescence image of HeLa spheroids treated with Example 1.

Hereinafter, with reference to the accompanying drawings, a compound according to one embodiment of the present invention, a composition containing the same, a photosensitizer and a composition for diagnosing or treating tumors including the composition, and a light using the composition for diagnosing or treating tumors The dynamic treatment method will be described in detail. The following is presented as an example, and the present invention is not limited thereby, and the present invention is only defined by the scope of the claims to be described later.

In this specification, the term "include" is used to indicate that other components may be added or/or intervened, not excluded, unless otherwise stated.

The term "combination thereof" as used herein means a mixture or combination with one or more of the recited components.

In the present specification, "pharmaceutically acceptable salt" is a concentration that has a relatively non-toxic and harmless effect on patients, and any organic or inorganic compound represented by Formula 1 or the like at which the side effects caused by the salt do not reduce the beneficial effect. means appendicitis. For these salts, inorganic and organic acids can be used as free acids, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, and fumarin as organic acids. Acids, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesul For example, phonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid may be used. In addition, these salts include alkali metal salts (sodium salt, potassium salt, etc.) and alkaline earth metal salts (calcium salt, magnesium salt, etc.) and the like. For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, bibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, malic Eight, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharide Lates, stearates, succinates, tartrates, tosylates, trifluoroacetates, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salts and the like may be included, among which hydrochloride or trifluoroacetate may be used.

In this specification, the term "nanoparticle" refers to a structural material having a size of 1 to 100 nanometers (nm) and exhibiting phenomena that are difficult to see in micro-sized structures.

A compound according to one embodiment may be represented by Formula 1 below:

<Formula 1>

In Formula 1,

R _1a and R _1b may be each independently a deuterium (-D), a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, or a combination thereof.

R ₂ and R ₃ are each independently hydrogen, deuterium (-D), a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkenyl group, a substituted or unsubstituted C ₁ -C ₂₀ alkoxy group, -N(Q ₁ )(Q ₂ ), -N ⁺ (Q ₁ )(Q ₂ )(Q ₃ ), or combinations thereof;

Q ₁ to Q ₃ may be each independently hydrogen, heavy hydrogen (-D), a substituted or unsubstituted C ₁ -C ₂₀ alkyl group, or a combination thereof.

For example, R _1a and R _1b may each independently be deuterium (-D), a substituted or unsubstituted C ₁ -C ₅ alkyl group, or a combination thereof.

For example, R ₂ and R ₃ are each independently hydrogen, heavy hydrogen (-D), a substituted or unsubstituted C ₁ -C 5 alkyl group, a substituted or unsubstituted C 1 -C ₅ alkenyl group, a substituted or unsubstituted C ₁ -C ₅ alkenyl group, an unsubstituted C ₁ -C ₅ alkoxy group, -N(Q ₁ )(Q ₂ ), -N ⁺ (Q ₁ )(Q ₂ )(Q ₃ ), or a combination thereof;

Q ₁ to Q ₃ may be each independently hydrogen, heavy hydrogen (-D), a substituted or unsubstituted C ₁ -C ₅ alkyl group, or a combination thereof.

For example, R ₂ and R ₃ are each independently hydrogen, heavy hydrogen (-D), a substituted or unsubstituted C ₁ -C ₅ alkyl group, or a group represented by Formulas 3-1 to 3-3 below. can:

In Chemical Formulas 3-1 to 3-3,

Me is a methyl group,

* is a binding site with a neighboring atom.

For example, R ₂ and R ₃ may be identical to each other.

For example, the compound represented by Formula 1 may be one of the following compounds 1 to 5:

Since the compound represented by Formula 1 includes R _1a and R _1b , it is possible to improve the emission ability and ROS generating ability of the compound in the far-infrared region by blocking the rotating non-radiative decay passage. In addition, the phenoxazine group in the compound represented by Chemical Formula 1 can realize an efficient ISC process by controlling ΔEST due to its steric volume.

A composition comprising micellar nanoparticles formed by conjugating the compound or a pharmaceutically acceptable salt thereof with an amphiphilic polymer has excellent light stability and biocompatibility. In addition, cancer cells can be effectively removed by generating intracellular ROS even under low-toxic green light (1 photon), and ROS can be generated in both aqueous solutions and living cells even under 2 photons.

Therefore, the compound represented by Chemical Formula 1 can be used as a photosensitizer having excellent emission and improved ROS generating ability, and as a composition for diagnosing or treating tumors.

A composition according to another embodiment, the above compound or a pharmaceutically acceptable salt thereof; and

It may be a composition containing at least one selected from a solvent, an acid, a base, and a buffer solution.

the aforementioned compound or a pharmaceutically acceptable salt thereof; And water, a buffer solution, or an organic solvent; may include a composition containing.

For example, the composition may have an energy gap (ΔE _S1T1 ) between the lowest singlet excited state and the lowest triplet state of the compound or a pharmaceutically acceptable salt thereof of less than 1.00 eV. For example, the energy gap ΔE _S1T1 may be greater than 0.00 eV and less than 0.50 eV.

For example, in the composition, the compound or a pharmaceutically acceptable salt thereof can generate singlet oxygen in an aqueous or organic solution.

For example, in the composition, the compound or a pharmaceutically acceptable salt thereof may emit light by intramolecular charge transfer in a far-infrared or near-infrared wavelength region.

For example, the composition may be micelle nanoparticles formed by conjugating the compound or a pharmaceutically acceptable salt thereof with an amphiphilic polymer.

For example, the average size of the micellar nanoparticles may be 60 nm or more and 150 nm or less. For example, the average size of the micellar nanoparticles is 60 nm or more and 140 nm or less, 60 nm or more and 130 nm or less, 60 nm or more and 120 nm or less, 60 nm or more and 110 nm or less, 60 nm or more and 100 nm or less, 60 nm or more and 90 nm or less 70 nm or more and 140 nm or less, 70 nm or more and 130 nm or less, 70 nm or more and 120 nm or less, 70 nm or more and 110 nm or less, 70 nm or more and 100 nm or less, 80 nm or more and 140 nm or less, 80 nm or more and 130 nm or less, 80 nm or more and 120 nm or less, 80 nm or more and 110 nm or less , or 80 nm or more and 100 nm or less.

A photosensitizer according to another embodiment may include the above-described composition.

A composition for diagnosing or treating tumors according to another embodiment may include the above-described composition.

For example, in the composition for diagnosing or treating tumors, the tumor is breast cancer, kidney cancer, testicular cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, vaginal cancer, fallopian tube cancer, rectal cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, small intestine cancer, pancreatic cancer, oral cancer, melanoma, or sarcoma.

A photodynamic therapy method according to another embodiment includes contacting the above-described composition for diagnosing or treating tumor with a non-human mammalian subject;

allowing time for the composition for diagnosing or treating tumors to be distributed in target cells; and

It may include irradiating light to a target cell region of the object.

The photodynamic therapy method may exhibit advantages such as spatiotemporal selectivity, non-invasiveness, and reduced side effects.

In this specification, substituents are derived by exchanging one or more hydrogen atoms or functional groups in an unsubstituted parent group. Unless otherwise stated, when a functional group is considered "substituted", it means that the functional group is an alkyl group of 1 to 40 carbon atoms, an alkenyl group of 2 to 40 carbon atoms, an alkynyl group of 2 to 40 carbon atoms, a cycloalkyl group of 3 to 40 carbon atoms It means that it is substituted with one or more substituents selected from an alkyl group, a cycloalkenyl group having 3 to 40 carbon atoms, and an aryl group having 7 to 40 carbon atoms. When a functional group is described as "optionally substituted", it is meant that the functional group may be substituted with the substituents described above.

In the present specification, a and b of "carbon number a to b" mean the carbon number of a specific functional group. That is, the functional group may include carbon atoms from a to b. For example, "an alkylene group having 1 to 4 carbon atoms" refers to an alkylene group having 1 to 4 carbon atoms, that is, -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -(CH ₃ ) ₂ C-, -CH ₂ CH ₂ CH ₂ CH ₂ -, -CH ₂ CH ₂ CH(CH ₃ )- and -(CH ₃ ) ₂ C-.

As used herein, the term “alkyl” refers to branched or unbranched aliphatic hydrocarbons. In one embodiment, an alkyl group can be substituted or unsubstituted. The alkyl group includes a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group, a hexyl group, a cyclopropyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, etc. It is not necessarily limited to these, and each of these may optionally be substituted or unsubstituted. In one embodiment, the alkyl group may have 1 to 6 carbon atoms. For example, the alkyl group having 1 to 6 carbon atoms may be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, etc., but is not necessarily limited thereto.

As used herein, the term "alkenyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon double bond. Non-limiting examples of alkenyl groups include vinyl, allyl, butenyl, isopropenyl, or isobutenyl.

As used herein, the term "alkynyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon triple bond. Non-limiting examples of alkynyl groups include ethynyl, butynyl, isobutynyl, isopropynyl, and the like.

As used herein, the term "cycloalkyl" refers to a monovalent saturated hydrocarbon monocyclic. Non-limiting examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and the like.

In this specification, the term "heterocycloalkyl" means a monovalent monocyclic containing at least one heteroatom selected from N, O, Si, P and S as a ring-forming atom. Non-limiting examples of the heterocycloalkyl group include a 1,2,3,4-oxatriazolidinyl group (1,2,3,4-oxatriazolidinyl), a tetrahydrofuranyl group, a tetrahydrothiophenyl group, and the like. there is.

As used herein, the term "cycloalkenyl" means monovalent monocyclic, having at least one double bond in the ring, or having no aromaticity. Non-limiting examples of the cycloalkenyl group include a cyclopentenyl group, a cyclohexenyl group, a cycloheptenyl group, and the like.

As used herein, the term "heterocycloalkenyl" refers to a monovalent monocyclic containing at least one heteroatom selected from N, O, Si, P and S as a ring-forming atom, wherein at least one double bond is present in the ring. means to have Non-limiting examples of the heterocycloalkenyl group include a 4,5-dihydro-1,2,3,4-oxatriazolyl group, a 2,3-dihydrofuranyl group, a 2,3-dihydrothiophenyl group, and the like. can be heard

As used herein, the term "aryl" refers to an aromatic ring whose ring backbone contains only carbon, a ring system (ie, two or more fused rings sharing two adjacent carbon atoms), or a plurality of aromatic rings. is a single bond, -O-, -S-, -C(=O)-, -S(=O) ₂ -, -Si(R _a )(R _b )-(R _a and R _b are independently of each other an alkyl group having 1 to 10 carbon atoms), a halogen-substituted or unsubstituted alkylene group having 1 to 10 carbon atoms, or a ring connected to each other by -C(=O)-NH-. When the aryl group is a ring system, each ring in the system is aromatic. For example, the aryl group includes, but is not limited to, a phenyl group, a biphenyl group, a naphthyl group, a phenanthrenyl group, a naphthacenyl group, and the like. The aryl group may be substituted or unsubstituted.

As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic organic compound containing one or more heteroatoms selected from N, O, P, or S, and the remaining ring atoms being carbon. do. The heteroaryl group may include, for example, 1-5 heteroatoms and 5-10 ring members. The S or N may be oxidized and have various oxidation states.

Non-limiting examples of “heteroaryl” include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxa Diazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl group, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5- Thiiadiazolyl, 1,3,4-thiadiazolyl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, oxazol-2-yl, oxazol-4- 1, oxazol-5-yl, isoxazol-3-yl, isoxazol-4-yl, isoxazol-5-yl, 1,2,4-triazol-3-yl, 1,2, 4-triazol-5-yl, 1,2,3-triazol-4-yl, 1,2,3-triazol-5-yl, tetrazolyl, pyrid-2-yl, pyrid-3- 1, 2-pyrazin-2-yl, pyrazin-4-yl, pyrazin-5-yl, 2-pyrimidin-2-yl, 4-pyrimidin-2-yl, or 5-pyrimidin-2-yl, etc. can be heard

Examples and comparative examples of the present invention are described below. However, the following examples are merely examples of the present invention and the present invention is not limited to the following examples.

[Example]

All experiments were performed under an inert nitrogen atmosphere. Anhydrous-grade solvents (Aldrich), spectral-grade solvents and all commercially available reagents were used as received. A deuterated solvent from Cambridge Isotope Laboratories was used. NMR spectra were recorded on a Bruker AM 300 (300.13 MHz for ¹ H, 75.48 MHz for ¹³ C) spectrometer at ambient temperature. Chemical shifts (ppm) were relative to external Me ₄ Si ( ¹ H, ¹³ C). Mass measurements were recorded on a Synapt G2-HDMS mass spectrometer.

Reaction Scheme 1 for the compound prepared by Synthesis Example 1 is as follows:

<Reaction Scheme 1>

Synthesis Example 1: Synthesis of BDP-6 (Compound 1)

1) Synthesis of Intermediate 1 (PXZ-CHO)

Fenoxazine (0.5g, 2.73mmol), 4-bromo-2,6-dimethylbenzaldehyde (0.64g, 3.00mmol), Pd(OAc) ₂ (0.03g, 0.14mmol), t-Bu ₃ PHBF ₄ (0.12 g, 0.41mmol) and K ₂ CO ₃ (1.12g, 8.10mmol) were dissolved in toluene (15ml) to obtain a solution. The solution was purged with N ₂ and stirred at 110 °C for 24 hours to obtain a reactant. The reaction was extracted with DCM (20 ml x 3). The extract was dried over anhydrous Na ₂ SO ₄ and the solvent was evaporated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography using dichloromethane / hexane (1: 1, v / v) as an eluent to obtain intermediate 1 (0.82 g, 95.27%).

¹ H NMR (CDCl ₃ ): δ 10.67 (s, 1H), 7.11 (s, 2H), 6.71 - 6.59 (m, 6H), 5.98 (d, J = 7.2Hz, 2H), 2.66 (d, J = 0.8Hz, 6H).

¹³ C NMR (75 MHz, CDCl ₃ ): δ 192.7, 144.4, 144.0, 132.6, 132.3, 131.4, 129.7, 123.3, 121.8, 115.6, 113.4, 20.6.

2) Synthesis of Compound 1 (BDP-6)

Intermediate 1 (0.5g, 1.59mmol) and 2,4-dimethylpyrrole (0.38g, 3.96mmol) were dissolved in anhydrous dichloromethane (40ml) under a nitrogen atmosphere to obtain a solution. A drop of trifluoroacetic acid (TFA) was added to the solution and stirred at room temperature for 3 hours to obtain a reactant. After confirming that the aldehyde completely disappeared using TLC, p-chloroanil (0.39 g, 1.59 mmol) was added to the reaction mixture to obtain a mixture. The mixture was stirred for 1 h, Et ₃ N (TEA) (3.09 mL, 2.25 g, 22.20 mmol) was added, and after 30 min BF ₃ OEt ₂ (2.74 mL, 3.15 g, 22.20 mmol) was added slowly. And the solvent was removed under reduced pressure to obtain a residue. The residue was purified by chromatography using dichloromethane/hexane (3:2, v/v) as an eluent to obtain compound 1 (BDP-6, 0.64 g, 76%).

¹ H NMR (CDCl ₃ ): δ 7.16 (s, 2H), 6.72-6.58 (m, 6H), 6.04 (s, 2H), 5.91 (dd, J = 7.7, 1.6 Hz, 2H), 2.59 (s, 6H), 2.23 (s, 6H), 1.55 (s, 6H).

¹³ C NMR (75 MHz, CDCl ₃ ): δ 155.9, 141.6, 139.8, 139.1, 134.7, 130.1, 123.2, 121.3, 112.9, 19.7, 14.7, 13.5. ESI-MS calcd. for C ₃₃ H ₃₁ BF ₂ N ₃ O [M+H] ⁺ : 534.2528, found: 534.2533.

Example 1: Synthesis of BDP-6 NPs

Compound 1 (BDP-6, 1.0 mg) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG (2000) biotin, 5 mg) were added in tetra After dissolving in hydrofuran (THF, 1 ml), it was slowly added dropwise to deionized water (10 ml). The mixture was stirred overnight to allow THF to evaporate. Then, the solution was filtered through a 0.20 μm disposable membrane to obtain a filtrate solution of BDP-6 NPs. After centrifuging the solution for 30 minutes, the supernatant was carefully removed with a pipette. 200 μL deionized water was added to the precipitate to obtain the BDP-6 NPs of Example 1.

Comparative Example 1: Synthesis of BDP-5 NPs

BDP-5 NP of Comparative Example 1 was obtained in the same manner as in Example 1, except that Compound A was used instead of Compound 1.

Analysis Example 1: DLS and TEM image measurement

Dynamic light scattering (DLS) was measured with a Nano-ZS (Malvern). TEM images were measured using a JSM-6700F field emission scanning electron microscope (JEOL).

1c is a DLS measurement result and a TEM image of Example 1 of a composition (Example 1) and Comparative Example 1 according to one embodiment. It can be seen that the composition of Example 1 is spherical and has an average size of about 90 nm.

Analysis Example 2: Measurement of UV-vis absorption and photoluminescence spectrum and ROS generating ability

UV-vis absorption and photoluminescence (PL) spectra were measured with Thermo Scientific Evolution 201 UV-vis spectrometer and FS-2 spectrophotometer (Scinco), respectively. A diluted sample solution (usually 10 μM) was prepared from a stock solution (1.0 mM) under ambient conditions. UV-vis and PL spectra of the solution (10 μM) were measured using a 1 cm quartz cuvette.

The ability to generate one-photon ROS was measured under green LED light irradiation in water using 2', 7'-dichlorofluorescein diacetate (DCFH-DA) as a probe. Two-photon ROS generating ability was measured under two-photon (TP) irradiation in an aqueous solution using DHR123 as a probe. A cuvette containing 10 μM BDP-6 NPs and 2.5 μM DHR123 was prepared by adding 30 μL of 1.03 mM BDP-6 NPs and 0.75 μL of 10 mM DHR123 to 2.97 mL H ₂ O. After measuring the initial fluorescence intensity of DHR123, the TP laser was exposed for 30 minutes. Changes in the increased fluorescence intensity after TP irradiation were measured by removing the initial fluorescence intensity of DHR123. The fluorescence intensity of DHR123 measured at 500 nm and the emission wavelength were recorded at 525 nm.

1d is a result of measuring UV-vis absorption spectrum and PL spectrum of Example 1 and Comparative Example 1.

Example 1 and Comparative Example 1 had a strong absorption peak centered at 500 nm. In particular, Example 1 had a maximum value at 620 nm and increased fluorescence intensity compared to Comparative Example 1. This is because the methyl group substituted at the ortho position of the phenylene group serves to block intramolecular rotation.

1e is a result of measuring ROS generating ability of Example 1 and Comparative Example 1.

It was found that Example 1 had a superior ROS generating ability than Comparative Example 1.

1f is a fluorescence intensity measurement result of Example 1 according to a two-photon excitation wavelength. 1g is a fluorescence intensity measurement result of Example 1 according to the irradiation time of the two-photon excitation wavelength.

TPs were irradiated at 10 nm intervals in the 740 nm to 840 nm wavelength range. Upon TP excitation at 100 mW power, rhodamine123 was formed and showed a strong emission signal at 526 nm. The response of DHR123 (dihydrorhodamine123) was the highest at 780 nm, and it was found that ROS were gradually generated when continuously exposed to TP for 160 minutes.

Analysis Example 3: Intracellular measurement

(1) Fluorescence image measurement

HeLa cells were cultured for 48 hours in Dulbecoo's Modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin (100 units mL ^-1 ). Before imaging, the cell culture medium was replaced with serum-free DMEM, then stained with a photosensitizer and cultured. Two-photon fluorescence images were obtained with a multiphoton and spectral confocal microscope (Leica TCS SP8 MP) using a x40 oil objective. The two-photon excited photosensitizer was excited at 780 nm with a mode-locked fs Ti:Sapphire laser source (Mai Tai HP) with 2.60 W output power corresponding to ~8.11 × 105 W cm ^-2 in the focal plane. The laser power delivered to the sample under these conditions was 3.73 mW.

Figure 2a is a fluorescence microscopy image of cancer cells (HeLa cells) and normal cells (WI38) before/after the treatment of Example 1, respectively. A red fluorescence signal was observed in cancer cells treated with Example 1, but almost no red fluorescence signal was observed in normal cells. Accordingly, it was found that the tumor targeting ability of Example 1 was excellent.

Figure 2b is a fluorescence microscopy image before/after treatment of Example 1 in the ROS probe (DCFH-DA) and the apoptotic cell marker (PI), respectively.

(2) Measurement of intracellular ROS generating ability

HeLa cells were stained with photosensitizer (5 μM) and DCHF-DA (20 μM) for 30 min. After incubation, cells were washed twice with serum-free DMEM. Cells were irradiated with green LED (20 min at 20/cm 2 ) or 780 nm TP light (0, 100, 200, 400 scans), respectively. The fluorescence intensity of DCHF-DA was measured after each TP scan. Imaging conditions for DCHF-DA were 488 nm excitation and 500–550 nm emission window.

Figure 2c is the result of analyzing the viability of HeLa cells treated with Example 1 at various concentrations. When not treated with Example 1, it was found that there was no cytotoxic effect upon irradiation with green LED. When treated with Example 1, it was found that the concentration of Example 1 had an IC ₅₀ (half-maximal inhibitory concentration) at 0.35 μM. This indicates that the composition of Example 1 can target cancer cells and improve the photodynamic therapy effect.

3 is a result of measuring the ROS generating ability of HeLa cells treated with DCFH-DA and/or Example 1.

HeLa cells treated with DCFH-DA showed no increase in fluorescence even when the number of TP scans increased, indicating insufficient DCF production. In HeLa cells treated with Example 1, it was found that Example 1 was distributed in the cytoplasm with constant fluorescence intensity. It was found that Example 1 was stable even under TP-induced PDT conditions. The fluorescence intensity of HeLa cells treated with both DCFH-DA and Example 1 increased as the number of TP scans increased, indicating that Example 1 efficiently generated ROS in living cells as the DCF intensity was improved.

(3) Hoechst 33442/PI analysis

HeLa cells were stained with photosensitizer (10 μM) and Hoechst 33342 (2 μM). After incubation, cells were washed twice with serum-free DMEM and irradiated with green LED (20 min at 20/cm 2 ) or TP light at 780 nm (0,100, 200, 300, 400, 500 scans). Cells were then stained with PI (10 μM) for 30 min. Imaging conditions were an emission window of 400 to 450 nm for Hoechst 33342 and excitation of 488 nm and an emission window of 605 to 625 nm for PI.

Figure 4a is an overlay image and a brightfield image of HeLa cells treated with Example 1 and Hoechst 33342. The region irradiated with TP showed fluorescence at PI (red spots) corresponding to apoptosis. As the number of scans increased, the fluorescence of Hoechst 33342 (blue spots) decreased and the number of PI-stained cells increased. In 500 scans, upon TP irradiation, cells treated with Example 1 are completely removed from the irradiated area, but in the non-irradiated area, it can be seen that there are live cells treated with Hoechst 33342, indicating TP induced PDT.

Figure 4b is a result of measuring the viability of cells irradiated at different laser powers after treatment with Example 1.

When the laser power was 6.3 mW, 50% of the cells were killed when TP was irradiated in 15 scans, and when the laser power was 2.4 mW, similar cell death was shown in 455 scans.

4C is a correlation between Example 1 and laser output.

Log (laser power ^-1 ) and log (LD ₅₀ ) showed linearity with a slope of 2.76 ± 0.38. This indicates that cell removal occurred upon TP irradiation through a nonlinear PDT process.

Figure 4d is a result of measuring cell viability after light excitation irradiation at 780 nm.

IC50 values for 100, 200, 400, and 500 scans were 8.3 μM, 5.6 μM, 4.1 μM, and 0.7 μM, respectively. This indicates that Example 1 exhibits an excellent PDT effect upon TP excitation.

(4) Cell spheroid image measurement

To make spheroids, HeLa cells were cultured for 48 hours in a 3D cell culture dish (MicroFIT). The cell culture medium was replaced with serum-free DMEM and incubated for 1 h in each PS (100 μM) and DHR123 (15 μM). After culturing, TP light of 780 nm was irradiated to measure ROS generating ability. After acquiring 3D spheroid fluorescence images by acquiring more than 100 section images using the xyz mode, each section image was combined using the LAS-X program. Imaging conditions for DHR123 were 488 nm excitation and 500–550 nm emission window.

Figure 5a is a 3D image of ROS generation of HeLa spheroids treated with DHR123 (dihydrorhodamine123).

Figure 5b is a 3D image of ROS generation of HeLa spheroids treated with Example 1.

5c is a fluorescence image of HeLa spheroids treated with DHR123.

5d is a fluorescence image of HeLa spheroids treated with Example 1.

5c and 5d, it can be seen that HeLa spheroids were stained with Example 1 and when TP excitation was performed at 780 nm, DHR123 exhibited bright green fluorescence, indicating that Example 1 effectively generated ROS in 3D cancer cell models upon TP irradiation. indicates that

From this, it can be seen that the compound according to one embodiment of the present invention can be used as a therapeutic composition for diagnosing or treating tumors.

Claims (16)

A compound represented by Formula 1 below:
<Formula 1>

In Formula 1,
R _1a and R _1b are each independently a C ₁ -C ₅ alkyl group;
R ₂ and R ₃ are each independently hydrogen, heavy hydrogen (-D), or a C ₁ -C ₅ alkyl group. delete delete delete According to claim 1,
Wherein R ₂ and R ₃ are the same as each other. According to claim 1,
The compound represented by Formula 1 is the following compound 1 or 2, a compound:

. The compound according to any one of claims 1, 5 and 6 or a pharmaceutically acceptable salt thereof; and
A composition for diagnosing or treating tumors comprising at least one selected from solvents, acids, bases, and buffer solutions,
The tumor includes breast cancer, kidney cancer, testicular cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, vaginal cancer, fallopian tube cancer, rectal cancer, lung cancer, stomach cancer, liver cancer, esophageal cancer, small intestine cancer, pancreatic cancer, oral cancer, melanoma, or sarcoma. To, a composition for diagnosing or treating tumors. According to claim 7,
An energy gap (ΔE _S1T1 ) between the lowest singlet excited state and the lowest triplet state of the compound or a pharmaceutically acceptable salt thereof is less than 1.00 eV, a composition for diagnosing or treating tumors. According to claim 7,
A composition for diagnosing or treating tumors, wherein the compound or a pharmaceutically acceptable salt thereof generates singlet oxygen in an aqueous solution or an organic solution. According to claim 7,
A composition for diagnosing or treating tumors, wherein the compound or a pharmaceutically acceptable salt thereof emits light by intramolecular charge transfer in the far-infrared or near-infrared wavelength region. According to claim 7,
The compound or a pharmaceutically acceptable salt thereof is a micellar nanoparticle formed by conjugation with an amphiphilic polymer, a composition for diagnosing or treating tumors. According to claim 11,
The average size of the micellar nanoparticles is 60 nm or more and 150 nm or less, a composition for diagnosing or treating tumors. A photosensitizer comprising the composition for diagnosing or treating tumors according to claim 7. delete delete contacting a non-human mammalian subject with the composition for diagnosing or treating tumors according to claim 7;
allowing time for the composition for diagnosing or treating tumors to be distributed in target cells; and
A photodynamic therapy method comprising: radiating light to a target cell region of the object.