KR102587700B1 - A compound having aggregation-induced emission properties and uses thereof - Google Patents

Abstract

It relates to compounds having aggregation-induced release properties and their uses. According to one aspect, the compound represented by Formula 1 not only does not fluoresce until it reaches the mitochondria and does not accumulate in lipophilic sites such as lipids, but also has aggregation-inducing properties in the near-infrared wavelength region, resulting in higher efficiency compared to background fluorescence. This is significantly higher. Through this, it can effectively target cancer cells, achieving efficient cancer treatment without toxicity or side effects. It has superior photostability and is not affected by changes in mitochondrial membrane potential, making it superior to existing fluorescent dyes. Therefore, the compound according to one aspect can be effectively used in fluorescence imaging, photodynamic diagnosis, and treatment.

Description

A compound having aggregation-induced emission properties and uses thereof}

It relates to compounds having aggregation-induced release properties and their uses.

Compared with chemotherapy, radiotherapy, and surgery, photodynamic therapy (PDT) is attracting attention as an ideal treatment strategy for malignant tumors because it can effectively solve the disadvantages of drug resistance, toxic side effects, and high cost. In the photodynamic therapy process, a photosensitizer (PS) is activated under light irradiation, and the excited photosensitizer interacts with oxygen molecules to generate reactive oxygen species (ROS) or radicals, which It can kill tumor cells by oxidizing biomolecules.

Recently, in the field of photodynamics, research on luminogen, AIEgen, based on Aggregation-Induced Emission (AIE) has attracted great interest. While most compounds show a tendency for their fluorescence to decrease when they aggregate in a low-concentration solution state, some specific compounds show higher photoemission efficiency in the aggregation or solid state than in the solution state, which is referred to as aggregation-induced emission. It is called a phenomenon.

However, most traditional AIEgens have limitations in that they exhibit undesirable aggregation in aqueous environments due to the “always on” fluorescence signal or do not guarantee activation efficiency in vivo . This is a serious obstacle to applications in biological imaging or phototherapy that require a high signal-to-noise ratio. In addition, most AIEgens have hydrophobic characteristics and tend to accumulate in lipophilic sites before reaching the target site, causing fluorescence due to spurious signals.

Therefore, there is a need for the development of AIEgen, which has high reactive oxygen species productivity and high photostability characteristics and can further increase the photodynamic treatment effect by increasing the accumulation rate at the target site.

The present inventors prepared AIEgen with excellent dispersibility in both lipophilic and hydrophilic systems through “step-by-step” molecular design, and the compound has amphiphilic properties in aqueous solution environments. It has "fluorescence-off" properties, can be dissolved, does not aggregate before binding to mitochondria, does not generate false positive signals due to unwanted aggregation, and successfully escapes from the lipophilic region of the cell. It was confirmed that it could come out. Through this, the present invention was completed by confirming that the compound can be usefully used in photodynamic therapy by overcoming the always-on fluorescence characteristic of traditional AIEgen and low targeting ability due to accumulation in lipophilic sites.

One aspect is to provide novel compounds or pharmaceutically acceptable salts thereof with aggregation-induced release properties.

Another aspect is to provide a photosensitizer containing the above compound or a pharmaceutically acceptable salt thereof.

Another aspect is to provide a composition for photodynamic diagnosis or treatment containing the above compound or a pharmaceutically acceptable salt thereof.

Another aspect is to provide a pharmaceutical composition for diagnosing or treating cancer containing the compound or a pharmaceutically acceptable salt thereof.

Another aspect is to provide a photodynamic diagnosis or treatment method using the compound or a pharmaceutically acceptable salt thereof.

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

All technical terms used in this specification, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art. In addition, the numerical values described in this specification are considered to include the meaning of “about” even if not specified.

In this specification, the term "include" is used to indicate that other components may be added or/and intervened, rather than excluding other components, unless specifically stated to the contrary.

As used herein, the term “combination thereof” means mixing or combining with one or more of the listed components.

As used herein, the term “interaction” may be direct or indirect, and may include direct binding or indirect binding, and the binding may be mediated by another molecule.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of Formula 1, etc., which is a concentration having an effective effect that is relatively non-toxic and harmless to patients, and side effects due to the salt do not reduce the beneficial efficacy of the compound represented by Formula 1, etc. It means any organic or inorganic addition salt of the compound represented by . For these salts, inorganic acids 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 fumarine can be used as organic acids. Acids, gluconic acid, methanesulfonic acid, glyconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethane sulfuric acid Fonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid, or malonic acid can be used. Additionally, these salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, bibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maly. ate, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharide Latex, stearate, succinate, tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salt, etc. may be included, and among these, it may be hydrochloride or trifluoroacetate.

In the present specification, a substituent is derived by exchanging one or more hydrogens with another atom or functional group in an unsubstituted mother group. Unless otherwise stated, when a functional group is considered to be “substituted,” it means that the functional group is an alkyl group with 1 to 40 carbon atoms, an alkenyl group with 2 to 40 carbon atoms, an alkynyl group with 2 to 40 carbon atoms, or a cyclo group with 3 to 40 carbon atoms. It means being 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 being “optionally substituted,” it means that the functional group may be substituted with the substituents described above.

In this specification, a and b of “carbon number a to b” refer to the carbon number of a specific functional group. That is, the functional group may include carbon atoms from a to b. For example, “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 a branched or unbranched aliphatic hydrocarbon. In one embodiment, the alkyl group may be substituted or unsubstituted. Alkyl groups include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, pentyl group, hexyl group, cyclopropyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group, etc. It is not necessarily limited to these, and each of them may or may not be optionally substituted. 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 to these.

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 “aryl” refers to an aromatic ring whose ring backbone contains only carbon, a ring system (i.e., two or more fused rings sharing two adjacent carbon atoms), or a plurality of aromatic rings. A single bond, -O-, -S-, -C(=O)-, -S(=O)2-, -Si(Ra)(Rb)-(Ra and Rb independently have 1 to 10 carbon atoms alkyl group), an alkylene group having 1 to 10 carbon atoms substituted or unsubstituted with halogen, or rings connected to each other by -C(=O)-NH-. If 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, phenyl group, biphenyl group, naphthyl group, phenanthrenyl group, naphthacenyl group, etc. 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 atom being carbon. do. The carbon number of the heteroaryl group is not particularly limited, but may have 2 to 60 carbon atoms. The heteroaryl group is, for example, a substituted or unsubstituted imidazolyl group, a substituted or unsubstituted triazolyl group, a substituted or unsubstituted tetrazolyl group, a substituted or unsubstituted oxadiazolyl group, or a substituted or unsubstituted group. oxatriazolyl group, substituted or unsubstituted cyatriazolyl group, substituted or unsubstituted benzimidazolyl group, substituted or unsubstituted benzotriazolyl group, substituted or unsubstituted pyridinyl group, substituted or unsubstituted Substituted pyrimidinyl group, substituted or unsubstituted triazinyl group, substituted or unsubstituted pyrazinyl group, substituted or unsubstituted pyridazinyl group, quinoline, substituted or unsubstituted isoquinoline, substituted or unsubstituted pro Thalazine, substituted or unsubstituted naphpyridine, substituted or unsubstituted quinoxaline, substituted or unsubstituted quinazoline, substituted or unsubstituted acridine, substituted or unsubstituted phenanthroline and substituted or unsubstituted It may be one or more structures selected from the group consisting of phenazine, or a combination thereof.

According to one aspect,

A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof is provided:

<Formula 1>

In Formula 1,

X can be O, S or N,

A may be a substituted or unsubstituted C1-C4 alkyl group or a substituted or unsubstituted C2-C4 alkenyl group,

CY ₁ may be an aryl amine group of C6-C20,

CY ₂ may be a cation of a heteroaryl group represented by the following formula (2).

<Formula 2>

In Formula 2,

R ₁ may be a C1-C10 alkyl phosphonium cation, and the phosphonium cation may be substituted with a substituted or unsubstituted C6-C20 aryl group,

R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ may each independently be hydrogen, a substituted or unsubstituted C1-C4 alkyl group, or a combination thereof.

For example, the substituted or unsubstituted aryl group of C6-C20 in the phosphonium cation of R ₁ of Formula 2 is C6-C30 substituted or unsubstituted by -CHO, -ORa, -NRa, -NHCORa, or -OCORa. It includes an aryl group, and Ra may be hydrogen, a C1-C20 alkyl group, or a C6-C20 aryl group.

R ₁ in Formula 2 may be a C1-C10 alkyl phosphonium cation represented by Formula 3 below:

<Formula 3>

In Formula 3 above,

R ₉ , R ₁₀ and R ₁₁ may each independently be a substituted or unsubstituted C6-C20 aryl group,

R ₁₂ may be a substituted or unsubstituted C1-C10 alkyl group,

* may be a portion connected to the nitrogen (N) element of Formula 2 above.

For example, the substituted or unsubstituted C6-C20 aryl group in R ₉ , R ₁₀ and R ₁₁ of Formula 3 is C6 substituted or unsubstituted with -CHO, -ORa, -NRa, -NHCORa, or -OCORa. It includes a -C30 aryl group, and Ra may be hydrogen, a C1-C20 alkyl group, or a C6-C20 aryl group.

In Formula 1, X may preferably be S. That is, the heterocycle containing X may be thiophene. The thiophene is CY ₁ in the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof; It may exist as a thiophene bridge connecting A and CY ₂ .

For example, CY ₁ may be triphenylamine (TPA). The triphenylamine is a hydrophobic portion in the compound and may be an electron donor.

In addition, A is a vinyl group, and CY ₂ may be a quinoline cation substituted with a C1-C10 alkyl phosphonium cation. The quinoline cation substituted with the alkyl phosphonium cation is a hydrophilic portion in the compound and may be an electron acceptor.

Preferably, CY ₂ may be a quinoline cation substituted with a pentyl triphenylphosphonium cation. Accordingly, the compound or a pharmaceutically acceptable salt thereof according to one embodiment may include a compound represented by Formula 4 below.

<Formula 4>

In the compound represented by Formula 4, the nitrogen (N) element of quinoline and the phosphorus (P) element of triphenylphosphine each have positive charges, so the entire compound may have a double positive structure. Due to these structural characteristics, the compound represented by Formula 4 can have excellent dispersibility in both lipophilic and hydrophilic systems based on its amphiphatic nature. Therefore, the compounds can be dissolved in aqueous solutions with desirable “fluorescence-off” properties, allowing them to remain non-aggregated before reaching the mitochondria. In addition, the compound represented by Formula 4 is 1) specifically accumulated in mitochondria within cancer cells as an alkyl phosphonium cation (e.g., pentyl triphenylphosphonium cation) is additionally introduced to the quinoline cation (synergistic effect due to double positive charge) ), 2) It may have near-infrared (NIR) emission ability due to a lower band gap than a molecule in which an alkyl phosphonium cation is not additionally introduced (e.g., TPA-S-Q).

The compound may have Aggregation-Induced Emission (AIE) properties.

Fluorescent substances that exhibit aggregation-induced emission have a chemical structure with rotational or vibrational freedom, so in low-concentration solutions, the molecules consume energy through rotational or vibrational movement rather than emitting energy as light. However, when the concentration of the substance increases and the molecules aggregate or crystallize, molecular movement is limited, resulting in a fluorescent material with high luminous efficiency. In one embodiment, the compound according to the above aspect has amphipathic properties, can be dissolved in an aqueous solution environment with “fluorescence-off” properties, and does not aggregate before binding to mitochondria, thereby causing unwanted aggregation. It was confirmed that no false positive signals were generated due to this and that it could successfully escape from the lipophilic region within the cell. Accordingly, the compound according to one aspect can overcome the always-on fluorescence characteristic of traditional AIEgen and the low targeting ability due to accumulation at lipophilic sites.

In the above compound, the triphenylphosphine group may target mitochondria within cancer cells.

Additionally, the compound may induce cancer cell death by generating reactive oxygen species (ROS) under light irradiation.

The reactive oxygen species may be singlet oxygen ( ¹ O ₂ ) and/or hydroxyl radical (·OH), but is not limited thereto.

The “cancer cell” refers to a cell that grows, divides, or proliferates abnormally, and may be used interchangeably with “tumor cell.”

According to different aspects,

A photosensitizer comprising the above-described compound or a pharmaceutically acceptable salt thereof is provided.

“Photosensitizer” is a chemical that changes oxygen molecules (O ₂ ) into reactive oxygen species such as singlet oxygen ( ¹ O ₂ ) when irradiated with light of a specific wavelength, creates new radicals, or It refers to a substance that creates new chemical species.

The range of the fluorescence excitation wavelength of the photosensitizer may be about 400 to 730 nm, preferably 430 to 700 nm, and more preferably 450 to 670 nm. Additionally, the range of the emission wavelength of the photosensitizer may be about 550 to 900 nm, preferably 580 to 880 nm, and more preferably 600 to 850 nm. Due to the luminescent properties, selective binding and/or accumulation in target tissues and/or cells is possible, which may enable in vivo or in vitro imaging.

The photosensitizer can generate reactive oxygen species (ROS) by irradiating light in a white light wavelength range of 400 to 700 nm, preferably 430 to 670 nm.

The photosensitizer is capable of fluorescence imaging and targeting mitochondria.

The photosensitizer may accumulate in cancer cells.

According to another aspect,

A composition for contrast medium containing the above-described compound or a pharmaceutically acceptable salt thereof is provided.

“Contrast agent” refers to a drug administered to the human body to make certain tissues or blood vessels visible during imaging tests or procedures. In one embodiment, it was confirmed that the above-described compound can emit fluorescence and, in particular, can specifically detect cancer cells. In particular, the above-mentioned compounds have no cytotoxicity to normal cells, enabling safe and meaningful diagnosis of cancer, and have toxicity to cancer cells, so they can achieve preventive or therapeutic effects against cancer.

When the above-described compound or a pharmaceutically acceptable salt thereof is used as a contrast agent, the range of the fluorescence excitation wavelength of the compound may be about 400 to 730 nm, preferably 430 to 700 nm, and more Preferably it may be 450 to 670 nm. Additionally, the range of the emission wavelength of the compound may be about 550 to 900 nm, preferably 580 to 880 nm, and more preferably 600 to 850 nm. The ranges of the fluorescence excitation wavelength and emission wavelength may change depending on the measurement target and may be adjusted differently depending on other substances included in the composition other than the above-mentioned compounds.

The composition may include a carrier and vehicle for contrast agents commonly used in the pharmaceutical field. These specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g. human serum albumin), buffering substances (e.g. various phosphates, glycine, sorbic acid, potassium sorbate, partial glycation of saturated vegetable fatty acids). hydride mixture), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, camberium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based matrix, polyethylene glycol, Including, but not limited to, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, or wool paper. The composition may further include lubricants, wetting agents, emulsifiers, suspending agents, excipients, diluents, or preservatives in addition to the above ingredients.

The composition can be prepared as an aqueous solution for parenteral administration, preferably using a buffered solution such as Hank's solution, Ringer's solution, or physically buffered saline. Aqueous injection suspensions can be supplemented with substances that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Additionally, the composition may be in the form of a sterile injectable preparation of a sterile injectable aqueous or oily suspension. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (e.g., solutions in 1,3-butanediol) in non-toxic, parenterally acceptable diluents or solvents. Vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils are typically used as solvents or suspending media. For this purpose, any non-volatile oil with little irritation, including synthetic mono- or diglycerides, can be used without limitation.

According to another aspect,

A photodynamic diagnostic or therapeutic composition comprising the above-described compound or a pharmaceutically acceptable salt thereof is provided.

“Photodynamic diagnosis or treatment” is a treatment that produces a therapeutic effect by administering a photosensitizer that reacts to light and then selectively accumulating light only in cells with a disease (e.g., cancer) when light of a specific wavelength is irradiated. means therapy.

The photodynamic diagnosis or treatment may be performed by irradiating near-infrared rays with a wavelength of 650 to 900 nm.

The composition may be prepared by adding the above-described compound or a pharmaceutically acceptable salt thereof to a solvent, buffer solution, or mixture thereof, and adding an acid and/or a base thereto. Additionally, the composition may additionally include other additives that can be used in the art. The contents of the solvent, acid, base, and buffer solution contained in the composition can be appropriately adjusted depending on the required performance. Alternatively, the composition may be mixed with a sample. The sample may be a biological sample containing one or more selected from microorganisms, cells, and tissues, but is not necessarily limited to these, and any sample that can be used as a biological sample in the art may be used. do.

The composition may contain 0.0001 to 50% by weight of the above-described compound or a pharmaceutically acceptable salt thereof based on the total weight, and may further include one or more active ingredients exhibiting the same or similar functions.

The composition can be formulated and used in various forms, such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injectable solutions, according to conventional methods to suit each purpose of use. It can be administered orally or through various routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Additionally, the composition may include one or more targeting ligands or moieties for targeting cancer tissue or target cells. Such targeting ligands may include small molecules (e.g., folate, dye, etc.), aptamers, antibodies, antibody fragments, compounds known to bind to specific receptors on the cell surface, etc., but are not limited thereto and are known in the art. Any known targeting ligand can be used without limitation.

According to another aspect,

A pharmaceutical composition for diagnosing or treating cancer comprising the above-described compound or a pharmaceutically acceptable salt thereof is provided.

In the present invention, “diagnosis” of cancer refers to confirming the presence or characteristics of a pathological state, and may specifically mean confirming the occurrence of cancer through the above-described compound or a pharmaceutically acceptable salt thereof.

In the present invention, “treatment” of cancer includes the act of administering the above-described compound or a pharmaceutically acceptable salt thereof, or a composition containing the same, to improve symptoms or cause necrosis and/or death of cancer cells, and other It can be used in combination with anticancer drugs, radiation therapy, surgery, etc.

The above cancers include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, and uterus. Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or any one or more selected from the group consisting of acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. However, it is not limited to this.

The pharmaceutical composition may additionally contain a carrier, excipient or diluent, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned.

In addition, the pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the pharmaceutical composition, such as starch, calcium carbonate, and sucrose. It can be formulated by mixing , lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may be used.

Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. may be included.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injectables may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, etc.

The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the It can be determined based on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition may be administered to a subject through various routes. There are no restrictions on the method of administration, and for example, it can be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.

In the present invention, the term "administration" means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or all general routes as long as it can reach the target tissue. Can be administered parenterally. Additionally, the composition may be administered using any device capable of delivering the active ingredient to target cells.

The term "subject" in the present invention is not particularly limited, but includes, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits or guinea pigs. It can be included.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route, and period, but can be appropriately selected by a person skilled in the art. Preferably, it can be administered at 0.001 to 100 mg/kg of body weight per day, and more preferably at 0.01 to 30 mg/kg of body weight per day. Administration may be administered once a day, or may be administered in multiple doses.

According to another aspect,

1) administering the above-described compound or a pharmaceutically acceptable salt thereof to an individual; and

2) A photodynamic diagnosis or treatment method is provided including the step of irradiating light to the object of step 1).

The step of irradiating light is 400 to 700 nm wavelength (white light wavelength), preferably 450 to 670 nm wavelength range for 1 minute to 45 minutes, preferably 5 minutes to 30 minutes, with an intensity of 50 to 300 mW/ cm ² , preferably 100 to 250 mW/cm ² , more preferably 100 to 200 mW/cm ² , but is not limited thereto, and those skilled in the art may appropriately vary the light irradiation conditions depending on the purpose. It can be applied. In one embodiment, when cells are irradiated with light with a wavelength of 400 to 700 nm and an intensity of 100 to 200 mW/cm ² for 5 to 20 minutes, for 30 minutes for animal models, the light is irradiated with a wavelength of 400 to 700 nm and an intensity of 100 to 200 mW. It was confirmed that a significant cancer cell killing effect (photodynamic therapy effect) can be achieved when irradiated with light having an intensity of /cm ² . The administering step may be performed orally or parenterally.

The method may further include allowing a predetermined time between steps 1) and 2) for the above-described compound or a pharmaceutically acceptable salt thereof to accumulate in the target cells and/or tissues of the subject.

Additionally, the method may further include an imaging step to confirm the location of the target cells and/or tissues of the subject after step 2). This can be done by detecting and visualizing fluorescence or luminescence images by light irradiation.

The subject may be a human or mammal.

The photodynamic diagnosis or treatment method may be used to detect or diagnose cancer and simultaneously prevent and treat cancer.

The method can be performed in vitro or in vivo . When the method is performed in vitro, the compound or a pharmaceutically acceptable salt thereof may be administered to tissues, cells, or cultures isolated from an individual.

The photodynamic diagnosis or treatment method may exhibit advantages such as spatiotemporal selectivity, non-invasiveness, and reduction of side effects.

According to one aspect, the compound represented by Formula 1 not only does not fluoresce until it reaches the mitochondria and does not accumulate in lipophilic sites such as lipids, but also has aggregation-inducing properties in the near-infrared wavelength region, resulting in higher efficiency compared to background fluorescence. This is significantly higher. Through this, it can effectively target cancer cells, achieving efficient cancer treatment without toxicity or side effects. It has superior photostability and is not affected by changes in mitochondrial membrane potential, making it superior to existing fluorescent dyes. Therefore, the compound according to one aspect can be effectively used in fluorescence imaging, photodynamic diagnosis, and treatment.

Figure 1 is a diagram showing the ¹ H NMR spectrum (a), ¹³ C NMR spectrum (b), and ESI-MS analysis results (c) of compound 3.
Figure 2 is a diagram showing the ¹ H NMR spectrum (a), ¹³ C NMR spectrum (b), and ESI-MS analysis results (c) of compound 5.
Figure 3 is a diagram showing the ¹ H NMR spectrum (a), ¹³ C NMR spectrum (b), and ESI-MS analysis results (c) of TPA-SD.
Figure 4 is a diagram showing the ¹ H NMR spectrum (a), ¹³ C NMR spectrum (b), and ESI-MS analysis results (c) of TPA-SQ.
Figure 5 is a diagram showing the ¹ H NMR spectrum (a), ¹³ C NMR spectrum (b), ³¹ P NMR spectrum (c), and ESI-MS analysis results (d) of compound 8.
Figure 6 is a diagram showing the ¹ H NMR spectrum (a), ¹³ C NMR spectrum (b), and ESI-MS analysis results (c) of TPA-S-TPP.
Figure 7 is a diagram showing the results of analyzing the photophysical properties of TPA-SQ (Compound 1) obtained in Synthesis Examples 1 to 6. A) Fluorescence intensity and maximum emission peak of TPA-SQ (10 μM) in various solvents (ACN, DMSO, EtOH, DW, and PBS), λex = 420 nm. B), C), D), and E) are TPA-SQ (10 μM) in the composition of ACN/DW (B), DMSO/DW (C), EtOH/DW (D), and DMSO/Toluene (E) mixtures, respectively. ) of fluorescence intensity and maximum emission peak.
Figure 8 is a diagram showing the results of analyzing the photophysical properties of TPA-SQ (Compound 1) obtained in Synthesis Examples 1 to 6. A) Fluorescence spectral change in mixed solutions of CH ₃ CN/DW (DW fraction f _DW : 0-95%) of TPA-SQ (10 μM), λex = 420 nm. B) Fluorescence spectral change in DMSO/DW (DW fraction f _DW : 0-95%) mixed solution of TPA-SQ (10 μM), λex = 420 nm. C) Fluorescence spectral change in EtOH/DW (DW fraction f _DW : 0-95%) mixed solution of TPA-SQ (10 μM), λex = 420 nm. D) Change in fluorescence spectrum of TPA-SQ (10 μM) in DMSO/Tol (Toluene fraction f _Tol : 0-95%) mixed solution, λex = 420 nm.
Figure 9 is a diagram showing A) dynamic light scattering (DLS) analysis results and B) TEM analysis results of TPA-SQ (10 μM) in DW.
Figure 10 is a diagram showing the results of analyzing the photophysical properties of TPA-SD (Compound 2) obtained in Synthesis Examples 1 to 6. A) Fluorescence intensity and maximum emission peak of TPA-SD (10 μM) in different solvents (ACN, DMSO, EtOH, DW, and PBS), λex = 530 nm. B) Fluorescence intensity and maximum emission peak of TPA-SD (10 μM) in DMSO/Toluene mixed solution.
Figure 11 is a diagram showing the results of analyzing the photophysical properties of TPA-SD (Compound 2) obtained in Synthesis Examples 1 to 6. A) Fluorescence spectral change in ACN/DW (DW fraction f _w : 0-90%) mixed solution of TPA-SD (10 μM), λex = 420 nm. B) Fluorescence spectral change in DMSO/DW (DW fraction f _w : 0-90%) mixed solution of TPA-SD (10 μM), λex = 530 nm. C) Fluorescence spectral change in EtOH/DW (DW fraction f _w : 0-90%) mixed solution of TPA-SD (10 μM), λex = 530 nm.
Figure 12 is a diagram showing A) dynamic light scattering (DLS) analysis results and B) TEM analysis results of TPA-SD (10 μM) in DW.
Figure 13 is a diagram showing the results of analyzing the photophysical properties of TPA-S-TPP (Compound 3) according to Synthesis Examples 1 to 6. A) Fluorescence intensity and maximum emission peak of TPA-S-TPP (10 μM) in various solvents (ACN, DMSO, EtOH, DW, and PBS), λex = 530 nm. B) Fluorescence spectral change of TPA-S-TPP (10 μM) in ACN/DW (DW fraction f _w : 0-90%) mixed solution, λex = 530 nm. C), D), and E) are changes in the fluorescence spectra of TPA-SD (10 μM) in the compositions of DMSO/DW (C), EtOH/DW (D), and DMSO/Toluene (E) mixtures, respectively. F) Maximum fluorescence intensity change of TPA-SD (10 μM) and TPA-S-TPP (10 μM) in DMSO/Toluene mixture (Toluene fraction f _Tol : 0–90%).
Figure 14 is a diagram showing the molar extinction coefficient (ε) of A) TPA-SD (10 μM) and B) TPA-S-TPP in DW.
Figure 15 shows the results of the Dyndall effect experiment of TPA-S-TPP (10 μM) in A) various solvents (DMSO, Tol, and DW) and B) the fluorescence image in toluene at an excitation wavelength of 559 nm.
Figure 16 shows A) absorption spectrum changes of ABDA (50 μM) in the presence of TPA-S-TPP (10 μM) under white light (25 mW/cm ² ) irradiation, B) ABDA by singlet oxygen ( ¹ O ₂ ). This diagram shows the decomposition process.
Figure 17 shows the results of stability analysis of AIEgen TPA-S-TPP (10 μM) under white light irradiation (25 mW/cm ² ) for various times (0-30 minutes).
Figure ¹⁸ shows A) Ce6 (10 μM), Rose Bengal (10 μM), TPA-SQ (10 μM; Compound 1), TPA-SD (10 μM; Compound 2) and Normalized degradation rate of ABDA (50 μM, absorbance at 378 nm) upon treatment with TPA-S-TPP (10 μM; Compound 3); B) Absorption spectrum changes of ABDA (50 μM) under white light irradiation (25 mW/cm ² ) for various times (0–300 s); C), D), E) and F) Ce6 (10 μM, C), TPA-SQ (10 μM, D), Rose under white light irradiation (25 mW/cm ² ) for various times (0-300 s). This diagram shows the change in absorption spectrum of ABDA (50 μM) when treated with Bengal (10 μM, E) and TPA-SD (10 μM, F).
Figure 19 is a diagram showing the electron spin resonance measurement results of TEMP + TPA-S-TPP under dark conditions and light irradiation.
Figure 20 shows A) the lifetime of the triplet state of TPA-S-TPP (10 μM) according to the nanosecond time-resolved transient absorption spectrum and B) the singlet oxygen generation mechanism under light irradiation. This is a diagram showing .
Figure 21 shows time-dependent fluorescence images of HeLa cells cultured for various times with TPA-SD (5 μM, AG) and TPA-S-TPP (5 μM, IO) (A, I: control; B, J: 0.5h; C, K: 1.0h; D, L: 1.5h; E, M: 2.0h; F, N: 3.0h; G, O: 4.0h). Figures 22H) and P) are diagrams showing fluorescence intensity in HeLa cell images. λex (excitation wavelength) = 559 nm, λem (emission wavelength) = 655-755 nm. Scale bar = 40 μm.
Figure 22 is a diagram showing the co-localization imaging results of AIEgen TPA-S-TPP using Hochest 33342 (Q1), Lyso-tracker green (R1), Mito-tracker green (S1), and BODIPY 493/503 (T1). Merged images: Q3, R3, S3 and T3. Pearson's coefficient: Q4, R4, S4 and T4. Scale bar = 10 μm. Confocal microscopy imaging results of HeLa cells to confirm mitochondrial membrane potential changes in response to CCCP (10 μM, treated for 20 minutes). UW) Fluorescence imaging of HeLa cells following different treatments (U: control; V: treated with Mito-tracker Red or TPA-S-TPP; W, after incubation with CCCP, treated with Mito-tracker Red or TPA-S-TPP. Scale bar = 30 μm. X, Y) Normalized fluorescence intensity analysis results (UW) based on HeLa cell imaging results. The pixel intensity of each channel (Mito-tracker Red, V1 and AIEgen TPA-S-TPP, V3) was defined as 1.0, respectively.
Figure 23 is a diagram showing the results of fluorescence imaging of HeLa cells treated with TPA-SQ. Ex = 405 nm, Em = 490-540 nm. Scale bar = 20 μm.
Figure 24 is a diagram showing the results of fluorescence imaging of HeLa cells treated with TPA-SQ at an excitation wavelength of 405 nm along the longitudinal Z axis (3961.54-3952.84 μm, step size = 0.58 μm) based on Figure 23. Scale bar = 20 μm.
Figure 25 shows TPA-SQ (Ex = 405 nm, Em = 490-540 nm) and A) mito-tracker deep red (Ex = 633 nm, Em = 655-755 nm), B) lyso-tracker deep red (Ex = 633 nm, Em = 655-755 nm) or C) nile red (Ex = 473 nm, Em = 575-675 nm). Scale bar = 10 μm.
Figure 26 is a diagram showing the fluorescence imaging results of HeLa cells pretreated with TPA-S-TPP and then added with CCCP. Scale bar = 30 μm.
Figure 27 shows A) confocal fluorescence images of HeLa cells treated with mito-tracker green and AIEgen TPA-S-TPP under continuous light irradiation (scale bar = 20 μm) and B) normalized fluorescence intensity of HeLa cells in different channels. This is a diagram showing (F/F0).
Figure 28 is a diagram showing the change in fluorescence intensity of mito-tracker (5 μm and 10 μM) in DMSO and DW. λem = 480 nm.
Figures 29A) to E) show the results of reactive oxygen species (ROS) production in HeLa cells after different treatments using DCF-DA dye. Scale bar = 30 μm. F) and G) of HeLa cells upon treatment with different concentrations of TPA-SQ and TPA-S-TPP (0, 1, 2, 3, 4, 5, and 6 μM) under dark conditions or white light irradiation. This diagram shows the results of MTT analysis. H) to K) are diagrams showing confocal fluorescence imaging results of HeLa cells co-stained with Annexin V-FITC and PI after different treatments (H: light irradiation only for 30 min; I: TPA-S-TPP + dark; J: TPA-S-TPP + light 10 min; K: TPA-S-TPP + light 30 min). Scale bar = 100 μm. L) Schematic diagram showing the photodynamic therapy mechanism of TPA-S-TPP, which shows improved photodynamic therapy effect in cells. M) to O) are diagrams showing the results of mitochondrial membrane potential measurement in HeLa cells using JC-1 dye. P) to S) show immunofluorescence imaging analysis results of HeLa cells exposed to AIEgen TPA-S-TPP with (Q: 5 min; R: 10 min; S: 20 min) or without (P) light irradiation. This is the diagram shown. Scale bar = 30 μm. T) This is a diagram showing the green fluorescence intensity according to immunofluorescence imaging (PS). U) This diagram shows the expression results of upregulated cleaved caspase 3 protein in cells according to Western blot. 1, blank + light (7 min); 2, blank + light (15 min); 3, TPA-S-TPP + dark; 4, TPA-S-TPP + light (7 min); 5, TPA-S-TPP + light (15 min).
Figure 30 is a diagram showing the results of analyzing fluorescence intensity from a fluorescence image for the generation of reactive oxygen species (ROS).
Figure 31 is a diagram showing the results of MTT analysis of HeLa cells treated with various concentrations (0, 2, 4, 6, 8, and 10 μM) of TPA-SD under dark conditions or white light irradiation.
Figure 32 is a diagram showing the results of annexin V-FITC and PI co-staining for HeLa cells subjected to different treatments. A, light irradiation only 30 min; B, TPP-S-TPP + dark; C, TPA-S-TPP + light 10 min; D, TPA-S-TPP + light 30 min. Scale bar = 100 μm.
Figure 33 is a diagram showing F _Green / F _red values according to the fluorescence image of JC-1.
Figure 34 is a diagram showing the results of Calcein AM and PI dye co-staining for HeLa cells subjected to different treatments (confocal fluorescence image). A, light irradiation only for 10 min; B, light irradiation only 30 min; C, TPP-S-TPP + dark; D, TPA-S-TPP + light 10 min; E, TPA-S-TPP + light 30 min. Scale bar = 80 μm.
35 shows A) in vivo images of Balb/c mice containing 4T1 tumor cells injected with AIEgen TPA-S-TPP and PBS (control), B) major organs (spleen: Sp; tumor: Tu; kidney: Ki; liver: Li; lung: Lu; heart: He) , C) Tumor volume changes in Balb/c mice subjected to different treatments (n = 5), D ) Pictures of tumors in mice after 14 days of treatment, E) Body weight changes in Balb/c mice subjected to different treatments, F) Survival rate of mice in each group, G) Major organ sections in each group after 14 days of treatment. This diagram shows the H&E staining results for (heart, liver, spleen, lung, and kidney).
Figure 36 shows the “step-by-step” molecular design strategy (a) to design probes TPA-SQ, TPA-SD, and TPA-S-TPP and AIEgen TPA anchored to mitochondria under light irradiation. -This is a diagram showing the tumor cell death mechanism (b) of S-TPP.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[Example]

All chemicals were purchased from commercial suppliers and used without further purification. The solvents used were purified by standard methods. ¹ H, ¹³ C and ³¹ P-NMR spectra were measured with a Bruker 300 MHz spectrophotometer. Chemical shifts (δ) are reported in ppm. The fluorescence spectrum was measured using an FS-2 fluorescence spectrophotometer (Scinco). Excitation and emission slit widths were appropriately changed to adjust the fluorescence intensity to an appropriate range. Absorption spectra were recorded with a Thermo Scientific Evolution 201 UV-Vis spectrophotometer. SYNAPT G2 (Waters, UK) was used to obtain mass spectra. Dynamic light scattering (DLS) was measured using Nano-ZS (Malvern). EPR analysis was performed with EMX-plus equipment (Bruker). TEM images were acquired with a JEOL-2100F electron microscope operating at 100 kV. Cell imaging was performed on an Olympus FV 1200 confocal laser scanning microscope (Olympus, Japan). Distilled deionized water (DW) was prepared by ultrafiltration.

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

<Reaction Scheme 1>

As in Reaction Scheme 1, the electron donor and hydrophobic triphenylamine (TPA, Compound 1) is connected to the electron acceptor and hydrophilic quinoline cation through a conjugated thiophene bridge to form the ampholyte. Maesung AIEgen (TPA-S-TPP) was synthesized.

Synthesis Example 1. Compound 3

Compound 1 (1.94 g, 6mM), compound 2 (1.12g, 7.2mM), K ₂ CO ₃ (2.48g, 18mM) and tetrakis(triphenylphosphine)palladium (346 mg, of Reaction Scheme 1) 0.3 mM) was added to the MeOH/toluene (v/v = 1:1, 100 mL) mixed solution under N ₂ atmosphere, stirred under reflux conditions for 24 hours, and then cooled to room temperature. The solvent was evaporated by reducing the pressure and purified by silica gel column chromatography to obtain Compound 3 as a yellow solid (1.15 g, yield 54%).

¹ H NMR (300 MHz, CDCl ₃ ): δ = 9.85 (s, 1H), 7.71 (d, J = 4.0 Hz, 1H), 7.57-7.47 (m, 2H), 7.35-7.23 (m, 5H), 7.18-7.00 (m, 8H). ¹³ C NMR (75 MHz, CDCl ₃ ): δ = 182.64, 154.61, 149.17, 146.99, 141.34, 137.78, 129.52, 127.28, 126.15, 125.20, 123.91, 122.90, 122. 39. ESI-MS: m/z calcd for C ₂₃ H ₁₇ NOS ⁺ [M] ⁺ 355.1031, found: 355.1026; m/z calcd for C ₂₃ H ₁₇ NONaS ⁺ [M+Na] ⁺ 378.0923, found: 378.0923.

Synthesis Example 2. Compound 5

Compound 4 (1.43 g, 10 mM) and 1-iododecane (2.68 g, 10 mM) of Reaction Scheme 1 were added to anhydrous CH ₃ CN (20 mL) under N ₂ atmosphere and stirred at 85°C for 12 hours. Afterwards, it was cooled to room temperature. The solvent was evaporated by reducing the pressure and purified by silica gel column chromatography to obtain compound 5 as a sticky liquid (2.75 g, yield 67%).

^1H NMR (300 MHz, DMSO- d6 ): δ = 9.46 (d, J = 6.1 Hz, 1H), 8.66-8.52 (m, 2H), 8.33-8.21 (m, 1H), 8.07 (ddd, J = 8.0, 6.5, 0.7 Hz, 2H), 5.02 (t, J = 7.5 Hz, 2H), 3.02 (s, 3H), 2.08-1.80 (m, 2H), 1.55-1.04 (m, 14H), 0.84 (t) , J = 6.7 Hz, 3H). ¹³ C NMR (75 MHz, DMSO- d6 ): δ = 159.00, 148.83, 137.19, 135.59, 130.07, 129.45, 127.66, 123.14, 119.87, 57.42, 55.45, 31.73, 29.93, 29.32, 29.11, 28.95, 26.21, 22.55, 20.24, 14.43. ESI-MS: m/z calcd for C ₂₀ H ₃ 0N ⁺ [M] ⁺ 284.2373, found: 284.2378.

Synthesis Example 3. TPA-S-D

Compound 3 (106.5 mg, 0.3mM), compound 5 (123mg, 0.3mM) and piperidine (1-3 drops) of Reaction Scheme 1 were added to 20mL of absolute ethanol under N ₂ atmosphere and stirred at 85°C overnight. Afterwards, it was cooled to room temperature. The solvent was evaporated by reducing the pressure and purified by silica gel column chromatography to obtain 61 mg of TPA-SD (yield 27%).

^1H NMR (300 MHz, CD ₂ Cl ₂ ): δ = 9.73 (d, J = 6.1 Hz, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.44 (d, J = 6.1 Hz, 1H) , 8.34-8.12 (m, 3H), 8.03-7.92 (m, 1H), 7.69-7.51 (m, 4H), 7.39-7.30 (m, 5H), 7.21-7.13 (m, 5H), 7.13-7.06 ( m, 3H), 5.03 (t, J = 7.5 Hz, 2H), 2.16-2.03 (m, 2H), 1.59-1.49 (m, 2H), 1.32 (m, 12H), 0.90 (t, J = 5.6 Hz) , 3H). ¹³ C NMR (75 MHz, CD ₂ Cl ₂ ): δ = 152.69, 150.15, 148.82, 147.01, 139.05, 138.01, 136.93, 135.59, 135.07, 129.47, 129.30, 129.07, 12 6.88, 126.75, 126.32, 126.15, 125.19, 123.89 , 123.74, 123.24, 122.30, 118.36, 116.21, 116.09, 57.34, 31.85, 29.86, 29.47, 29.40, 29.25, 29.13, 26.54, 22.66, 13.88. ESI-MS: m/z calcd for C ₄₃ H ₄₅ N ₂ S ⁺ [M] ⁺ 621.3298, found: 621.3303.

Synthesis Example 4. TPA-S-Q

The solutions of Compound 3 (710 mg, 2 mM), Compound 4 (572 mg, 4 Mm), and benzoyl chloride (394 mg, 2 mM) in Reaction Scheme 1 were added to 5 mL of anhydrous DMF under N ₂ atmosphere and incubated for 8 hours at 60°C. Stirred for an hour. Then, the reaction mixture was added to water, extracted with DCM, and dried over anhydrous Na ₂ SO ₄ . The solvent was evaporated by reducing the pressure and purified by silica gel column chromatography to obtain 163 mg of TPA-SQ (yield 17%).

¹ H NMR (300 MHz, CDCl ₃ ): δ = 8.90 (d, J = 4.7 Hz, 1H), 8.28-8.14 (m, 2H), 7.74 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.59 (ddd, J = 11.4, 7.4, 3.2 Hz, 3H), 7.53-7.39 (m, 3H), 7.34-7.23 (m, 4H), 7.16 (m, 6H), 7.12-7.03 (m, 4H). ¹³ C NMR (75 MHz, CDCl ₃ ): δ = 149.81, 148.41, 147.86, 147.35, 145.17, 142.79, 142.43, 140.35, 129.97, 129.85, 129.75, 129.54, 129. 44, 128.25, 128.19, 127.64, 126.61, 126.19, 124.79 , 123.43, 123.29, 122.85, 120.99, 116.36. ESI-MS: m/z calcd for C ₃₃ H ₂₅ N ₂ S ⁺ [M+H] ⁺ 481.1733, found: 481.1737.

Synthesis Example 5. Compound 8

Compound 6 (2.28 g, 10 mM) and compound 7 (2.62 g, 10 mM) of Reaction Scheme 1 were added to 20 mL of anhydrous toluene under N ₂ atmosphere, stirred at 110°C overnight, and cooled to room temperature. The solvent was evaporated by reducing the pressure and purified by column chromatography to obtain 2.55 g of compound 8 (yield 53%).

¹ H NMR (300 MHz, CDCl ₃ ): δ = 7.81 (m, 6H), 7.77-7.72 (m, 3H), 7.66 (m, 6H), 3.87-3.72 (m, 2H), 3.31 (t, J = 6.4 Hz, 2H), 1.81 (m, 4H), 1.67-1.60 (m, 2H). ¹³ C NMR (75 MHz, CDCl ₃ ): δ = 135.11, 135.07, 133.74, 133.60, 130.63, 130.47, 118.76, 117.62, 33.51, 32.00, 28.92, 23.08, 21.88. ³¹ P NMR (122 MHz, CDCl ₃ ): δ = 24.28. ESI-MS: m/z calcd for C ₂₃ H ₂₅ BrP ⁺ [M] ⁺ 411.0877, found: 411.0877.

Synthesis Example 6. TPA-S-TPP

TPA-SQ (192 mg, 0.4 mM) and Compound 8 (147 mg, 0.3 mM) were added to 5 mL of anhydrous 1,2-dichlorobenzene under N ₂ atmosphere, stirred at 130°C overnight, and cooled to room temperature. The solvent was evaporated by reducing the pressure and purified by column chromatography to obtain 55 mg of TPA-S-TPP (yield 19%).

^1H NMR (300 MHz, MeOD): δ = 9.10 (d, J = 6.6 Hz, 1H), 8.69 (d, J = 8.6 Hz, 1H), 8.39 (d, J = 9.0 Hz, 1H), 8.17 ( dt, J = 15.9, 7.7 Hz, 3H), 7.94-7.79 (m, 7H), 7.79-7.67 (m, 10H), 7.54-7.43 (m, 3H), 7.36-7.23 (m, 5H), 7.11- 7.05 (m, 6H), 6.95 (d, J = 8.3 Hz, 2H), 4.90 (t, J = 7.4 Hz, 2H), 3.56-3.43 (m, 2H), 2.08 (br, 2H), 1.76 (br , 4H). ¹³ C NMR (75 MHz, MeOD): δ = 153.05, 149.83, 148.63, 147.03, 146.19, 139.24, 138.12, 136.45, 135.09, 134.89, 134.85, 133.56, 133.52 , 133.43, 133.38, 130.24, 130.08, 129.28, 129.03, 126.65, 126.58, 126.43, 126.01, 124.90, 123.70, 123.62, 121.97, 119.15, 119.02, 118.65, 118.00, 117.87, 116.44, 115.19, 5 6.49, 28.76, 27.21, 21.87, 20.96. ESI-MS: m/z calcd for C ₅₆ H ₄₉ BrN ₂ PS ⁺ [M] ⁺ 891.2532, found: 891.2537.

Analysis Example 1. Photophysical property analysis

Fluorescence emission spectrum analysis was performed on TPA-S-Q (compound 1), TPA-S-D (compound 2), and TPA-S-TPP (compound 3) obtained in Synthesis Examples 1 to 6. Fluorescence emission spectra were recorded with an FS-2 spectrophotometer (Scinco).

Referring to Figure 7 (a), TPA-S-Q showed bright fluorescence in organic solvents (CAN, DMSO, and EtOH), while it was quenched in aqueous solutions (DW and PBS), which is typical of coherent quenching. It can be confirmed that it is consistent with the (aggregation-caused quenching: ACQ) phenomenon.

These properties of TPA-S-Q were confirmed in binary solvents such as ACN/DW, DMSO/DW, and EtOH/DW with various water volume fractions (fDW). Referring to Figures 7 (b) to (d), it can be seen that the fluorescence intensity of TPA-S-Q significantly decreased as the DW volume fraction increased. Additionally, referring to (a) to (c) of Figures 8, it can be seen that the emission peak has shifted to blue. Meanwhile, referring to Figure 7 (e) and Figure 8 (d), it can be seen that in the case of the DMSO/Tol mixed solution, the emission peak of TPA-S-Q is the same as the blue shift, but there is no significant change in fluorescence intensity. You can.

TEM image analysis and dynamic light scattering (DLS) analysis were performed on TPA-S-Q (Compound 1) to confirm the formation of nanoparticles in an aqueous solution. Referring to Figures 9 (a) and (b), it can be seen that the ACQ phenomenon of TPA-S-Q is due to the dominance of the π-ð stacking effect of planar quinoline in the above process.

Unlike TPA-S-Q, AIEgen TPA-S-D with quinoline cation introduced showed “fluorescence-off” properties in various solutions. Referring to Figure 10 (a), it can be seen that TPA-S-D does not show fluorescence in both organic solvents and aqueous solutions. Additionally, referring to Figures 11 (a) to (c), it can be seen that fluorescence is not observed in TPA-S-D even when the DW volume fraction increases in ACN/DW, DMSO/DW, and EtOH/DW. Referring to Figures 12 (a) and (b), it can be confirmed that this is due to the loose aggregation state of TPA-S-D within the DW without limited rotational movement.

Considering that AIEgen is insoluble in low polar solvents such as toluene, n-hexane, and ether, AIE properties were analyzed in DMSO/Tol mixed solution. Referring to (b) of Figure 10, it can be seen that AIE fluorescence occurs when the fDW of the mixed solvent exceeds 80%.

TPA-S-TPP with triphenylphosphine cation introduced showed increased fluorescence-quenching properties due to enhanced amphiphilicity. Referring to (a) of Figure 13, TPA-S-TPP has an increased extinction effect, and referring to (a) and (b) of Figure 14, the molar extinction coefficient in DW is 1.65 Х It can be seen that the photon absorption capacity has increased by increasing from 10 ⁴ to 2.60 Х 10 ⁴ .

Referring to Figures 13 (b) to (d), it can be seen that fluorescence is not observed in TPA-S-TPP even when the DW volume fraction increases in ACN/DW, DMSO/DW, and EtOW/DW. This means that AIEgen has amphipathic properties, excellent dispersion ability, and rotational freedom. In addition, referring to (e) of Figure 13, it can be seen that TPA-S-TPP forms hard nanoaggregates when fTol exceeds 80%, and the intrinsic near-infrared (NIR) fluorescence signal rapidly increases. there is. Referring to Figures 15(a) and (b), it can be seen that this effect is also confirmed through Dyndall effect experiments and fluorescence microscopy imaging. Referring to Figure 13(f), it can be seen that the fluorescence intensity of TPA-S-TPP was significantly increased compared to TPA-S-D due to its increased hydrophilic properties.

Analysis Example 2. Singlet oxygen in cells ( ^One O ₂ ) detection imaging

Using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA, 50 μM) as a singlet oxygen indicator, Ce6 (10 μM), Rose Bengal (10 μM), and The degree of singlet oxygen production of TPA-SQ (10 μM; Compound 1), TPA-SD (10 μM; Compound 2), and TPA-S-TPP (10 μM; Compound 3) was analyzed. The sample mixture (ABDA + probe in solution) was irradiated with white light (25 mW/cm ² ) at 10-second intervals for 30 seconds, and then the absorbance of ABDA (50 μM) was measured using a Thermo Scientific Evolution 201 UV-visible spectrophotometer. In addition, the decay rate of the photosensitization process was analyzed by measuring the absorbance of ABDA (50 μM) at 378 nm at various irradiation times (0-300 s).

Referring to Figures 16 (a) and (b), it can be seen that as the irradiation time increases, ABDA undergoes a decomposition process due to singlet oxygen and the absorption peak rapidly decreases. On the other hand, referring to Figure 17, the absorption peak of TPA-S-TPP did not show any significant change, confirming that TPA-S-TPP has high singlet oxygen production ability and high photostability. there is.

Additionally, other compounds, including commercial photosensitizers Ce6 and Rose Bengal (RB), were tested for their singlet oxygen generation ability. Referring to Figures 18 (a) to (f), it can be seen that TPA-S-TPP exhibits significantly superior photodynamic effects compared to commercial photosensitizers.

Analysis Example 3. Electron spin resonance and instantaneous absorption spectrum

TPA under light irradiation using 2,2,6,6-tetramethyl-4-piperidinol (TEMP) as a singlet oxygen trapper. The singlet oxygen production ability of -S-TPP was confirmed through electron spin resonance (ESR) analysis. Referring to Figure 19, it can be seen that the ESR signal of the TPA-S-TPP + TEMP solution is detected significantly higher in light conditions compared to dark conditions.

To prove the singlet oxygen generation mechanism of AIEgen, laser flash photolysis analysis was performed to confirm the lifetime of the triplet state of TPA-S-TPP. Specifically, the femtosecond transient absorption spectrum of AIEgen TPA-S-TPP was measured using a home-made ultrafast pump-probe setup. The wavelength of the pump beam was selected at 355 nm, and the sample was used at a concentration of 10 μM in DMSO at room temperature. As a result, referring to Figure 20(a), it can be seen that the triplet lifetime of TPA-S-TPP is measured to be 11.43 μs. As can be seen in Figure 20(b), this is due to the favored energy transfer from AIEgen to chemicals, resulting in increased singlet oxygen production efficiency under light irradiation.

Analysis Example 4. In vitro ( in vitro ) Cell imaging

4-1. cell culture

HeLa (human cervix adenocarcinoma) cells were purchased from Korean Cell Line Bank (Seoul, Korea). 4T1 breast cancer cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were cultured in confocal culture dishes using medium supplemented with 10% FBS (Invitrogen) at 37°C, humidified, and 5% CO ₂ for 24 hours.

4-2. cell imaging

_HeLa cells ( ¹ The culture medium was removed, and Hank's Balanced Salt Solution (HBSS) containing TPA-SD (5 μM) and TPA-S-TPP (5 μM) was added for 0 and 0.5 hours at 37°C, humidified, 5% CO ₂ conditions. Added for 1.0 hours, 1.5 hours, 2.0 hours, 3.0 hours and 4.0 hours and then washed three times with DPBS. Confocal fluorescence images of AIEgen were obtained using a 60 × lens, excitation at 559 nm and emission at 655-755 nm (FV 1200, Olympus).

Referring to Figure 21, it can be seen that when HeLa cells are treated with amphipathic TPA-S-TPP, a NIR fluorescence signal appears with a high signal-to-noise ratio after 2 hours of incubation (M, P in Figure 21). This shows that TPA-S-TPP successfully penetrated into the cells. On the other hand, HeLa cells treated with TPA-S-D showed a low fluorescence signal, confirming that TPA-S-D had difficulty penetrating into cancer cells (Figure 21, A to H). This low cellular uptake rate of TPA-S-D was expected to be due to the long hydrophobic alkyl group restricting passage through the cell membrane.

4-3. Co-localization analysis

_HeLa cells ( ¹ The culture medium was removed, Hank's Balanced Salt Solution (HBSS) containing TPA-S-TPP (5 μM) was added for 1.5 hours at 37°C, humidified, 5% CO ₂ conditions, and then commercial Hochest 33342, Lyso- Tracker green and Mito-tracker green were added to the cells, respectively. After an additional 30 min of incubation, HeLa cells were washed twice with DPBS and imaged with an FV 1200 confocal microscope.

Referring to Figure 22, it can be seen that the red fluorescence signal from the amphipathic TPA-S-TPP (S2 in Figure 22) and the green fluorescence signal from the mitochondrial dye (S1 in Figure 22) almost completely overlap (Figure 22). S3). At this time, the Pearson coefficient is 0.86, which means the nucleus (P=0.32; Q1-Q4 in Figure 22), lysosomes (P=0.45; R1-R4 in Figure 22), and lipid droplets (P=0.34; T1-T4 in Figure 22). ), and is due to the synergistic effect of the double positive charges of triphenylphosphine and quinoline selectively accumulated in mitochondria. Through the above results, it was confirmed that TPA-S-TPP has excellent targeting properties to mitochondria.

On the other hand, referring to Figures 23 and 24, it can be seen that HeLa cells treated with TPA-SQ show bright yellow spots when observed under a confocal microscope, which is due to the lipophilicity of TPA-SQ (high Log P value). observed) was expected to be due to accumulation in cytoplasmic lipid droplets. Referring to Figure 25, it can be seen that the above prediction is verified by the imaging analysis results following treatment with mitochondrial dye (Mito-tracker), dye for lysosomes (lyso-tracker), which are acidic endoplasmic reticulum, and Nile Red dye for lipid droplets.

Through the above results, it was found that TPA-S-TPP (Compound 3) according to one aspect can function between suborganelles of living cells, from lysosomes to mitochondria.

Meanwhile, the oil-water partition coefficient (Log P ) was measured using the shake-flask method. Water and octanol were mixed and shaken thoroughly to achieve equilibrium, and then the two layers were separated to obtain octanol saturated with water and water saturated with octanol. The sample was dissolved in 1 mL of octanol saturated with water, and then 9 mL of water saturated with octanol was added thereto. The mixture was shaken vigorously at 37°C for 24 hours. Then, the concentration of the sample was recorded using UV-Vis spectroscopy and calculated using the following formula.

Log P _o/w = Log([solute]octanol/[solute]water)

Analysis Example 5. Mitochondrial localization of AIEgen by CCCP

Singlet oxygen produced by photosensitizers decreases the mitochondrial membrane potential (MMP), which causes off-targets of photosensitizers and results in anticancer effects in photodynamic therapy. decreases. Therefore, to determine whether AIEgen TPA-S-TPP can be permanently located in mitochondria, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which depolarizes the mitochondrial membrane potential, was used to Localization experiments were performed.

Specifically, HeLa cells (1 x 10 ⁵ cells/mL) were seeded on a culture plate and placed in an incubator at 37°C, humidified, 5% CO ₂ conditions for 24 hours. The culture medium was removed, Hank's Balanced Salt Solution (HBSS) containing TPA-S-TPP (5 μM) was added for 2 hours at 37°C, humidified, 5% CO ₂ conditions, and then CCCP (10 μM) was added. was added and incubated for an additional 20 minutes. Cells were washed twice with DPBS and images were obtained with an FV 1200 confocal microscope. The same experiment was performed, except that the order was changed so that CCCP was added first and then the probe was added.

Referring to Figure 22, when HeLa cells were treated with Mito-tracker Red for 20 minutes, a fluorescent signal was clearly observed (V1 in Figure 22). On the other hand, when CCCP was treated first and then mito-tracker Red was treated, it could be seen that the fluorescence signal was significantly reduced to an almost undetectable level (W1, X in Figure 22). Through the above results, it can be confirmed that commercial mitochondrial dyes have a high dependence on mitochondrial membrane potential. In contrast, similar to untreated cells, it can be seen that significant fluorescence emission was observed in HeLa cells first cultured with CCCP and then treated with TPA-S-TPP (W3, Y in Figure 22).

Meanwhile, referring to Figure 26, surprisingly, the fluorescence intensity is maintained in the case of HeLa cells first cultured with TPA-S-TPP and then treated with CCCP, which is different from the group treated only with TPA-S-TPP (V3 in Figure 22). It was confirmed that the results were consistent. In other words, it was found that TPA-S-TPP was not affected by reduced mitochondrial membrane potential. This was expected to be due to the unique properties of TPA-S-TPP, which can not only be tightly anchored to the mitochondrial membrane, but also have a double charge effect.

Through the above results, while existing photosensitizers targeting mitochondria had a limitation of leakage from mitochondria due to a decrease in mitochondrial membrane potential, TPA-S-TPP according to one aspect overcomes this limitation and penetrates into mitochondria. It was confirmed that it can be permanently fixed, enabling tumor cell-specific photodynamic therapy.

Analysis Example 6. Photostability evaluation

The photostability of TPA-S-TPP was evaluated under continuous laser irradiation conditions. As a control, a commercial mitochondrial dye was used.

Referring to Figure 27, after continuous light irradiation for about 7 minutes, no fluorescence signal was detected from the mitochondrial dye (green) due to excessive photobleaching. Additionally, referring to Figure 28, it can be seen that its unique ACQ characteristics are not helpful for bioimaging. On the other hand, in the case of TPA-S-TPP, almost no loss of fluorescence intensity was observed even after continuous light irradiation for 20 minutes, and in contrast to the fluorescence intensity of the mitochondrial dye, which decreased to 15% of the original, even after continuous light irradiation, it remained at 100% of the original. It was confirmed that the strength was maintained. Through the above results, it was confirmed that TPA-S-TPP has high photostability characteristics.

Analysis Example 7. Singlet oxygen in cells ( ^One O ₂ ) and cell viability analysis

7-1. Detection of intracellular singlet oxygen

The ability to produce intracellular singlet oxygen is a factor that determines the effectiveness of photodynamic therapy using AIEgen photosensitizer. 2,7-Dichlorodihydrofluorescein Diacetate (DCF-DA) was used to measure intracellular singlet oxygen under white light irradiation. HeLa cells were seeded on a confocal culture plate and placed in an incubator at 37°C, humidified, 5% CO ₂ conditions for 24 hours. The culture medium was removed, Hank's Balanced Salt Solution (HBSS) containing TPA-S-TPP (5 μM) was added for 1 hour at 37°C, humidified, 5% CO ₂ conditions, and then DCF-DA (10 μM) was added. ) was further incubated for 20 minutes. Cells were washed twice with DPBS and exposed to white light (60 mW/cm ² ) for various times (0, 5 min, 10 min). In addition, NaN ₃ (50 μM) was added before light irradiation to measure the amount of reactive oxygen species (ROS) generation consumption. As a control group, cells treated only with DCF-DA (10 μM) were used. Confocal fluorescence images were obtained with excitation at 473 nm and emission at 490–540 nm (FV 1200, Olympus).

Referring to Figure 29, as a result of DCF-DA analysis, it can be seen that compared to the control group (A, B of Figure 29), HeLa cells pretreated with TPA-S-TPP under light irradiation show a distinct green fluorescence signal ( Figure 29 C, D). In addition, it can be seen that in the presence of NaN ₃ , a representative scavenger of singlet oxygen, the green fluorescence signal of HeLa cells weakens (Figure 29E). Referring to Figure 30, it can be seen that this is due to NaN ₃ effectively inhibiting singlet oxygen production in HeLa cells. Through the above results, it was confirmed that amphipathic TPA-S-TPP has cell penetrating ability and singlet oxygen generation ability controlled by light irradiation, and thus cell death ability.

7-2. Cell viability analysis

MTT assay was performed to confirm the cytotoxicity of AIEgen. Specifically, HeLa cells (1 x 10 ⁵ cells/mL) were seeded in a 96-well plate in 100 μL medium containing 10% FBS and left for 24 hours to attach to the plate. After washing the plate with 100 μL/well PBS, the cells were cultured in medium containing various concentrations of probes and without FBS at 37°C for 2 hours. Afterwards, the medium was replaced with 10% FBS supplemented and white light (100 mW/cm ² ) was irradiated for 30 minutes. After further culturing for 24 hours, MTT solution (5 mg/mL) was added to each well for 4 hours at 37°C, humidified, 5% CO ₂ conditions. Afterwards, the medium was carefully removed, the purple crystal was dissolved in 100 μL DMSO for 15 to 20 minutes, and the absorbance of the solution was measured at 650 nm using a microplate reader. Cell viability was expressed as a percentage of the control culture value and was calculated using the following formula.

Cell viability (%) = (OD _dye - OD _blank )/(OD _control - OD _blank ) Х 100

Referring to Figure 29, as the AIEgen concentration increases, the cell survival rate of HeLa cells is maintained at a very high level of about 90%, but it can be seen that the cell survival rate is significantly reduced when irradiated with white light for 30 minutes (G in Figure 29 ). This means that AIEgen itself has low cytotoxicity, but has high cytotoxicity to cancer cells when irradiated with light. Through the above results, it was confirmed that TPA-S-TPP according to one aspect can be usefully used as an AIE photosensitizer in photodynamic therapy.

Meanwhile, the same experiment was performed on TPA-S-Q (compound 1) and TPA-S-D (compound 2). Referring to Figures 29 and 31, it can be seen that when HeLa cells are irradiated with TPA-S-Q and light, the cell survival rate decreases to about 78.49% (f of Figure 29) and about 78.74% (Figure 31). Through this, it was confirmed once again that TPA-S-TPP (Compound 3) can exhibit an ideal photodynamic effect at the cellular level.

Analysis Example 8. Annexin V-FITC/PI staining

When cell death occurs, phosphatidyl serine present inside the cell membrane is exposed to the outside of the cell membrane, and this can be detected with Annexin V-FITC, which has high affinity for phosphatidyl serine. PI can pass through the cell membrane of necrotic cells or late apoptotic cells whose cell membranes are completely damaged and stain the nuclei. Accordingly, cell death during photodynamic therapy was evaluated through Annexin V-FITC/PI staining.

Specifically, HeLa cells were cultured in confocal culture dishes at 37°C, humidified, and 5% CO ₂ conditions. Then, the cells were treated as follows: group 1, irradiated with white light for 30 minutes (Control); Group 2, incubated with TPA-S-TPP (5 μM) at 37°C for 2 h (Dark); Group 3, incubated with TPA-S-TPP (5 μM) at 37°C for 2 hours and then irradiated with white light for 10 minutes (TPA-S-TPP + light 10 min); Group 4, incubated with TPA-S-TPP (5 μM) at 37°C for 2 hours and then irradiated with white light for 30 minutes (TPA-S-TPP + light 30 min). After treatment, HeLa cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit and confocal fluorescence images were obtained (FV 1200, Olympus).

Referring to Figure 29, compared to the control group (Figure 29H), HeLa cells pretreated with TPA-S-TPP did not show a fluorescence signal in dark conditions (Figure 29I), but the Annexin V-FITC channel under light irradiation It can be seen that there is a strong green fluorescence signal (J1, K1 in Figure 29) and a strong red signal (J2, K2 in Figure 20) in the PI channel. In addition, referring to Figure 32, it can be seen that the cell membrane of HeLa cells pretreated and irradiated with TPA-S-TPP was completely destroyed as a result of confocal microscopy imaging. Through the above results, it was confirmed that TPA-S-TPP effectively induces apoptosis of cancer cells under light irradiation.

Analysis Example 9. Mitochondrial membrane potential (MMP) analysis

9-1. JC-1 staining

HeLa cells were cultured in confocal culture plates at 37°C, humidified, 5% CO ₂ conditions, washed with DPBS, and incubated with Hank's Balanced Salt Solution (HBSS) containing TPA-S-TPP (5 μM) for 2 hours. cultured for a while. The cells were washed once again with DPBS, fresh cell culture medium was added, and white light (100 mW/cm ² ) was irradiated for 20 minutes. After incubation for 30 minutes, HeLa cells were treated with fresh cell culture medium containing the fluorescent probe JC-1 (2 μg/mL) at 37°C. Cells were washed three times with DPBS and confocal fluorescence images were obtained (FV 1200, Olympus).

JC-1 is red when the mitochondrial membrane potential is normal and green when it is decreased. Referring to Figure 29, it can be seen that upon light irradiation, the green fluorescence signal of HeLa cells cultured with TPA-S-TPP becomes stronger (M2 in Figure 29) and the red fluorescence signal becomes weaker (N2 in Figure 29). Referring to Figure 33, this means that the green-to-red fluorescence intensity ratio increased by 6.14 times compared to the group treated with TPA-S-TPP under dark conditions ("TPA-S-TPP + dark"), which shows the above results. It was confirmed that MMP was reduced by pretreatment with TPA-S-TPP and light irradiation.

9-2. Cleaved caspase 3 immunofluorescence analysis

Mitochondrial damage promotes the release of cytochrome C, initiating an apoptotic cascade such as activation of caspase 3. Therefore, caspase 3 activation was analyzed using cleaved caspase 3 antibody.

Specifically, HeLa cells were cultured on a slide for 24 hours, treated with TPA-S-TPP for 2 hours, and then irradiated with light (0, 5, 10, 20 minutes). This was washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then permeabilized with PBS containing 0.1% Triton X-100 at room temperature for 15 minutes. Then, the cells were cultured for 1 hour using blocking buffer (PBS supplemented with 10% normal goat serum) and incubated with primary antibody (Cell Signaling Tech.) in 1.5% normal goat serum overnight at 4°C. To remove excess primary antibody, cells were washed three times with PBS at 5-minute intervals. Secondary antibodies conjugated with Alexa Fluor 488 (Cell Signaling Tech.) were added to the cells and incubated in the dark at room temperature for 1 hour. Then, HeLa cells were washed three times with PBS at 5-minute intervals and mounted using UltraCruz Mounting Medium with DAPI (Santa Cruz Biotech.). Fluorescence images were obtained with DAPI (ex. 405 nm/em. 430-455 nm) and Alexa Fluor 488 (ex. 473 nm/em. 490-590 nm) using a confocal microscope (FV1200, Olympus).

Referring to Figure 29, it can be seen that the green fluorescence signal of HeLa cells cultured with AIEgen TPA-S-TPP (Q in Figure 29) is significantly stronger than that of the control group cultured in dark conditions (P in Figure 29). Additionally, their average fluorescence intensity was consistent with the cell imaging results (P to T in Figure 29).

9-3. western blotting

The results were confirmed by Western blotting. Specifically, HeLa cells were cultured in 6 well-plates for 24 hours and treated as follows: group 1, blank + light (7 min); Group 2, blank + light (15 minutes); Group 3, TPA-S-TPP (3 μM) + dark; Group 4, TPA-S-TPP (3 μM) + light (7 min); Group 5, TPA-S-TPP (3 μM) + light (15 min). Then, rinse with PBS and dissolve with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton After being placed on ice, it was centrifuged at 4°C, 13,000 rpm, for 10 minutes. The supernatant was collected and protein concentration was quantified using the BCA protein assay kit (Thermo Scientific). Equal amounts of protein (35 μg) were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with blocking buffer (5% nonfat dry milk in TTBS (0.1% Tween 20-TBS)) for 1 hour, washed with TTBS, treated with primary antibody dissolved in blocking buffer, and incubated overnight at 4°C. Afterwards, the membrane was washed three times with TTBS, incubated with secondary antibody for 2 hours, and protein bands were confirmed using Pierce ECL Western blotting substrate (Thermo Scientific). Primary antibodies, caspase-3, β-actin, and anti-rabbit IgG HRP-linked secondary antibodies were all purchased from Cell signaling Tech.

Referring to Figure 29, it can be seen that intracellular cleaved caspase 3 expression was upregulated in the “TPA-S-TPP + light” group (U in Figure 29). Through the above results, TPA-S-TPP reduces the mitochondrial membrane potential by generating singlet oxygen under light irradiation, promoting the release of cytochrome C, and subsequently activating caspase 3, efficiently causing mitochondria-mediated cell death. It was confirmed that it could be induced.

Analysis Example 10. Survival/Death Analysis

HeLa cells were cultured in confocal culture plates at 37°C in humidified, 5% CO ₂ conditions and then treated as follows: Group 1, irradiated with white light for 10 min (Control + light 10 min); Group 2, irradiated with white light for 30 minutes (Control + light 30min); Group 3, incubated with TPA-S-TPP (5 μM) at 37°C for 2 h (TPA-S-TPP + Dark); group 4; Incubate with TPA-S-TPP (5 μM) at 37°C for 2 hours and then irradiate with white light for 10 minutes (TPA-S-TPP + light 10 min); Group 5, incubated with TPA-S-TPP (5 μM) at 37°C for 2 hours and then irradiated with white light for 30 minutes (TPA-S-TPP + light 30 min). After treatment, HeLa cells were stained with Calcein AM (3 μM, Ex = 473 nm, Em = 490-540 nm) and Propidium Iodide (15 μg, Ex = 559 nm, Em = 575-675 nm) and confocal fluorescence images. was obtained (FV 1200, Olympus).

Referring to Figure 34, a strong green fluorescence signal was observed in both the control group and the “TPA-S-TPP + dark” group, confirming that there was no damage to the cells (A2, B2, C2 in Figure 34). On the other hand, cancer cells irradiated with TPA-S-TPP treatment were killed by light irradiation, and PI entered the nucleus across the seriously damaged cell membrane and generated a red fluorescent signal (D3, E3 in Figure 34). At this time, the green fluorescence signal was significantly reduced (D2, E2 in Figure 34). Through the above results, it was confirmed once again that TPP-S-TPP can effectively induce cell death by generating singlet oxygen during photodynamic treatment.

Analysis Example 11. In vivo imaging and phototherapy effect analysis

Animal experiments were performed with the approval of the Animal Ethics Committee of Dalian University of Technology (Certificate number/Ethics approval no. 2018-043). Female BALB/c mice aged 6 to 8 weeks were purchased from the SPF Laboratory Animal Center of Dalian Medical University, and a mouse tumor model transplanted with 4T1 tumor cells (1 x 10 ⁶ cells/mouse) was prepared and used.

When the tumor volume grew to approximately 150 mm ³ , in vivo imaging and phototherapy were performed. Mice were anesthetized and in situ injected with AIEgen TPA-S-TPP (200 μM, 100 μL), and then imaged at various times (1, 2, 3, 4, 6, 12, 24 and 48 hours) NightOWL II LB983) Images were obtained with an excitation laser at 550 nm and an emission filter at 700 nm. For phototherapy, mice were randomly divided into four groups (5 per group): 1, “PBS + dark”; 2, “PBS + light”; 3, “TPA-S-TPP + dark”; 4, “TPA-S-TPP + light”. After intratumor injection of TPA-S-TPP (200 μM, 100 μL), the tumor area was exposed to 550 ^nm Tumor volumes were measured daily. Tumor volume was calculated using the following formula.

V (mm ³ ) = a (mm) Х a (mm) Хb (mm)/2

(V, tumor volume; a, tumor width; b, tumor length.)

Referring to Figure 35, it can be seen that the NIR fluorescence signal in the tumor site of the mouse gradually increased compared to the control group and reached its peak after 12 hours, and a clear fluorescence signal was observed even after 48 hours (A in Figure 35). The same results were confirmed in ex vivo organ imaging (Figure 35B). In addition, in the case of groups 1, 2, and 3, tumor growth was confirmed to increase consistently after 14 days of treatment, showing that light irradiation itself or treatment with TPA-S-TPP alone did not have a cancer treatment effect ( 35C). On the other hand, when phototherapy was performed with AIEgen, tumor growth was significantly inhibited. The same results as above were confirmed when checking the size of the tumor with the naked eye (D in Figure 34). Through the above results, it was confirmed that TPA-S-TPP can significantly inhibit tumor growth when used in phototherapy and achieve high photodynamic treatment effects.

Analysis Example 12. H&E staining

After the experiment was performed in the same manner as in Analysis Example 11, the mice were euthanized and the major organs (heart, liver, spleen, lung, and kidney) were obtained and made into paraffin sections, and then histological analysis was performed through H&E staining. Mouse body weight was measured using an electronic balance (analytical balance).

Referring to Figure 35, it can be seen that there was no significant difference in mouse body weight between the "TPA-S-TPP + light" group and the other groups (E in Figure 35), and that no mouse death occurred during the treatment process. (F in Figure 35). In addition, as a result of histological analysis, it was confirmed that no noticeable inflammation or cell necrosis lesions were observed in any organ (G in Figure 35). Through the above results, it was confirmed that TPA-S-TPP has high biological stability and can therefore be used as an efficient AIE photosensitizer with excellent biocompatibility.

From the above analysis examples, it was confirmed that the compound according to one aspect has excellent photostability as it stays in the mitochondria of cancer cells for a long time, and that effective treatment is possible without side effects only on the target area (cancer cells) when irradiated with light. In addition, due to the aggregation-inducing properties in the near-infrared wavelength region, the efficiency is significantly higher compared to background fluorescence, and it is not affected by changes in mitochondrial membrane potential, so it was confirmed that it can overcome the shortcomings of existing fluorescent dyes and photosensitizers. Therefore, the compound according to one aspect or a pharmaceutically acceptable salt thereof is expected to be useful in fluorescence imaging, photodynamic diagnosis and treatment, and treatment of various diseases, including cancer.

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

Claims (22)

A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
<Formula 1>

In Formula 1,
X is O or S,
A is a substituted or unsubstituted C1-C4 alkyl group or a substituted or unsubstituted C2-C4 alkenyl group,
CY ₁ is triphenylamine (TPA),
CY ₂ is a cation of a heteroaryl group represented by the following formula (2),
<Formula 2>

In Formula 2,
R ₁ is a C1-C10 alkyl phosphonium cation, and the phosphonium cation is substituted with a substituted or unsubstituted C6-C20 aryl group,
R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are each independently hydrogen, a substituted or unsubstituted C1-C4 alkyl group, or a combination thereof,
The substituents of each of the substituted C1-C4 alkyl group, substituted C2-C4 alkenyl group, and substituted C6-C20 aryl group are C1-C40 alkyl group, C2-C40 alkenyl group, C2-C40 alkynyl group, It is selected from a C3-C40 cycloalkyl group, a C3-C40 cycloalkenyl group, and a C7-C40 aryl group. In claim 1,
R ₁ of Formula 2 is a C1-C10 alkyl phosphonium cation represented by Formula 3 below, or a pharmaceutically acceptable salt thereof.
<Formula 3>

In Formula 3 above,
R ₉ , R ₁₀ and R ₁₁ are each independently a substituted or unsubstituted C6-C20 aryl group,
R ₁₂ is a substituted or unsubstituted C1-C10 alkyl group,
* is a portion connected to the nitrogen (N) element of Formula 2,
The substituents of each of the substituted C6-C20 aryl group and the substituted C1-C10 alkyl group are C1-C40 alkyl group, C2-C40 alkenyl group, C2-C40 alkynyl group, C3-C40 cycloalkyl group, C3- It is selected from a cycloalkenyl group at C40 and an aryl group at C7-C40. In claim 1,
Wherein X is S, a compound or a pharmaceutically acceptable salt thereof. delete In claim 1,
A is a vinyl group, and CY ₂ is a quinoline cation substituted with a C1-C10 alkyl phosphonium cation, or a pharmaceutically acceptable salt thereof. In claim 1,
The CY ₂ is a quinoline cation substituted with a pentyl triphenylphosphonium cation, and the compound is represented by the following formula (4), or a pharmaceutically acceptable salt thereof.
<Formula 4>
In claim 1,
The compound or a pharmaceutically acceptable salt thereof has Aggregation-Induced Emission (AIE) properties. In claim 1,
The compound is an amphipathic compound or a pharmaceutically acceptable salt thereof. In claim 6,
The compound or pharmaceutically acceptable salt thereof, wherein the pentyl triphenylphosphonium cation targets mitochondria in cancer cells. In claim 1,
The compound is a compound or a pharmaceutically acceptable salt thereof that induces cancer cell death by generating reactive oxygen species (ROS) under light irradiation. A photosensitizer comprising the compound according to any one of claims 1 to 3 and 5 to 10 or a pharmaceutically acceptable salt thereof. In claim 11,
The photosensitizer is a photosensitizer that generates reactive oxygen species by irradiation with light in the wavelength range of 400 to 700 nm. In claim 11,
The photosensitizer targets fluorescence imaging and mitochondria. The photosensitizer according to claim 11, wherein the photosensitizer accumulates in cancer cells. A composition for contrast medium containing the compound according to any one of claims 1 to 3 and 5 to 10 or a pharmaceutically acceptable salt thereof. In claim 15,
The composition is a composition for contrast medium for specifically detecting cancer. delete delete A pharmaceutical composition for diagnosis or treatment of cancer targeting mitochondria, comprising the compound according to any one of claims 1 to 3 and 5 to 10 or a pharmaceutically acceptable salt thereof. In claim 19,
The above cancers include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, and uterus. Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or any one or more selected from the group consisting of acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. A pharmaceutical composition for diagnosis or treatment of cancer targeting mitochondria. 1) administering the compound according to any one of claims 1 to 3 and 5 to 10 or a pharmaceutically acceptable salt thereof to an entity other than a human; and
2) A method for diagnosing or treating cancer comprising the step of irradiating light to an object other than a human in step 1),
The above cancers include lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colon cancer, breast cancer, fallopian tube carcinoma, and uterus. Endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or any one or more selected from the group consisting of acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. , method of diagnosing or treating cancer. In claim 21,
A method of diagnosing or treating cancer, wherein the light is near-infrared rays with a wavelength of 650 to 900 nm.