Composition for cancer diagnosis targeting tumor and / or tumor microenvironment and use thereof

Abstract

The present invention relates to a composition for cancer diagnosis targeting a tumor and / or a tumor microenvironment and use thereof and, more specifically, provides a fluorescent contrast agent composition targeting biomolecules overexpressed in cancer cells and a tumor microenvironment. According to the present invention, a fluorescent contrast agent capable of targeting both cancer (folate receptor alpha-expressing cancer) cells and tumor-related macrophages in a tumor microenvironment can be provided.

Description

Composition for cancer diagnosis targeting tumor and / or tumor microenvironment and use thereof

[0001] The present invention relates to a cancer diagnostic composition targeting a tumor and / or a tumor microenvironment and its use, and more particularly, to a cancer diagnostic composition targeting a substance overexpressed in a tumor and / or a cancer microenvironment and its use.

[0002] The tumor microenvironment (TME) refers to the complex and diverse set of elements surrounding cancer cells, including other cells, extracellular matrix, growth hormones, and signaling molecules. The tumor microenvironment plays an important role as a culture medium for the growth of cancer cells.

[0003] Among the various immune cells that recruit to tumor sites, macrophages are particularly abundant throughout the entire cancer development process. Clinical and mouse studies have shown that they play a cancer-promoting role. Macrophages stimulate tumor angiogenesis and increase cancer cell invasion, migration, and intravascular invasion. During metastasis, macrophages prepare metastatic sites and promote cancer cell extravasation, survival, and sustained growth. These cancer-promoting activities are driven by distinct subpopulations of macrophages, suggesting that macrophages could be valuable targets for cancer therapy.

[0004] Research is underway on the role of tumor-associated macrophages in the cancer microenvironment, targeting them as cancer biomarkers and therapeutic targets. Tumor growth is promoted by tumor-associated macrophages in various cancer tissues, and the abundance of tumor-associated macrophages is associated with lower survival rates and poorer prognosis in cancer patients. Therefore, targeting tumor-associated macrophages is considered a novel strategy for cancer treatment. Therefore, tumor-associated cells can serve as important diagnostic and prognostic markers as well as therapeutic targets. Therefore, angiography targeting tumor-associated macrophages as a biomarker can visualize tumor location and the interface between cancer and normal tissue in real time during surgery.

[0005] With the growing importance of intraoperative cancer detection imaging, various near-infrared fluorescent contrast agents capable of passive or active targeting have been developed. However, passive targeting using ICG has the disadvantage of low fluorescence under near-infrared light and rapid clearance from the liver due to binding to blood albumin when injected in vivo, resulting in low cancer detection efficiency. Active targeting also faces ongoing stability issues in the human body. Most existing targeted fluorescent contrast agents are synthesized targeting proteins already widely known to be overexpressed in cancer, and therefore rely on mechanistic studies of specific proteins. However, the cancer microenvironment, which refers to the surrounding environment of a cancer, is composed of diverse immune cells regardless of the type or mutation of the cancer and maintains a relatively similar environment. Therefore, the development of a fluorescent contrast agent that can target the cancer microenvironment is expected to enable universal use regardless of the type or mutation of the cancer.

[0006] Patent documents related to the present invention include Korean Patent Publication No. 10-2020-0027895. Non-patent documents related to the present invention include Yang C et al., ZW800-PEG: A Renal Clearable Zwitterionic Near-Infrared Fluorophore for Potential Clinical Translation. Angew Chem Int Ed Engl. 2021 Jun 14;60(25):13847-13852.

[0007] The present invention relates to a cancer diagnostic composition targeting cancer and / or a cancer microenvironment and its use, and more specifically, to a cancer diagnostic composition targeting folate receptor alpha of cancer cells, folate receptor beta of tumor-associated macrophages, and / or mannose receptor of tumor-associated macrophages.

[0008] However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the relevant technical field from the description below.

[0009] In order to solve the above problem, the present invention can provide a compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof:

[0010] [Chemical Formula 1]

[0011]

[0012] In the above chemical formula 1,

[0013] R 1 and R 2 are the same or different from each other, and each independently represents hydrogen, a hydroxyl group, a C1-C5 alkoxy group, (Y) m X, (Y) m COO - , (Y) m SO3 - , (Y) m PO3H -, and at least one selected from the group consisting of combinations thereof,

[0014] R 3 and R 4 are identical or different from each other, and each is independently (Y) n H, (Y) n (CO)R 5 and at least one selected from the group consisting of combinations thereof,

[0015] R 5 is a hydroxyl group or And,

[0016] A is a C2-C6 heterocycloalkyl group containing at least one heteroatom selected from the group consisting of substituted or unsubstituted N, O and S,

[0017] X is a halogen group,

[0018] Y is CH2 or CH2CH2O,

[0019] Z is halogen or And,

[0020] B is hydrogen or And,

[0021] a, b, m and n are the same or different and can each independently be an integer from 0 to 5.

[0022] As one embodiment of the present invention, R in the chemical formula 1 1 and R 2 are each independently hydrogen or (Y) m SO3 - It could be.

[0023] As another embodiment of the present invention, R in the chemical formula 1 3 and R 4 are each independently (Y) n (CO)R 5 And,

[0024] R 5 is a hydroxyl group or And,

[0025] A may be a C2-C6 heterocycloalkyl group containing one or more heteroatoms selected from the group consisting of substituted or unsubstituted N, O and S.

[0026] As another embodiment of the present invention,

[0027] The above A is It could be.

[0028] As another embodiment of the present invention,

[0029] In the above chemical formula 1, Z is Cl, , or

[0030] It could be.

[0031] In another embodiment of the present invention, the compound may be at least one selected from the group consisting of compounds represented by the following [Chemical Formula 1-1] to [Chemical Formula 1-6].

[0032] [Chemical Formula 1-1]

[0033] ;

[0034] [Chemical Formula 1-2]

[0035] ;

[0036] [Chemical Formula 1-3]

[0037] ;

[0038] [Chemical Formula 1-4]

[0039] ;

[0040] [Chemical Formula 1-5]

[0041] ; and

[0042] [Chemical Formula 1-6]

[0043] .

[0044] In addition, the present invention can provide a contrast agent composition for cancer diagnosis comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0045] As one embodiment of the present invention, the contrast agent composition can target a tumor and / or a tumor microenvironment.

[0046] In another embodiment of the present invention, the contrast agent composition can bind to at least one of folate receptor alpha of cancer cells, folate receptor beta of tumor-associated macrophages (TAMs) in the tumor microenvironment, and CD206 of TAMs.

[0047] As another embodiment of the present invention, the contrast agent composition can simultaneously target a tumor and a tumor microenvironment.

[0048] Additionally, the present invention provides a method for diagnosing cancer, comprising a step of targeting a tumor and / or a tumor microenvironment using any one of the above compositions.

[0049] The present invention relates to a composition for cancer diagnosis or cancer imaging that targets tumor-associated macrophages in a cancer microenvironment, and comprises folic acid and a fluorescent substance, and more specifically, provides a fluorescent contrast agent composition that targets folate receptor alpha of tumor cells and / or folate receptor beta of tumor-associated macrophages as biomarkers. According to the present invention, a fluorescent contrast agent that can react more specifically than fluorescent contrast agents that react to simple characteristics of the cancer microenvironment, such as temperature, pH, and hypoxia, can be provided, and can specifically target tumors (folate receptor alpha-expressing tumors) and tumor-associated macrophages in the tumor microenvironment.

[0050] Figure 1 shows the absorbance and fluorescence analysis results of compound 1.

[0051] Figure 2 shows the absorbance and fluorescence analysis results of compound 2.

[0052] Figure 3 shows the absorbance and fluorescence analysis results of compound 4.

[0053] Figure 4 shows the absorbance and fluorescence analysis results of compound 6.

[0054] Figure 5a shows the fluorography results obtained by the compound of the present invention according to folate receptor expression.

[0055] Figure 5b shows the fluorescence intensity of the fluorescence photography experiment.

[0056] Figure 6 shows the cell viability when compound 6 (FMK-2) was treated at various concentrations in two lung cancer cell lines, A549 and H522.

[0057] Figure 7a shows the fluorescence results of a mouse model experiment for compound 6 of the present invention.

[0058] Figure 7b shows the ratio of near-infrared fluorescence signals appearing in tumors compared to normal tissues.

[0059] Figure 8 shows the tissues of a mouse model administered with compound 6 of the present invention, frozen and sliced, and analyzed by near-infrared fluorescence analysis, H&E staining, and immunohistochemical analysis.

[0060] Figure 9 shows the results of detecting fluorescence by extracting organs after injecting the compound of the present invention into a mouse.

[0061] Figure 10 is a half-life graph of FMK-2.

[0062] Figure 11 shows the results of fluorescence analysis confirmed after intravenous injection of FMK-2 into an inflammation model in a mouse.

[0063] Figure 12 shows the results of a comparative experiment on staining ZW800-PEG and human lung cancer cell lines.

[0064] Figure 13 shows the results of fluorescent imaging of cancer tissue after intravenous injection of OCTL14 and FMK-2 into a mouse cancer model.

[0065] Figure 14 is a schematic diagram of the tumor microenvironment.

[0066] FIG. 15 is a drawing showing the mechanism by which a composition for cancer imaging manufactured according to one embodiment of the present invention exhibits fluorescence.

[0067] Hereinafter, embodiments are described in detail with reference to the attached drawings. However, the embodiments may be modified in various ways, and the scope of the patent application is not limited or restricted by these embodiments. It should be understood that all modifications, equivalents, or alternatives to the embodiments are included within the scope of the patent application.

[0068] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0069] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0070]

[0071] In addition, when describing with reference to the attached drawings, identical components will be assigned the same reference numerals regardless of the drawing numbers, and redundant descriptions thereof will be omitted. When describing embodiments, if a detailed description of a related known technology is judged to unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.

[0072] Components included in one embodiment and components with common functions will be described using the same names in other embodiments. Unless otherwise stated, the descriptions given in one embodiment can be applied to other embodiments, and detailed descriptions will be omitted to the extent of overlap.

[0073]

[0074] Hereinafter, the cancer diagnostic composition targeting cancer and / or the cancer microenvironment of the present invention and its use will be described in detail with reference to examples and drawings. However, the present invention is not limited to these examples and drawings.

[0075]

[0076] The present invention provides a compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof:

[0077] [Chemical Formula 1]

[0078]

[0079] In the above chemical formula 1,

[0080] R 1 and R 2 are the same or different from each other, and each independently represents hydrogen, a hydroxyl group, a C1-C5 alkoxy group, (Y) m X, (Y) m COO - , (Y) m SO3 - , (Y) m PO3H -, and at least one selected from the group consisting of combinations thereof,

[0081] R 3 and R 4 are identical or different from each other, and each is independently (Y) n H, (Y) n (CO)R 5 and at least one selected from the group consisting of combinations thereof,

[0082] R 5 is a hydroxyl group or And,

[0083] A is a C2-C6 heterocycloalkyl group containing at least one heteroatom selected from the group consisting of substituted or unsubstituted N, O and S,

[0084] X is a halogen group,

[0085] Y is CH2 or CH2CH2O,

[0086] Z is halogen or And,

[0087] B is hydrogen or And,

[0088] a, b, m and n are the same or different and can each independently be an integer from 0 to 5.

[0089] In the present invention, the term “substitution” refers to a reaction in which an atom or atomic group included in a molecule of a compound is replaced with another atom or atomic group.

[0090] In the present invention, the term “chain alkyl group” refers to a group derived from a straight-chain or branched-chain saturated aliphatic hydrocarbon having a specific number of carbon atoms and at least one valence. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 2-butyl, 3-butyl, pentyl, n-hexyl, and the like.

[0091] In the present invention, the term “halogen group” refers to elements belonging to group 17 of the periodic table, such as fluorine (F), chloride (Cl), bromine (Br), or iodine (I).

[0092] In the present invention, the term “alkoxy group” refers to an atomic group C formed by bonding an oxygen atom to an alkyl group. n H 2n +O-, examples of such alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, or phthaloxy.

[0093] In the present invention, the term “heterocycloalkyl” refers to a stable 3- to 18-membered saturated or partially unsaturated radical consisting of 2 to 20, preferably 2 to 15, preferably 2 to 10, preferably 2 to 6 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, for example 1 to 5 heteroatoms, 1 to 4 heteroatoms, 1 to 3 heteroatoms, or 1 to 2 heteroatoms.

[0094] In the present invention, the term “pharmaceutically acceptable salt” means a formulation of a compound that does not cause serious irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salt can be obtained by reacting the compound of the present invention with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., a sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, etc., an organic carboxylic acid such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc. In addition, the compound of the present invention can be obtained by reacting it with a base to form a salt such as an alkali metal salt such as an ammonium salt, a sodium or potassium salt, an alkaline earth metal salt such as a calcium or magnesium salt, a salt of an organic base such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and a salt of an amino acid such as arginine or lysine.

[0095] In addition, the present invention can provide a contrast agent composition for cancer diagnosis comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0096] In the present invention, "including as an active ingredient" means that the ingredient is included in an amount necessary or sufficient to realize a desired biological effect. In actual application, the amount included as an active ingredient can be determined by considering the amount for treating or diagnosing the target disease and not causing other toxicity, and may vary depending on various factors such as the disease or condition being treated, the form of the composition being administered, the size of the subject, or the severity of the disease or condition. A person of ordinary skill in the art to which the present invention pertains can empirically determine the effective amount of an individual composition without undue experimentation.

[0097] In the present invention, the term "contrast agent" refers to a substance administered into the body to strongly and specifically contrast or image cancer cells, etc. in the body, and is currently widely used in the medical and diagnostic fields to enhance images of tissues and cells. The term "contrast agent" in the present invention is not limited to the scope of contrast agents for magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET), and is used to mean imaging agents for ultrasound image analysis, imaging agents for fluorescence image analysis, etc.

[0098] In one embodiment of the present invention, the contrast agent composition can target a tumor and / or a tumor microenvironment. The target may be formed by including a ligand within the composition that binds to a biomolecule overexpressed in the tumor and / or the tumor microenvironment.

[0099] The above term “overexpressed biomolecule” may be a receptor, biomarker, exosome, protein, genetic material and enzyme that is expressed at a higher level in cancer cells and cancer microenvironment than in normal cells, preferably a receptor, and most preferably a folate receptor and a mannose receptor.

[0100] In the present invention, the term “ligand” refers to a substance that forms a complex by binding to a biomolecule overexpressed in cancer and / or a cancer microenvironment, and may specifically be a protein, peptide, polypeptide, sugar, organic acid, fatty acid, and glycoprotein, and may preferably be a substrate that binds to a receptor, and most preferably may be folic acid or mannose.

[0101] The above ligand may preferably be independently substituted at the -NH2 terminal and the -COOH terminal of the compound of the above [Chemical Formula 1-3]. According to the most preferred embodiment of the present invention, folic acid may be substituted at the -NH2 terminal, and mannose may be substituted at the -COOH terminal.

[0102] According to one aspect, the -NH2 terminal can be modified into a ligand that binds to folate receptor alpha (FR α) of cancer cells and / or folate receptor beta (FR β) of tumor-associated macrophages (TAMs).

[0103] According to one side, the ligand is folic acid and can be represented by the following [chemical formula 1-4].

[0104] [Chemical Formula 1-4]

[0105]

[0106] Folate is an essential nutrient for the division and growth of both cancer cells and normal cells, and is absorbed and metabolized within cells through folate receptors. Folate receptors are overexpressed in various cancer cells, such as renal cancer, ovarian cancer, pituitary tumors, and colon cancer, and can be used as molecular targets for diagnosis and treatment. Folate can act as a targeting ligand that enables the uptake of folate conjugates into the target cells by specifically binding to folate receptors overexpressed on the surfaces of target cancer cells and inflammatory cells. More specifically, it can target folate receptor alpha, which is overexpressed in cancer cells, and folate receptor beta, which is overexpressed in tumor-associated macrophages. Based on these characteristics, the compound of the present invention can have the effect of simultaneously targeting cancer cells and tumor-associated macrophages.

[0107] In another embodiment of the present invention, the contrast agent composition can bind to at least one of folate receptor alpha of cancer cells, folate receptor beta of tumor-associated macrophages (TAMs) in the tumor microenvironment, and CD206 of TAMs.

[0108] The folate receptor functions to reduce folate derivatives by binding to folic acid, and is a receptor that mediates the intracellular transport of folate after binding to folic acid or a folic acid derivative. The folate receptor may be expressed to a limited extent in normal tissues, but is overexpressed in tumors, allowing it to be used as a target for selective drug delivery to tumor tissue.

[0109] Folate receptors exist in three isomeric forms, folate receptor alpha, beta, and gamma, of which alpha and beta are typically bound to the cell membrane by a glycosyl phosphatidylinositol (GPI) anchor, allowing recycling between the extracellular and endocytic compartments and transport of folate into the cell.

[0110] The folate receptor beta (FR β, FR-beta, FR B, FOLR-2, or FOLR2) is overexpressed in M2-type polarized tumor-associated macrophages, and can target tumor-associated macrophages within the cancer microenvironment. The folate receptor beta can bind folic acid and reduced folate derivatives and mediate the intracellular transport of 5-methyl tetrahydrofolate (5-methyl tetrahydrofolate) and folate analogs, and has a high affinity for folate and folate analogs at neutral pH. Furthermore, exposure to slightly acidic pH after receptor endocytosis can induce a conformational change that significantly reduces the affinity for folate and mediates its release.

[0111] The above tumor-associated macrophages (TAMs) may preferably have an M2 type macrophage phenotype and exist in the cancer microenvironment, but are not specifically limited to the M2 type. The above macrophages are one of the phenotypes of the final differentiation of myeloid cells, and refer to myeloid cells that mainly exist in the bone marrow that migrate to the cancer microenvironment via the blood and differentiate, and can be polarized into M1 macrophages and M2 macrophages under various physiological and pathological conditions in vivo. Tumor-associated macrophages play a crucial role in linking inflammation and cancer, and can promote tumor angiogenesis by increasing the proliferation, invasion, and metastasis of cancer cells, and can play a role in inducing tumor progression by inhibiting the anti-cancer immune response mediated by T cells, and can be used as a potential therapeutic target for cancer and a biomarker for the diagnosis and prognosis of cancer.

[0112] The tumor microenvironment (TME) refers to the environment surrounding a tumor or cancer. It is a comprehensive term for the complex and diverse components surrounding cancer cells, including other cells, the extracellular matrix, growth hormones, and signaling molecules. Regardless of the type or mutation of the cancer, the cancer microenvironment is known to be composed of diverse immune cells and maintains a relatively similar environment.

[0113] According to one aspect, the -COOH terminal of the compounds of the above [Chemical Formula 1-3] and [Chemical Formula 1-4] may be modified into a ligand targeting cancer cells or a tumor-associated macrophage (TAM) receptor substrate.

[0114] The above tumor-associated macrophage receptor can enable the diagnostic composition for cancer to bind more specifically to M2 type macrophage tumor-associated macrophages.

[0115] The above tumor-associated macrophage (TAM) receptor substrate can specifically bind to the tumor-associated macrophage (TAM) receptor and effectively target tumor-associated macrophages (TAM).

[0116] According to one aspect, the tumor-associated macrophage (TAM) receptor may preferably be CD206. The CD206 is a mannose receptor, which refers to a type I transmembrane protein with an extracellular N-terminus and an intracellular C-terminus, and when applied to a contrast agent containing a fluorescent substance, it can induce tumor-associated macrophages (TAM) polarized to the M2 type and express folate receptor beta.

[0117] According to one aspect, the tumor-associated macrophage (TAM) receptor substrate may be a single molecule, preferably mannose. The mannose is a receptor substrate that specifically binds to CD206, a tumor-associated macrophage receptor within the tumor microenvironment, and the compound modified as described above may be represented by the following [Chemical Formula 1-5] or [Chemical Formula 1-6].

[0118] [Chemical Formula 1-5]

[0119] ; and

[0120] [Chemical Formula 1-6]

[0121] .

[0122]

[0123] As another embodiment of the present invention, the contrast agent composition can simultaneously target a tumor and a tumor microenvironment.

[0124] Additionally, the present invention provides a method for diagnosing cancer, comprising a step of targeting a tumor and / or a tumor microenvironment using any one of the above compositions.

[0125] Another aspect of the present invention is to provide a cancer diagnosis method that simultaneously targets folate receptor alpha-expressing cancer and the tumor microenvironment.

[0126] According to one embodiment, the folate receptor alpha-expressing cancer may be any one selected from the group consisting of lung cancer, mesothelioma, ovarian cancer, renal cancer, brain cancer, cervical cancer, nasopharyngeal cancer, squamous cell carcinoma of the head and neck, endometrial cancer, breast cancer, bladder cancer, pancreatic cancer, bone cancer, pituitary cancer, colorectal cancer, and medullary thyroid cancer.

[0127] The folate receptor alpha targets cancer tumor cells, and the folate receptor beta targets tumor-associated macrophages.

[0128] As used herein, the term “diagnosis” includes determining the susceptibility of an individual to a specific disease or condition, determining whether an individual currently has a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, therametrics (e.g., monitoring the condition of an individual to provide information on the efficacy of a treatment), or theranostics (e.g., diagnosing a disease using a substance that targets a lesion and simultaneously delivering a drug only to the affected area to treat the disease). Specifically, in the present invention, “diagnosis” means determining whether lung cancer cells have invaded the visceral pleural elastin layer in an individual.

[0129] In the present invention, the term “subject” is not limited to a mammal such as a livestock or human that requires diagnosis, but may preferably be a human.

[0130] The contrast agent composition according to the present invention can be administered via various routes, including oral, transdermal, subcutaneous, intravenous, or intramuscular. The dosage of the active ingredient can be appropriately selected based on various factors, such as the route of administration, the patient's age, sex, weight, and severity of the condition. Furthermore, the composition of the present invention can be administered in combination with known compounds capable of enhancing the desired effect.

[0131] The present invention also provides an imaging method comprising the step of treating a biological sample with the contrast agent composition. The present invention also provides an imaging method comprising the step of administering the contrast agent composition to a subject.

[0132] In the present invention, the term "imaging," also known as "imaging," refers to all methods for visualizing a target object. In the present invention, imaging may preferably be optical imaging using light.

[0133] The above “imaging” may be, but is not limited to, one or more selected from the group consisting of fluorescence, bioluminescence, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and combinations thereof.

[0134] The compounds of [Chemical Formula 1-1] to [Chemical Formula 1-6] of the present invention can function as fluorescent substances in a dark environment. The fluorescent substance is a substance that emits color in response to light of a specific wavelength, and includes molecules, metal ions, complex compounds, organic dyes, conductors, semiconductors, insulators, quantum dots, etc. that emit light in an excited state, and examples thereof include, but are not particularly limited to, cyanine series, pyrene series, NIR series, Alexa series, and ZW series.

[0135] The fluorescent material preferably refers to a contrast agent used in fluorescence imaging, one of the imaging diagnostic methods used in cancer diagnosis, and uses a material that emits fluorescence when exposed to exited site light of a specific wavelength. In this case, the body is exposed to exited site light outside the body, and the fluorescence emitted from the fluorescent contrast agent inside the body is detected.

[0136] The composition for cancer imaging may be a fluorescent contrast agent, and the fluorescent material used in the fluorescent contrast agent may preferably be a material that exhibits absorption in the near-infrared light range, from 700 to 1300 nm, and most preferably, may be excited at a wavelength of 779 nm and emit a wavelength of 807 nm. When a fluorescent material in the visible light range corresponding to 400 to 600 nm, which is outside this wavelength range, is used, the light transmittance through biological tissue is very low, and it is almost impossible to detect lesions deep in the body, whereas near-infrared light exhibits high transmittance through biological tissue and can pass through a skull measuring about 10 cm.

[0137] The fluorescence wavelength of the compound of the present invention has a wavelength similar to that of Indocyanine Green (ICG), a fluorescent substance conventionally used in cancer surgery, and thus has the advantage of being able to use the fluorescence imaging equipment currently used in clinical practice.

[0138] The fluorescent contrast agent may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0139] Hereinafter, the present invention will be described in detail with reference to the following examples and comparative examples. However, the technical concept of the present invention is not limited or restricted thereby.

[0140]

[0141] Manufacturing Example 1. Synthesis of Precursor 1

[0142] All chemicals and solvents used were of American Chemical Society or HPLC purity. HPLC-grade methanol (MeOH), ethanol (EtOH), and distilled water (DW) were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other chemicals, including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate (EA), and N,N-diisopropylethylamine (DIEA), were purchased from Fisher Scientific (Pittsburgh, PA, USA) and Sigma-Aldrich (St. Louis, MO, USA). The purity of all compounds was determined using liquid chromatography-mass spectrometry (LC-MS) consisting of an Alliance e2695 separation module (Waters), a 2998PDA detector (Waters, 212–800 nm), and an Acquity QDA detector (Waters, m / z range: 50–1,239). An XBridge C18 (4.6'150 mm, 5 μm) reversed-phase HPLC column (Waters) was used for LC-MS. The final compound was purified using preparative HPLC consisting of a Waters 2489 UV / Visible detector and a Waters 1525 Binary HPLC pump. An XBridge Prep C18 (19'150 mm, 5 μm) reversed-phase HPLC column (Waters) was used, and the purity of the final compound was greater than 90%, as measured by a photodiode array (PDA) at 254 / 750 nm absorbance.

[0143] [Precursor 1]

[0144]

[0145] Precursor 1 (1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indol-1-ium-5-sulfonate, (SC-COOH)) was synthesized as follows.

[0146] A mixture of potassium 2,3,3-trimethyl-3H-indole-5-sulfonate (10.0 g, 36 mmol) and 3-bromopropanoic acid (6.3 g, 42 mmol) in toluene (150 mL) was heated at 110°C for 24 h under a nitrogen atmosphere. The mixture was cooled to room temperature and the solvent was decanted. Methanol (20 mL) was added to the mixture and stirred for 30 min to dissolve it. The mixture was filtered and collected. The undissolved solid was removed by filtration. Then, the mixed solution was slowly added to ethyl acetate (700 mL) using a dropping funnel. The precipitate was filtered, dried, and identified by LC-MS. The product was collected as a pink solid and used in the next step without further purification (3.9 g, 35% yield). The product 1 H NMR data and 13 C NMR data are as follows. Accurate mass TOF MS m / z [M]+ calculated for [C14H17NO5S]+ 311.08, found [M+H]+ 312.08. 1 H NMR (500 MHz, DMSO-d6) δ 8.01 - 7.90 (m, 2H), 4.74 (t, J = 8.6 Hz, 1H), 2.91 (d, J = 8.7 Hz, 1H), 1.71 (s, 3H). 13 C NMR (125 MHz, DMSO-d6) δ 193.78, 175.40, 150.07, 140.22, 139.61, 130.80, 123.47, 117.50, 45.10, 44.17, 35.80, 26.13, 15.51.

[0147]

[0148] Manufacturing Example 2. Synthesis of Compound 1 (KBSF2)

[0149] [Chemical Formula 1-1]

[0150]

[0151] Compound 1 (3-(2-((E)-2-((E)-3-(2-((E)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propanoate, 3-(2-((E)-2-((E)-3-(2-((E)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propanoate) was synthesized as follows.

[0152] A mixture of bromide salt, precursor 1 (5.0 g, 16.1 mmol), Vilsmeier-Haack reagent (2.5 g, 7.1 mmol), and anhydrous sodium acetate (2.0 g, 24.1 mmol) in anhydrous ethanol (50 mL) was heated under reflux at 85°C and stirred for 2 h in a nitrogen atmosphere. To obtain the best purity and yield, the molar ratio of precursor 1 to Vilsmeier-Haack reagent was set higher than 2:1. The mixture was checked 2.5 h after the reaction to confirm the completion of the reaction. After the reaction, it was cooled to room temperature, filtered, washed with ethanol, collected, and dried to obtain compound 1 as a dark green solid (4.4 g, 81% yield). The absorbance and fluorescence analysis results of compound 1 are shown in Figure 1. The prepared product 1 H NMR data and 13 C NMR data are as follows. Accurate mass TOF MS m / z [M]+ calculated for [C36H39ClN2O10S2]+, found [M+H]+ 759.18. 1H NMR (500 MHz, DMSO-d6) δ 8.09 - 8.02 (m, 2H), 7.97 (dd, J = 8.1, 2.2 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.82 (s, 1H), 7.65 (dd, J = 8.0, 2.1 Hz, 1H), 7.06 - 6.97 (m, 2H), 6.91 (dt, J = 15.1, 0.8 Hz, 1H), 6.69 - 6.58 (m, 2H), 4.84 (t, J = 8.5 Hz, 2H), 3.95 (t, J = 5.7 Hz, 2H), 2.88 (d, J = 8.6 Hz, 1H), 2.76 - 2.59 (m, 5H), 1.75 (s, 4H), 1.67 - 1.58 (m, 2H), 1.62 (s, 5H). 13 C NMR (125 MHz, DMSO-d6) δ 176.20, 174.84, 171.84, 159.01, 148.89, 148.24, 144.16, 140.65, 139.47, 138.14, 136.77, 136.33, 135.09, 132.79, 131.26, 130.75, 129.06, 122.91, 122.63, 122.01, 121.31, 112.39, 96.28, 45.51, 45.19, 43.48, 43.35, 35.75, 32.46, 28.09, 28.06, 27.19, 27.09, 23.02.

[0153]

[0154] Manufacturing Example 3. Synthesis of Compound 2 (MAN-KBSF2)

[0155] [Chemical Formula 1-2]

[0156]

[0157] Compound 2 (2-((E)-2-((E)-2-chloro-3-(2-((E)-3,3-dimethyl-1-(3-oxo-3-(((3R,4R, 5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)propyl)-5-sulfoindolin-2-ylidene)ethylidene)cyclohex-1-en-1 -yl)vinyl)-3,3-dimethyl-1-(3-oxo-3-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-2-yl)amino)propyl)-5-sulfo-3H-indol-1-ium, 2-((E)-2-((E)-2-chloro-3-(2-((E)-3,3-dimethyl-1-(3-oxo-3-(((3R,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)propyl)-5-sulfoindolin-2-ylidene)e thylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-oxo-3-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)propyl)-5-sulfo-3H-indol-1-ium) was synthesized as follows.

[0158] A mixture of compound 1 (1.0 g, 1.32 mmol), (3R,4R,5R,6S)-2-amino-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (0.6 g, 3.30 mmol) and DMTMM (1.83 g, 6.6 mmol) was sonicated in anhydrous methanol and reacted for 6 h in a nitrogen atmosphere. After the reaction, the mixture was precipitated in EA, filtered, washed with EtOH, collected and dried to obtain compound 2 as a dark green solid (0.7 g, 48% yield). The absorbance and fluorescence analysis results of compound 2 are shown in Figure 2. The prepared product 1 H NMR data and 13C NMR 데이터는 다음과 같다. Accurate mass TOF MS m / z [M]+ calculated for [C48H61ClN4O18S2]+, found [M+H]+ 1081.31. 1 H NMR (500 MHz, DMSO-d6) δ 9.22 (d, J = 8.2 Hz, 1H), 8.09 - 8.02 (m, 1H), 8.00 - 7.90 (m, 1H), 7.83 - 7.75 (m, 1H), 7.65 (dd, J = 8.0, 2.1 Hz, 1H), 7.06 - 6.97 (m, 1H), 6.91 (dt, J = 15.1, 0.9 Hz, 1H), 6.69 - 6.58 (m, 1H), 5.70 (d, J = 3.2 Hz, 1H), 4.94 (td, J = 4.4, 3.2 Hz, 1H), 4.85 - 4.76 (m, 1H), 4.66 (d, J = 4.7 Hz, 1H), 4.48 - 4.40 (m, 2H), 3.91 (t, J = 5.1 Hz, 1H), 3.81 - 3.68 (m, 2H), 3.63 (ddd, J = 12.1, 4.5, 3.2 Hz, 1H), 3.48 (tt, J = 8.1, 4.7 Hz, 1H), 3.42 - 3.36 (m, 1H), 3.11 (td, J = 7.2, 4.6 Hz, 1H), 2.82 - 2.65 (m, 3H), 2.60 - 2.48 (m, 1H), 1.75 (s, 2H), 1.67 - 1.58 (m, 1H), 1.62 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 173.00, 172.72, 171.84, 159.06, 148.89, 148.50, 143.38, 140.65, 139.60, 138.14, 136.77, 136.33, 135.09, 132.79, 131.26, 130.75, 129.06, 122.91, 122.63, 122.02, 121.24, 112.37, 96.28, 94.53, 94.48, 75.40, 72.12, 72.09, 70.68, 61.63, 55.75, 55.72, 45.50, 45.33, 44.21, 43.35, 34.92, 34.36, 28.09, 28.06, 27.19, 27.08, 23.02.

[0159]

[0160] Manufacturing Example 4. Synthesis of Compound 3 (KBSF2-PEG-NH2)

[0161] [Chemical Formula 1-3]

[0162]

[0163] Compound 3(2-((E)-2-((E)-2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)thio)-3-(2-((E)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfo- 3H-indol-1-ium, 2-((E)-2-((E)-2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)thio)-3-(2-((E)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium) was synthesized as follows.

[0164] Compound 1 (1.0 g, 1.32 mmol) was added to 8 mL of distilled water in a 100 mL round-bottom flask. DIEA (0.4 mL; 2.34 mmol; 2.0 molar equivalents) was pipetted into the flask and mixed. tert-Butyl(2-(2-(2-mercaptoethoxy)ethoxy)ethyl)carbamate (390 mg; 1.47 mmol; 1.11 molar equivalents) in 2 mL of DMSO was added to the reaction solution, which was heated in an oil bath at 60 °C for 40 min. The reaction progress was monitored using an LC-MS system at the 40-min time point. 1 mL of 6 M HCl was added at 60 °C and the reaction was continued for 0.5 h for deprotection. After the reaction, it was cooled to room temperature and slowly poured into 100 mL of a mixture of acetone / EA (1:1) and stirred at room temperature for 30 minutes. The suspension was filtered to collect the precipitate. The precipitate was dried overnight at room temperature and compound 3 was collected as a dark green solid (0.9 g, 57% yield). The product thus prepared was 1 H NMR data and 13 C NMR data are as follows. Accurate mass TOF MS m / z [M]+ calculated for [C54H75N5O20S3]+, found [M+H]+ 1210.62. 1H NMR (500 MHz, DMSO-d6) δ 8.09 - 8.02 (m, 1H), 8.00 - 7.90 (m, 1H), 7.83 - 7.75 (m, 1H), 6.27 - 6.20 (m, 1H), 5.70 (d, J = 3.2 Hz, 1H), 4.94 (td, J = 4.4, 3.2 Hz, 1H), 4.85 - 4.76 (m, 1H), 4.66 (d, J = 4.7 Hz, 1H), 4.48 - 4.40 (m, 2H), 3.91 (t, J = 5.1 Hz, 1H), 3.77 (dd, J = 8.4, 7.1, 4.5, 1.5 Hz, 1H), 3.76 - 3.67 (m, 2H), 3.63 (s, 1H), 3.67 - 3.59 (m, 1H), 3.53 - 3.36 (m, 2H), 3.15 - 3.02 (m, 2H), 2.86 - 2.74 (m, 1H), 2.77 - 2.70 (m, 2H), 2.70 (td, J = 7.1, 0.9 Hz, 1H), 2.60 - 2.48 (m, 1H), 1.75 (s, 1H), 1.62 (s, 2H), 1.64 - 1.56 (m, 1H). 13 C NMR (125 MHz, DMSO-d6) δ 173.50, 172.88, 172.72, 160.01, 148.50, 145.14, 144.05, 143.38, 140.65, 140.57, 139.60, 138.14, 136.77, 134.54, 131.26, 130.31, 129.06, 122.91, 122.63, 121.24, 120.13, 112.37, 95.98, 94.53, 94.48, 75.40, 72.97, 72.12, 72.09, 71.01, 70.68, 69.99, 69.30, 61.63, 55.75, 55.72, 45.50, 45.33, 44.21, 43.35, 41.72, 34.92, 34.36, 31.78, 28.09, 27.76, 27.08, 26.62, 22.86.

[0165]

[0166] Manufacturing Example 5. Synthesis of Compound 4 (FA-KBSF2)

[0167] [Chemical Formula 1-4]

[0168]

[0169] Compound 4(2-((E)-2-((E)-2-((1-(4-(((2-amino-4-oxo-1,4-dihydropteridin-7-yl)methyl)amino)phenyl)-3-carboxy-1,6-dioxo-10,13-dioxa-2,7-diazapentadecan-15-yl)thio)-3-(2-((E)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium, 2-((E)-2-((E)-2-((1-(4-(((2-amino-4-oxo-1,4-dihydropteridin-7-yl)methyl)amino)phenyl)-3-carboxy-1,6-dioxo-10,13-dioxa-2,7-diazapentadecan-15-yl)thio)-3-(2- ((E)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(2-carboxyethyl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium) It was synthesized as follows.

[0170] Folic acid (4 mg, 9.1 nmol), EDC (2 mg), and HOBt (1.4 mg) were added to 1 mL of DMSO in a 1.5 mL EP tube. DIEA (5 μl) was added to the reaction solution under vortexing for 2 h at room temperature. 7.2 mg of compound 3 (8.1 nM) was added, and the reaction was continued for 4 h. The suspension was filtered to collect the precipitate. The precipitate was dried overnight at room temperature, and compound 4 was collected as a dark green solid (4.8 mg, 45% yield). The absorbance and fluorescence analysis results of compound 4 are shown in Figure 3. 1H NMR 데이터 및 13 C NMR 데이터는 다음과 같다. Accurate mass TOF MS m / z [M]+ calculated for [C61H70N10O17S3]+, found [M+H] / 2+ 655.50. 1 H NMR (500 MHz, DMSO-d6) δ 8.60 (t, J = 0.8 Hz, 1H), 8.53 (d, J = 8.7 Hz, 1H), 8.09 - 8.02 (m, 2H), 7.97 (dd, J = 8.1, 2.2 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.83 - 7.75 (m, 2H), 7.75 - 7.70 (m, 2H), 7.65 (dd, J = 8.0, 2.1 Hz, 1H), 7.09 - 7.01 (m, 2H), 6.96 (d, J = 14.6 Hz, 1H), 6.85 - 6.79 (m, 2H), 6.63 - 6.58 (m, 3H), 6.27 - 6.20 (m, 2H), 4.84 (t, J = 8.5 Hz, 2H), 4.59 (dd, J = 6.0, 0.9 Hz, 2H), 4.32 (dt, J = 8.7, 6.5 Hz, 1H), 3.95 (t, J = 5.7 Hz, 2H), 3.70 (t, J = 5.5 Hz, 2H), 3.64 (s, 4H), 3.57 (t, J = 4.3 Hz, 2H), 3.42 (dt, J = 5.1, 4.3 Hz, 2H), 3.05 (d, J = 10.8 Hz, 1H), 2.89 (t, J = 8.5 Hz, 2H), 2.77 - 2.70 (m, 4H), 2.63 (d, J = 5.7 Hz, 1H), 2.28 (dt, J = 16.3, 8.7 Hz, 1H), 2.16 (dt, J = 16.4, 8.8 Hz, 1H), 2.03 (m, 1H), 1.81 (m, 1H), 1.75 (s, 3H), 1.62 (s, 5H), 1.64 - 1.56 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 176.20, 174.84, 174.54, 174.03, 172.88, 167.01, 163.86, 159.92, 156.75, 154.64, 150.23, 148.24, 146.50, 146.31, 145.14, 144.16, 144.05, 140.65, 140.57, 139.47, 138.14, 136.77, 134.54, 131.26, 130.42, 130.31, 129.90, 129.06, 124.74, 122.91, 122.63, 121.31, 120.14, 112.55, 112.39, 95.98, 72.95, 69.74, 69.34, 69.30, 53.15, 45.89, 45.51, 45.19, 43.48, 43.36, 40.06, 35.75, 32.46, 32.13, 31.78, 28.09, 27.76, 27.09, 26.62, 22.86.

[0171]

[0172] Manufacturing Example 6. Synthesis of Compound 5 (MAN-KBSF2-PEG-NH2)

[0173] [Chemical Formula 1-5]

[0174]

[0175] Compound 5(2-((E)-2-((E)-2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)thio)-3-(2-((E)-3,3-dimethyl-1-(3-oxo-3-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)propyl)-5-sulfoindolin-2-ylidene)ethylidene) cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-oxo-3-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3 -yl)amino)propyl)-5-sulfo-3H-indol-1-ium, 2-((E)-2-((E)-2-((2-(2-(2-aminoethoxy)ethoxy)ethyl)thio)-3-(2-((E)-3,3-dimethyl-1-(3-oxo-3-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)propyl)-5-sulfoin dolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-oxo-3-((2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)amino)propyl)-5-sulfo-3H-indol-1-ium) It was synthesized as follows.

[0176] The compound 2 (MAN-KBSF2; 1.0 g, 0.92 mmol) was dissolved in 8 mL of distilled water and added to a 100 mL round-bottomed flask. DIEA (0.4 mL; 2.34 mmol; 2.5 molar equivalents) was added to the flask and mixed well. Tert-butyl(2-(2-(2-mercaptoethoxy)ethoxy)ethyl)carbamate (270 mg; 1.02 mmol; 1.11 molar equivalents) dissolved in 2 mL of DMSO was added to the reaction solution, which was then heated in an oil bath at 60 °C for 40 min. After 40 min, the progress was monitored using an LC-MS system. 1.5 mL of 6 M HCl was added at 60 °C and the reaction was continued for 30 min for deprotection. After the reaction, cool to room temperature, slowly pour into 100 mL of a mixture of acetone / EA (1:1), stir at room temperature for 30 minutes, filter the suspension, and collect the precipitate. The precipitate was dried overnight at room temperature and collected as a dark green solid (0.68 g, 61% yield). The product thus prepared was 1 H NMR data and 13 C NMR data are as follows. Accurate mass TOF MS m / z [M]+ calculated for [C54H75N5O20S3]+, found [M+H] / 2+ 605.25. 1H NMR (500 MHz, DMSO-d6) δ 8.16 - 8.07 (m, 1H), 7.83 - 7.69 (m, 1H), 6.27 - 6.20 (m, 1H), 5.70 (d, J = 3.2 Hz, 1H), 4.94 (td, J = 4.4, 3.2 Hz, 1H), 4.85 - 4.76 (m, 1H), 4.66 (d, J = 4.7 Hz, 1H), 4.48 - 4.40 (m, 2H), 3.91 (t, J = 5.1 Hz, 1H), 3.81 - 3.67 (m, 2H), 3.63 (s, 1H), 3.67 - 3.59 (m, 1H), 3.53 - 3.37 (m, 2H), 3.15 - 3.02 (m, 2H), 2.86 - 2.74 (m, 1H), 2.77 - 2.71 (m, 2H), 2.73 - 2.67 (m, 1H), 2.60 - 2.48 (m, 1H), 1.75 (s, 2H), 1.62 (s, 2H), 1.64 - 1.56 (m, 1H). 13 C NMR (125 MHz, DMSO-d6) δ 173.00, 172.88, 172.72, 160.01, 148.79, 145.14, 144.05, 143.38, 140.69, 140.57, 138.94, 138.14, 136.77, 134.54, 132.45, 130.31, 129.06, 123.57, 122.68, 122.63, 120.14, 112.37, 95.98, 94.53, 94.48, 75.40, 72.97, 72.12, 72.09, 71.01, 70.68, 69.99, 69.30, 61.63, 55.75, 55.72, 45.50, 45.33, 44.21, 43.27, 41.72, 34.92, 34.36, 31.78, 28.09, 27.76, 27.09, 26.62, 22.84.

[0177]

[0178] Manufacturing Example 7. Synthesis of Compound 6 (FMK-2)

[0179] [Chemical Formula 1-6]

[0180]

[0181] Compound 6(2-((E)-2-((E)-2-((1-(4-(((2-amino-4-oxo-1,4-dihydropteridin-7-yl)methyl)amino)phenyl)-3-carboxy-1,6-dioxo-10,13-dioxa-2,7-diazapentadecan-15-yl)thio)-3-(2-((E)-3,3-dimethyl-1-(3)-oxo-3-(((3R,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)propyl)-5-sulfoindoline-2 -ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-oxo-3-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)propyl)-5-sulfo-3H-indol-1-ium, 2-((E)-2-((E)-2-((1-(4-(((2-amino-4-oxo-1,4-dihydropteridin-7-yl)methyl)amino)phenyl)-3-carboxy-1,6-dioxo-10,13-dioxa- 2,7-diazapentadecan-15-yl)thio)-3-(2-((E)-3,3-dimethyl-1-(3-oxo-3-(((3R,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)te trahydro-2H-pyran-2-yl)amino)propyl)-5-sulfoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-o xo-3-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)amino)propyl)-5-sulfo-3H-indol-1-ium) It was synthesized as follows.

[0182] Folic acid (4 mg, 9.1 nmol), EDC (2 mg), and HOBt (1.4 mg) were added to 1 mL of DMSO in a 1.5 mL EP tube. DIEA (5 μl) was added to the reaction solution under vortexing for 2 h at room temperature. 10 mg of compound 5 (MAN-KBSF2-PEG-NH2 (8.3 nmol)) was added, and the reaction was continued for 4 h. The suspension was filtered to collect the precipitate, which was dried overnight at room temperature and collected as a dark green solid (5.7 mg, 42% yield). The absorbance and fluorescence analysis results of compound 6 are shown in Figure 4, and the product prepared 1 H NMR data and 13 C NMR data are as follows. Accurate mass TOF MS m / z [M]+ calculated for [C61H70N10O17S3]+, found [M+H] / 2+ 655.50. 1H NMR (500 MHz, DMSO-d6) δ 8.60 (t, J = 0.8 Hz, 1H), 8.53 (d, J = 8.7 Hz, 1H), 8.09 - 8.02 (m, 2H), 7.97 (dd, J = 8.1, 2.2 Hz, 1H), 7.92 (d, J = 2.2 Hz, 1H), 7.83 - 7.75 (m, 2H), 7.75 - 7.70 (m, 2H), 7.65 (dd, J = 8.0, 2.1 Hz, 1H), 7.09 - 7.01 (m, 2H), 6.96 (d, J = 14.6 Hz, 1H), 6.85 - 6.79 (m, 2H), 6.63 - 6.58 (m, 3H), 6.27 - 6.20 (m, 2H), 4.84 (t, J = 8.5 Hz, 2H), 4.59 (dd, J = 6.0, 0.9 Hz, 2H), 4.32 (dt, J = 8.7, 6.5 Hz, 1H), 3.95 (t, J = 5.7 Hz, 2H), 3.70 (t, J = 5.5 Hz, 2H), 3.64 (s, 4H), 3.57 (t, J = 4.3 Hz, 2H), 3.42 (dt, J = 5.1, 4.3 Hz, 2H), 3.05 (d, J = 10.8 Hz, 1H), 2.89 (t, J = 8.5 Hz, 2H), 2.77 - 2.70 (m, 4H), 2.63 (d, J = 5.7 Hz, 1H), 2.28 (dt, J = 16.3, 8.7 Hz, 1H), 2.16 (dt, J = 16.4, 8.8 Hz, 1H), 2.03 (m, 1H), 1.81 (m, 1H), 1.75 (s, 3H), 1.62 (s, 5H), 1.64 - 1.56 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 176.20, 174.84, 174.54, 174.03, 172.88, 167.01, 163.86, 159.92, 156.75, 154.64, 150.23, 148.24, 146.50, 146.31, 145.14, 144.16, 144.05, 140.65, 140.57, 139.47, 138.14, 136.77, 134.54, 131.26, 130.42, 130.31, 129.90, 129.06, 124.74, 122.91, 122.63, 121.31, 120.14, 112.55, 112.39, 95.98, 72.95, 69.74, 69.34, 69.30, 53.15, 45.89, 45.51, 45.19, 43.48, 43.36, 40.06, 35.75, 32.46, 32.13, 31.78, 28.09, 27.76, 27.09, 26.62, 22.86.

[0183]

[0184] Example 1. Fluorescent contrast observation

[0185] Among the compounds manufactured in the above manufacturing examples, compound 1 (KBSF2), compound 2 (MAN-KBSF2), compound 4 (FA-KBSF2), and compound 6 (FMK-2) were used to manufacture fluorescent contrast agents, and then the agents were treated to A549 cancer cells that do not express folate receptors and H522 cancer cells that express folate receptors, respectively, and the results are shown in Fig. 5. As shown in the figure, compounds 1, 2, and 4 of the present invention expressed fluorescence in cancer cells regardless of whether folate receptors were expressed, but it was confirmed that compound 6 (FMK-2) was capable of fluorescence imaging specifically for cells expressing folate receptors. In particular, as shown in Fig. 6 below, when compound 6 (FMK-2) was treated at various concentrations to two lung cancer cell lines, A549 and H522, it was confirmed that cell viability did not decrease. Therefore, it was confirmed that FMK-2 had no cytotoxicity. It was assumed that the fact that the cell viability was slightly increased compared to when not treated was due to the effect of FA (folic acid) promoting cell growth.

[0186]

[0187]

[0188] Example 2. Mouse fluorescence experiment

[0189] Compound 6 (FMK-2) was intravenously injected into cancer-induced mice at doses of 25 nmol, 50 nmol, and 100 nmol, and the near-infrared fluorescence signals were observed at different time points. The results are shown in Fig. 7. As can be seen in the figure, a strong near-infrared fluorescence signal was detected in cancer tissues compared to normal tissues. In addition, the ratio of fluorescence signal to normal tissue was the highest at a concentration of 50 nmol, and the strongest luminescence signal was measured 4 hours after injection.

[0190]

[0191] Example 3. Immunochemical analysis

[0192] In Example 2, cancer tissue and surrounding normal tissue of mice intravenously injected with compound 6 (FMK-2) were obtained, frozen, and sliced ​​(cryosection). The near-infrared fluorescence signal was confirmed under a fluorescence microscope to confirm the distribution of compound 6, and immunohistochemistry analysis was performed to confirm the distribution of tumor-associated macrophages (TAMs) along with H&E staining, and the results are shown in Fig. 8. In the H&E staining image, the cancer tissue was identified on the left side of the yellow dotted line in the lower right corner, and the normal tissue was identified on the right side. Looking at the immunochemical staining results, it was confirmed that the folate receptor alpha, which is known to be overexpressed in cancer cells, was distributed in brown in the cancer tissue, and the folate receptor beta, CD206, and F4 / 80, which are known as markers of TAMs, were all distributed in large quantities at the border between the cancer and normal tissues. In particular, it was confirmed that the fluorescence distribution in the area where the folate receptor beta was mainly expressed was similar to the near-infrared fluorescence image. Therefore, it was confirmed that compound 6 (FMK-2) of the present invention can effectively target TAM and folate receptors, and can be used as a fluorescent contrast agent to distinguish between cancer tissue and normal tissue.

[0193]

[0194] Example 4. Mouse tissue staining experiment

[0195] In order to confirm in which organ each compound of the present invention accumulates and is excreted, each fluorescent substance was injected into normal mice, and 4 hours later each organ was removed and fluorescent images were taken, and the results are shown in Fig. 9. Each graph represents the fluorescent signal of each organ. In the case of KBSF2 of the present invention, the signal was higher in the liver and kidney compared to other organs, and the signal was confirmed in the kidney and small intestine for FA-KBSF2, and the overall signal was confirmed in all organs for MAN-KBSF2. In particular, the signal of FMK-2 was measured to be high only in the kidney. Therefore, it was confirmed that FMK-2 was excreted through the kidney without nonspecific binding to other organs.

[0196]

[0197] Example 5. Pharmacokinetic analysis of compound 6

[0198] Pharmacokinetic parameter analysis was performed on compound 6 and the results are shown in Fig. 10 and Table 1. The half-lives of distribution (T 1 / 2α ) was measured as 1.1 min, and the half-lives of elimination phase (T 1 / 2β ) was measured as 26.9 min. The area under the curve (AUC) value was measured as 100.6, and the urine excretion 4 hours after intravenous injection of 100 nmol FMK-2 was measured as 42%.

[0199] [Table 1]

[0200]

[0201] Example 6. Comparison experiment of inflamed tissue and staining

[0202] To verify how specifically compound 6 of the present invention can target cancer tissues compared to inflamed tissues, the following experiment was conducted. Inflammation was induced in mice or cancer models were established, and FMK-2 was intravenously injected into each mouse. Fluorescence images were taken over time, and the results are shown in Fig. 11. In the case of the inflammation model, the signal weakened significantly over time and there was no specific binding to inflamed tissues compared to normal tissues, whereas in the cancer model, the signal was strong for up to 24 hours, and the signal contrast between cancer and normal tissues was also strong. The graph shows the value of the fluorescence signal ratio of inflamed or cancer tissues compared to normal tissues. In the cancer tissue staining, the highest fluorescence emission was confirmed at 4 hours, and a high level of fluorescence was maintained for up to 24 hours. In contrast, inflamed tissues emitted a similar level of fluorescence for 2 to 24 hours, and the intensity was measured to be lower than that of cancer tissues.

[0203]

[0204] Comparative Example 1. Comparative experiment with ZW800-PEG

[0205] In order to confirm the difference in effect between ZW800-PEG (Non-patent Document 1), which is known to have a conventional zwitterionic near-infrared fluorophore, and the compound 1 (KBSF2) of the present invention, a comparative experiment was conducted as follows.

[0206] The human lung cancer cell line H522 was purchased from ATCC (Manassas, VA). Cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (p / s) at 37°C in a humidified 5% CO2 atmosphere. To assess the binding affinity of the probes to cells, lung cancer cells were incubated in Hank's balanced salt solution (HBSS) containing 10% FBS without phenol red, either without dye or in the presence of 5 μM ZW800-PEG and compound 1 (KBSF2) for 1 h at 37°C. After washing with FBS-free HBSS, cells were imaged using a fluorescence microscope (EVOS FL Auto Imaging System; Life Technologies, Carlsbad, CA). All NIR fluorescence images for a specific fluorophore were normalized to the same values ​​across conditions throughout the experiment. That is, the image with the strongest signal was optimized for brightness and contrast, and the same was applied to all other images acquired for a specific fluorophore. The results are shown in Fig. 12. As can be seen in the figure, the fluorescence intensity of KBSF2 of the present invention was confirmed to be more than twice as strong in the presence of 5 μM of the probe compared to the prior art ZW800-PEG, confirming that the fluorescence contrast effect in the near-infrared region was clearly superior when treated with 5 μM.

[0207]

[0208]

[0209] Comparative Example 2. Comparative Experiment with OCTL14

[0210] In vivo and ex vivo comparative experiments were conducted on OCTL14, a conventionally known contrast agent, and compound 6 (FMK-2) of the present invention. OCTL14 and FMK-2 were intravenously injected into a mouse cancer model at different times, and after a certain period of time, the cancer tissues were photographed by fluorescence images, which are shown in Fig. 13. OCTL14 has a fluorescence wavelength of 700 nm, so fluorescence images were taken at that wavelength, and FMK-2 was photographed at 800 nm. OCTL14, which is known to accumulate in cancer tissues, was confirmed to accumulate inside the cancer tissues (tumor stroma), and FMK-2 was confirmed to accumulate in the tumor microenvironment (tumor nest).

[0211]

[0212] In addition, a schematic diagram of a tumor microenvironment, which is an example to which the compound of the present invention and a contrast agent composition comprising the same can be most appropriately applied, is shown in Fig. 14, and a schematic diagram of a fluorescence expression mechanism is shown in Fig. 15.

[0213]

[0214] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0215] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

1. A compound represented by the following [chemical formula 1] or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R 1 and R 2 are the same or different from each other, and each independently represents hydrogen, a hydroxyl group, a C1-C5 alkoxy group, (Y) m X, (Y) m COO - , (Y) m SO3 - , (Y) m PO3H - , and at least one selected from the group consisting of combinations thereof, R 3 and R 4 are identical or different from each other, and each is independently (Y) n H, (Y) n (CO)R 5 and at least one selected from the group consisting of combinations thereof, R 5 is a hydroxyl group or And, A is a C2-C6 heterocycloalkyl group containing at least one heteroatom selected from the group consisting of substituted or unsubstituted N, O and S, X is a halogen group, Y is CH2 or CH2CH2O, Z is halogen or And, B is hydrogen or And, a, b, m and n are equal to or different from each other and are each independently an integer from 0 to 5.

2. In paragraph 1, In the above chemical formula 1, R 1 and R 2 are each independently hydrogen or (Y) m SO3 - A compound or a pharmaceutically acceptable salt thereof, characterized in that:

3. In paragraph 1, In the above chemical formula 1, R 3 and R 4 are each independently (Y) n (CO)R 5 And, R 5 is a hydroxyl group or And, A compound or a pharmaceutically acceptable salt thereof, characterized in that A is a C2-C6 heterocycloalkyl group containing at least one heteroatom selected from the group consisting of substituted or unsubstituted N, O and S.

4. In paragraph 3, The above A is A compound or a pharmaceutically acceptable salt thereof, characterized in that:

5. In paragraph 1, In the above chemical formula 1, Z is Cl, , or A compound or a pharmaceutically acceptable salt thereof, characterized in that:

6. In paragraph 1, A compound or a pharmaceutically acceptable salt thereof, characterized in that the compound is at least one selected from the group consisting of compounds represented by the following [Chemical Formula 1-1] to [Chemical Formula 1-6]. [Chemical Formula 1-1] ; [Chemical Formula 1-2] ; [Chemical Formula 1-3] ; [Chemical Formula 1-4] ; [Chemical Formula 1-5] ; and [Chemical Formula 1-6] .

7. A contrast agent composition for cancer diagnosis comprising a compound of any one of claims 1 to 6 as an active ingredient.

8. In paragraph 7, A contrast agent composition for cancer diagnosis, characterized in that the contrast agent composition targets a tumor and / or a tumor microenvironment.

9. In paragraph 7, The above contrast agent composition is a contrast agent composition for diagnosing cancer, which binds to at least one of folate receptor alpha of cancer cells, folate receptor beta of tumor-associated macrophages (TAM) in a tumor microenvironment, and CD206 of TAM.

10. In paragraph 7, A contrast agent composition for cancer diagnosis, characterized in that the contrast agent composition simultaneously targets a tumor and a tumor microenvironment.

11. A method for diagnosing cancer, comprising a step of targeting a tumor and / or a tumor microenvironment using a composition according to any one of claims 7 to 10.

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