Complex drug conjugate for preventing or treating integrin-overexpressing cancer

Abstract

The present invention can effectively prevent, alleviate, or treat integrin-overexpressing cancer even if a complex drug conjugate is administered at a low dose.

Description

Complex drug conjugate for the prevention or treatment of integrin-overexpressing cancer

[0001] The present invention relates to a complex drug conjugate for preventing, improving, or treating cancer that overexpresses integrin.

[0002] Endocytosis is a general term defining the process by which a cell absorbs and / or releases bound extracellular substances, such as molecules, viruses, particles, and microorganisms, and delivers them to specific organelles within the cytoplasm. Endocytosis is broadly divided into phagocytosis and pinocytosis; pinocytosis can be further classified into macro-pinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and clathrin- and caveolin-independent endocytosis.

[0003] Endoclonalization of extracellular substances plays a crucial role in molecular biology fields such as virology, drug discovery, and gene delivery, specifically in the internalization of drugs into target cells that can specifically bind to molecules (e.g., receptors) widely expressed on the cell membrane. In particular, efficient endocytosis can be induced by introducing molecules (e.g., ligands) that specifically bind to molecules (e.g., receptors) widely expressed on the cell membrane as a method for effectively internalizing drugs into target cells (Marsh M. et al., Cell 2006; 124:729-40; Smith AE et al., Science 2004; 304:237-42). In this process, the drug-bound receptor is collected at the invagination site via surface diffusion; if this collection does not occur smoothly, endocytosis does not take place effectively and within a short period of time.

[0004] Meanwhile, integrins are heterodimer receptors essential for intercellular and cell-extracellular matrix (ECM) interactions, consisting of one α-subunit and one β-subunit. These cell adhesion molecules act as transmembrane links connecting extracellular ligands with the cytoskeleton, performing various biological functions by regulating important signaling pathways in most cells. Integrins play a key role in several processes, including cell migration, angiogenesis, wound healing, hemostasis, and oncogenic changes. Given that many integrins are associated with pathological conditions, they are considered promising therapeutic targets. In particular, integrin α V β3 and α V β5 is involved in the angiogenesis and metastasis of solid tumors and is evaluated as a potential candidate for the treatment of cancers with high unmet medical needs, such as glioblastoma, head and neck cancer, colorectal cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, pancreatic cancer, prostate cancer, and brain metastasis.

[0005] Among the aforementioned integrin-overexpressing solid tumors, glioblastoma (GBM) in particular exhibits very high resistance to radiation and chemotherapy compared to other cancers, resulting in an average survival period of only about one year once diagnosed. Notably, temozolomide (TMZ), used as a first-line treatment for glioblastoma, has a fundamental limitation in that it cannot effectively cross the blood-brain barrier (BBB). Reports indicate that only about 1% of intravenously or orally administered temozolomide crosses the BBB to reach brain tissue. Consequently, high-dose administration is required to achieve sufficient drug concentrations; however, this leads to increased side effects such as systemic toxicity, bone marrow suppression, vomiting, fatigue, and immunosuppression, thus limiting the ability to secure clinically effective concentrations.

[0006] As a result of diligent efforts, the inventors of the present invention have developed a novel complex drug conjugate capable of efficiently delivering a drug into the brain even at low doses by targeting glioblastoma cells overexpressing integrins and promoting endocytosis. Furthermore, the drug conjugate of the present invention is applicable not only to glioblastoma but also to integrin α V β3 and / or α V It was experimentally confirmed that β5 exhibits selective internalization into cells and antitumor activity in various solid tumor cells (e.g., breast cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, etc.) that are overexpressed.

[0007] Accordingly, the present invention provides a new anticancer therapeutic agent that overcomes the problems of BBB permeability and toxicity of conventional temozolomide by using a delivery vehicle capable of passing through the blood-brain barrier, and at the same time achieves an effective therapeutic effect even at low doses by selectively targeting integrin-overexpressing tumor cells.

[0008] One objective of the present invention is to provide a novel complex drug conjugate for the prevention, improvement, or treatment of integrin overexpression cancer.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010] According to one embodiment of the present invention, the invention relates to a pharmaceutical composition for the prevention, improvement, or treatment of integrin-overexpressing cancer comprising as an active ingredient: a first compound selected from a compound represented by any one of the following chemical formulas 1 to 4, and a pharmaceutically acceptable salt, optical isomer, hydrate, and solvate thereof; and a second compound selected from a compound represented by the following chemical formula 5, and a pharmaceutically acceptable salt, optical isomer, hydrate, and solvate thereof.

[0011] [Chemical Formula 1]

[0012]

[0013] [Chemical Formula 2]

[0014]

[0015] [Chemical Formula 3]

[0016]

[0017] [Chemical Formula 4]

[0018]

[0019] [Chemical Formula 5]

[0020]

[0021] In the above chemical formulas 2 and 4, n is a natural number from 1 to 30, a natural number from 1 to 20, a natural number from 1 to 10, a natural number from 2 to 8, or a natural number from 3 to 6, and preferably can be 4.

[0022] The chemical formula of the compound represented by the above chemical formula 1 is C 129 H 159 FN 30 O 30 and the molecular weight is 2628.82. The chemical formula of the compound (n=4) represented by the above chemical formula 2 is C 137 H 175 FN 30 O 30 and the molecular weight is 2805.03. The chemical formula of the compound represented by the above chemical formula 3 is C 132 H 162 FN 31 O 33 and the molecular weight is 2729.88. The chemical formula of the compound represented by the above chemical formula 4 (n=4) is C 140 H 178 FN 31 O 37 and the molecular weight is 2906.1. The chemical formula of the compound represented by the above chemical formula 5 is C 62 H 90 N 22 O 17It is, and the molecular weight is 1415.52.

[0023] In general, when developing a drug conjugate, the selection of the targeting substance, the payload, and the linker connecting the two is important. Depending on each component constituting the drug conjugate, the efficacy, stability, and overall therapeutic effect of the entire drug conjugate can be significantly affected. As a result of diligent efforts, the inventors of the present invention discovered that compounds with structures represented by Chemical Formulas 1 to 5 have excellent in vivo stability, and that when the first compound represented by Chemical Formulas 1 to 4 and the second compound represented by Chemical Formula 5 are administered in combination, the level of cell internalization increases significantly, leading to the present invention.

[0024] The compounds represented by Chemical Formulas 1 to 4 and the compound represented by Chemical Formula 5 have a cyclic RGD (arginine(R)-glycine(G)-aspartic acid(D)) structure, preferably, the compounds include a D-[c(RGDfK)]2 structure in which two cyclic RGDfK (arginine(R)-glycine(G)-aspartic acid(D)-phenylalanine(f)-lysine(K)) are linked by aspartic acid (D), thereby integrin α that is highly expressed in cancer cells, particularly brain cancer, breast cancer, prostate cancer, and pancreatic cancer cells. V β3 and α V It can bind to β5 with high affinity.

[0025] Here, the aforementioned "integrin" refers to a transmembrane protein that promotes adhesion between cells and between cells and the extracellular matrix; upon ligand binding, it activates signaling pathways that mediate cellular signals, such as cell cycle regulation, intracellular cytoskeletal organization, and the translocation of new receptors to the cell membrane. Integrins are composed of the combination of alpha and beta subunits, and various types of integrins can be formed through diverse combinations of these subunits. Among them, α V β3 acts as a receptor for vitronectin, is expressed by platelets, or functions as a receptor for phagocytosis in macrophages or dendritic cells. α V β3 plays a crucial role in the process of angiogenesis and is associated with the blood supply required for excessive cell proliferation in cancer and other diseases. α V β5 binds to the extracellular matrix and proteolytic enzymes to promote angiogenesis.

[0026] The compounds of Chemical Formulas 1 to 4 above include an adibo group and an azide group of Chemical Formula 5 above. When a first compound of any one of Chemical Formulas 1 to 4 and a second compound represented by Chemical Formula 5 are administered into the body, the adibo group of Chemical Formulas 1 to 4 and the azide group of Chemical Formula 5 can be combined with each other by a click reaction.

[0027] The compounds represented by the above chemical formulas 1 to 4 include the structure of exatecan as a payload and can induce apoptosis by inhibiting DNA topoisomerase I of the target cancer cells and interfering with DNA replication of the cancer cells.

[0028] The compounds of Chemical Formulas 1 to 4 above include a linker that connects the D-[c(RGDfK)]2 and the payload. In a drug conjugate, the linker must be stable in the body and prevent non-specific binding of the targeting substance and the payload during the treatment process. In addition, the linker must be able to efficiently degrade in a specific environment inside the cell to release the payload. Therefore, the selection of the linker is very important when designing a drug conjugate, and the linker must be selected differently depending on the type and structure of the targeting substance and the payload. Depending on which linker is selected, the drug conjugate may exhibit completely different effects in terms of in vivo stability and intracellular payload release.

[0029] The compounds represented by Chemical Formulas 1 and 2 of the present invention include a valine (Val)-lysine (Lys)-glycine (Gly) tripeptide (VKG) as the linker, thereby enabling the payload to be stably delivered to target cancer cells and effectively hydrolyzed by enzymes within the cancer cells to increase the release rate of the payload. The compound represented by Chemical Formula 2 further includes a -(PEG)n- linker (where n is an integer from 1 to 30, preferably 4) between the targeting substance and the VKG linker to increase the in vivo stability of the drug conjugate, reduce non-specific binding of the drug conjugate, and enable the payload to be effectively delivered to the desired target.

[0030] In addition, the compounds represented by Chemical Formulas 3 and 4 include glutamic acid (Glu)-valine (Val)-citrulline (Cit) tripeptide (EVCit) as a linker connecting the D-[c(RGDfK)]2 and the payload, thereby enabling the payload to be stably delivered into the target cancer cells, and within the cancer cells, it can be effectively hydrolyzed by enzymes to increase the release rate of the payload. The compound represented by Chemical Formula 4 further includes a -(PEG)n- linker between the targeting substance and the EVCit linker to increase the in vivo stability of the drug conjugate, reduce non-specific binding of the drug conjugate, and enable the payload to be effectively delivered to the desired target.

[0031] As described above, the first compounds of chemical formulas 1 to 4 and the second compound of chemical formula 5 both contain cyclic RGD and can bind to integrins, which are surface ligands of cancer cells; furthermore, the first compounds and the second compounds can be combined with each other via a click reaction. That is, when the first compound bound to the cancer cell and the second compound bound to the cancer cell are combined with each other, a cluster is formed. The cluster formed by multiple drug conjugates promotes endocytosis within the cell, thereby increasing the efficiency of the drug conjugate's internalization. The internalized drug conjugate moves to a lysosome or endosome, where the linker is degraded by a hydrolytic enzyme, releasing the payload exatecan, which can produce effective anticancer activity.

[0032] In the present invention, when a complex drug conjugate comprising the first compound and the second compound is administered to a target individual, as the degree of cellular internalization of exatecan in the target cancer cells of the individual increases, an excellent anticancer effect can be obtained even with only a small amount administered compared to when exatecan is administered alone.

[0033] Furthermore, the complex drug conjugate composed of the first and second compounds of the present invention can efficiently cross the blood-brain barrier (BBB) ​​through the promotion of endocytosis resulting from the aforementioned cluster formation. Therefore, by administering the complex drug conjugate of the present invention, an enhanced therapeutic effect can be expected even in brain cancer, where it was difficult to obtain a significant therapeutic effect with existing drugs, at a low dose.

[0034] In this specification, the term "individual" refers to an individual that has developed or is suspected of developing integrin overexpression cancer. The types of individuals include all animals, including humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, which may develop the disease, but individuals that can be treated with the active substance provided in the present invention are included without limitation.

[0035] The first compound and the second compound may exist outside the body in an unbound state. The first compound and the second compound may be administered individually into the body and bind within the body via a click reaction.

[0036] In the present invention, the cancer that highly expresses the integrin may be a solid tumor such as brain cancer, head and neck cancer, colorectal cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, pancreatic cancer, or prostate cancer.

[0037] The above-mentioned cancer is, among integrins, particularly integrin α V β3 or α V It may be a cancer overexpressing β5, and preferably integrin α V It may be a cancer that overexpresses β3, and more preferably, it may be a brain cancer.

[0038] The above brain cancer may be selected from the group consisting of astrocytoma, glioblastoma, ependymoma, oligodendroglioma, mixed glioma, brain stem glioma, optic nerve glioma, pituitary adenoma, craniopharyngioma, medulloblastoma, primitive neuroectodermal tumors, pineal tumors, meningioma, schwannoma, metastatic brain tumors, CNS lymphoma, neurofibromatosis, pseudotumor cerebri, and tuberous sclerosis, and preferably may be glioblastoma. there is.

[0039] In the present invention, the brain cancer that is the subject of prevention, improvement, or treatment may be primary brain cancer that occurs directly in the brain, or secondary brain cancer in which cancer originating in another part of the body has metastasized to the brain.

[0040] In the present invention, the first and second compounds may include, in addition to the compounds represented by each chemical formula, pharmaceutically acceptable salts thereof. The pharmaceutically acceptable salt is a salt generally considered by those skilled in the art to be suitable for medical application (e.g., because such salt is not harmful to the subjects to whom it may be treated with said salt), or a salt that causes acceptable side effects within each treatment. Generally, the pharmaceutically acceptable salt is a salt considered acceptable by regulatory authorities such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or the Pharmaceuticals and Medical Devices Agency (PMDA) of the Japanese Ministry of Health, Labour and Welfare. However, in principle, the present invention also includes, for example, a salt of a compound according to the present invention that is not pharmaceutically acceptable in itself, as an intermediate in the preparation of a compound according to the present invention or a physiologically active derivative thereof, or as a pharmaceutically acceptable salt of a compound according to the present invention or an intermediate in the preparation of a physiologically active derivative thereof. The salt comprises a water-insoluble salt, and in particular, comprises a water-soluble salt.

[0041] In each case, a person skilled in the art can easily determine whether a specific compound according to the present invention or its physiologically functional derivative can form a salt, that is, whether the compound according to the present invention or its physiologically functional derivative has a charged group such as, for example, an amino group, a carboxylic acid group, etc.

[0042] Exemplary salts of the compounds of the present invention are acid addition salts or salts with bases, in particular pharmaceutically acceptable inorganic and organic acid addition salts and salts with bases commonly used in pharmaceuticals, which are water-insoluble or particularly water-soluble acid addition salts. Depending on the substituents of the compounds of the present invention, salts with bases may also be suitable. Acid addition salts may be formed, for example, by mixing a solution of the compounds of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Likewise, pharmaceutically acceptable base addition salts are alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); It may include salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed using counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfons, and aryl sulfons).Exemplary examples of pharmaceutically acceptable salts include acetate, adipate, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphosulfonate, camsylate, carbonate, chloride, citrate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edicylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate. Hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, mandelate, methanesulfonate (mesylate), methyl sulfate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subverate, succinate, tannate, tartrate, tosylate, undecanoate, valerate, etc. are included but not limited thereto.

[0043] Salts that are not pharmaceutically acceptable in the present invention and, for example, may be obtained as process products during the manufacture of compounds according to the present invention on an industrial scale are also included in the present invention, and if desired, can be converted into pharmaceutically acceptable salts by methods known to those skilled in the art.

[0044] Meanwhile, since the compounds according to the present invention may have asymmetric carbon centers, they may exist as R or S isomers or racemic compounds, and all of these optical isomers and mixtures may be included within the scope of the present invention.

[0045] In addition, the compounds of the present invention as well as their salts may contain varying amounts of solvent, for example, when separated into crystalline forms. Accordingly, solvates of the compounds of the present invention, particularly hydrates, as well as solvates of the salts of the compounds of the present invention, particularly hydrates, may be included within the scope of the present invention. More particularly, the present invention may include hydrates of compounds, salts, and / or physiologically functional derivatives according to the present invention, comprising one, two, or half molecules of water with respect to stoichiometry.

[0046] In the present invention, the term "prevention" may be included without limitation as long as it is any act that can block, suppress, or delay symptoms caused by integrin overexpressing cancer or related diseases using the composition of the present invention.

[0047] In the present invention, the term "treatment" may be included without limitation as long as it is any act that uses the composition of the present invention to improve or benefit from symptoms caused by integrin overexpressing cancer or related diseases.

[0048] In the present invention, the term "improvement" may be included without limitation as long as it is any act in which symptoms caused by integrin overexpressing cancer or related diseases are improved or beneficially altered using the composition of the present invention.

[0049] The above pharmaceutical compositions are not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, topical preparations, suppositories, and sterile injectable solutions according to conventional methods. Preferably, the above pharmaceutical compositions may be formulated for intratracheal administration or inhalation administration; or for use as an injectable, but are not limited thereto.

[0050] The above pharmaceutical composition may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavors, etc. For injectables, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., mixed and used. For topical administration, it may include bases, excipients, lubricants, preservatives, etc. The formulations of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it may be prepared in the form of unit dosing ampoules or multi-dose formulations. Additionally, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0051] Examples of carriers, excipients, and diluents suitable for the above formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0052]

[0053] According to another embodiment of the present invention, the invention relates to a method for preventing, improving, or treating integrin-overexpressing cancer, comprising the step of administering a pharmaceutical composition according to the present invention to a subject.

[0054] The above subject may be an individual that has developed or has a high probability of developing integrin overexpression cancer and requires appropriate treatment.

[0055] The above subject may preferably be a human individual, but may also include non-human animal individuals. Suitable animal individuals may be mammals, including but not limited to primates such as humans, monkeys, apes, etc.; bovines, such as cattle, oxen, etc.; ovines, such as sheep, etc.; caprines, such as goats, etc.; porcines, such as pigs, hogs, etc.; equines, such as horses, donkeys, zebras, etc.; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, etc.; or rodents, such as mice, rats, etc.

[0056] The routes of administration of the above pharmaceutical composition are not limited to but include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. It may be administered orally or parenterally, preferably orally, but is not limited thereto.

[0057] The above parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0058] The above pharmaceutical composition may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the above pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0059] The first compound and the second compound may be administered to an individual simultaneously or sequentially. For example, the first compound and the second compound may be administered to an individual simultaneously, the first compound may be administered to the individual first followed by the second compound, or the second compound may be administered after the second compound. In the case where the first compound and the second compound are administered sequentially, the administration interval may be within 2 days, within 1 day, within 18 hours, within 12 hours, within 6 hours, within 3 hours, within 2 hours, within 1 hour, within 50 minutes, within 40 minutes, within 30 minutes, within 20 minutes, within 5 minutes, within 3 minutes, or within 1 minute, but is not limited thereto.

[0060] The present invention may include, but is not limited to, the following examples.

[0061] First embodiment: A first compound selected from a compound represented by any one of the above formulas 1 to 4, and its pharmaceutically acceptable salts, optical isomers, hydrates, and solvates; and a second compound selected from a compound represented by the above formula 5, and its pharmaceutically acceptable salts, optical isomers, hydrates, and solvates; the present invention relates to a pharmaceutical composition for the prevention or treatment of integrin-overexpressing cancer comprising as active ingredients: a first compound selected from a compound represented by any one of the above formulas 1 to 4, and its pharmaceutically acceptable salts, optical isomers, hydrates, and solvates.

[0062] Second Example: In the first example, the pharmaceutical composition may include a compound represented by Formula 1 and a compound represented by Formula 5.

[0063] Third Example: In the first example, the pharmaceutical composition may include a compound represented by Formula 2 and a compound represented by Formula 5.

[0064] Fourth Example: In the first example, the pharmaceutical composition may include a compound represented by Formula 3 and a compound represented by Formula 5.

[0065] Fifth Example: In the first example, the pharmaceutical composition may include a compound represented by Formula 4 and a compound represented by Formula 5.

[0066] 6th Example: In at least one of the 1st to 5th Examples, the 1st compound and the 2nd compound may exist separately outside the body.

[0067] 7th Example: In at least one of the 1st to 6th Examples, the 1st compound and the 2nd compound can be combined in the body via a click reaction.

[0068] Example 8: In Example 7, the cluster formed by the click reaction linkage of the first compound and the second compound promotes cell endocytosis, thereby increasing the efficiency of cell internalization of the drug conjugate.

[0069] 9th Example: In the 7th or 8th Example, the first compound and the second compound can move into the lysosome or endosome of the cancer cell.

[0070] 10th Example: In at least one of the 7th to 9th Examples, exatecan may be released from the first compound within the cancer cell.

[0071] 11th Example: In the 10th Example, exatecan may be released from the first compound by an enzyme within the lysosome or endosome of the cancer cell.

[0072] 12th Example: In at least one of the 1st to 11th Examples, the first compound and the second compound may be administered individually.

[0073] 13th Example: In at least one of the 1st to 12th Examples, the first compound and the second compound may be administered simultaneously or sequentially.

[0074] 14th Example: In the 13th Example, the first compound may be administered first, and the second compound may be administered.

[0075] 15th Example: In the 13th Example, the first compound and the second compound are administered sequentially, and the administration interval may be within 1 hour.

[0076] 16th Example: In at least one of the 1st to 15th Examples, the integrin overexpressing cancer may be brain cancer, head and neck cancer, colorectal cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, pancreatic cancer, or prostate cancer.

[0077] Example 17: In at least one of Examples 1 to 15, the pharmaceutical composition may be for the prevention, improvement, or treatment of brain cancer.

[0078] Example 18: In at least one of Examples 1 to 15, the pharmaceutical composition may be for the prevention, improvement, or treatment of glioblastoma.

[0079] Example 19: In at least one of Examples 1 to 15, the pharmaceutical composition may be for the prevention, improvement, or treatment of breast cancer.

[0080] 20th Example: In at least one of the 1st to 15th Examples, the pharmaceutical composition may be for the prevention, improvement, or treatment of pancreatic cancer.

[0081] 21st Example: In at least one of the 1st to 15th Examples, the pharmaceutical composition may be for the prevention, improvement, or treatment of prostate cancer.

[0082] 22nd Example: In at least one of the 1st to 16th Examples, the first compound and the second compound can pass through the blood-brain barrier (BBB).

[0083] 23rd Example: The present invention relates to a method for preventing or treating integrin-overexpressing cancer, comprising the step of administering to a subject a pharmaceutical composition comprising as an active ingredient a first compound selected from a compound represented by any one of the formulas 1 to 4, a pharmaceutically acceptable salt, optical isomer, hydrate, and solvate thereof; and a second compound selected from a compound represented by the formula 5, a pharmaceutically acceptable salt, optical isomer, hydrate, and solvate thereof.

[0084] 24th Example: In the 23rd Example, the pharmaceutical composition may include a compound represented by Formula 1 and a compound represented by Formula 5.

[0085] 25th Example: In the 23rd Example, the pharmaceutical composition may include a compound represented by Formula 2 and a compound represented by Formula 5.

[0086] Example 26: In Example 23, the pharmaceutical composition may include a compound represented by Formula 3 and a compound represented by Formula 5.

[0087] 27th Example: In the 23rd Example, the pharmaceutical composition may include a compound represented by Formula 4 and a compound represented by Formula 5.

[0088] Example 28: In at least one of Examples 23 to 27, the first compound and the second compound may exist separately outside the body.

[0089] Example 29: In at least one of Examples 23 to 28, the first compound and the second compound can be combined in the body via a click reaction.

[0090] 30th Example: In the 29th Example, the cluster formed by the click reaction linkage of the first compound and the second compound promotes cell endocytosis, thereby increasing the efficiency of cell internalization of the drug conjugate.

[0091] Example 31: In Example 29 or Example 30, the first compound and the second compound can move into the lysosome or endosome of a cancer cell.

[0092] 32nd Example: In at least one of the 29th to 31st Examples, exatecan may be released from the first compound within the cancer cell.

[0093] 33rd Example: In the 32nd Example, exatecan may be released from the first compound by an enzyme within the lysosome or endosome of the cancer cell.

[0094] Example 34: In at least one of Examples 23 to 33, the first compound and the second compound may be administered individually to the subject.

[0095] Example 35: In at least one of Examples 23 to 34, the first compound and the second compound may be administered to the subject simultaneously or sequentially.

[0096] Example 36: In Example 35, the first compound may be administered to the subject first, and the second compound may be administered.

[0097] Example 37: In Example 35, the first compound and the second compound are administered sequentially, and the administration interval may be within 1 hour.

[0098] 38th Example: In at least one of the 23rd to 37th Examples, the integrin overexpressing cancer may be brain cancer, head and neck cancer, colorectal cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, pancreatic cancer, or prostate cancer.

[0099] 39th Example: In at least one of the 23rd to 37th Examples, the method may be for the prevention, improvement, or treatment of brain cancer in a subject.

[0100] 40th Example: In at least one of the 23rd to 37th Examples, the method may be for the prevention, improvement, or treatment of glioblastoma in a subject.

[0101] 41st Example: In at least one of the 23rd to 37th Examples, the method may be for the prevention, improvement, or treatment of breast cancer in a subject.

[0102] 42nd Example: In at least one of the 23rd to 37th Examples, the method may be for the prevention, improvement, or treatment of pancreatic cancer in a subject.

[0103] 43rd Example: In at least one of the 23rd to 37th Examples, the method may be for the prevention, improvement, or treatment of prostate cancer in a subject.

[0104] Example 44: In at least one of Examples 23 to 38, the first compound and the second compound can pass through the blood-brain barrier (BBB) ​​of the subject.

[0105] When the complex drug conjugate according to the present invention is administered into the body, not only can both drug conjugates bind to integrins that are highly expressed in cancer cells, but the two drug conjugates can also bind to each other. The cluster formed in this way has increased retention within the cell due to endocytosis. Therefore, the complex drug conjugate of the present invention can exhibit a very high cancer treatment effect even when administered in a small amount.

[0106] Figure 1 shows the results of HPLC analysis of HD(RGD dimer)-ADIBO synthesized in Preparation Example 1.

[0107] Figure 2 shows the results of HPLC analysis of the VKG_01 compound synthesized in Example 1.

[0108] Figure 3 shows the results of mass spectrum analysis of the VKG_01 compound synthesized in Example 1.

[0109] Figure 4 shows the results of HPLC analysis of the VKG_02 compound synthesized in Example 2.

[0110] Figure 5 shows the results of mass spectrum analysis of the VKG_02 compound synthesized in Example 2.

[0111] Figure 6 shows the results of HPLC analysis of the EVCit-01 compound synthesized in Example 3.

[0112] Figure 7 shows the results of mass spectrum analysis of the EVCit-01 compound synthesized in Example 3.

[0113] Figure 8 shows the results of HPLC analysis of the EVCit-02 compound synthesized in Example 4.

[0114] Figure 9 shows the results of mass spectrum analysis of the EVCit-02 compound synthesized in Example 4.

[0115] Figure 10 shows the results of HPLC analysis of the two-drug compound synthesized in Example 5.

[0116] Figure 11 shows the results of mass spectrum analysis of the two compounds synthesized in Example 5.

[0117] Figure 12 is a graph showing the results of measuring the change in cell viability after treating U87 MG and U251 cells with drug conjugate 2 alone or with drug conjugate 1 and 2 together in Experimental Example 2.

[0118] Figure 13 shows a confocal microscope image observed after fluorescent staining following treatment of U87 MG cells with drug conjugate 1 and 2 (Cy5.5) in Experimental Example 3.

[0119] Figure 14 shows a confocal microscope image observed after fluorescent staining following treatment of U251 cells with drug conjugate 1 and 2 (Cy5.5) in Experimental Example 3.

[0120] Figure 15 shows a photograph of the change in fluorescence intensity of cells observed using a confocal microscope after treating U87 MG cells with drug conjugate 1 and 2 (Cy5.5) at various concentrations in Experimental Example 4.

[0121] Figure 16 is a graph showing the results of measuring the level of reduction in fluorescence intensity compared to the untreated control group after treating the in-vitro blood-brain barrier (BBB) ​​mimicking Transwell with drug conjugate 2 alone or with drug conjugate 1 and 2 together in Experimental Example 5.

[0122] Figure 17 is a graph showing the results of observing changes in tumor volume over time after sequentially administering drug conjugates 1 and 2 to a glioblastoma xenograft mouse model in Experimental Example 6 and administering exatecan alone as a control.

[0123] Figure 18 is a graph showing the results of observing changes in tumor volume over time after sequentially administering drug conjugate 1 and 2 to a glioblastoma xenograft mouse model in Experimental Example 7, and administering exatecan alone in an amount equal to that of exatecan contained in drug conjugate 1 as a control.

[0124] Fig. 19 is a reference example showing integrin α in U87 MG cells and U251 cells. V This shows the results of comparing the expression levels of β3 using Western blot.

[0125] Figure 20 shows a confocal microscope image observed after fluorescent staining of MDA-MB-231 cells treated with drug conjugate 1 and 2 (Cy5.5) in Experimental Example 8.

[0126] Figure 21 shows a confocal microscope image observed after fluorescent staining following treatment of Panc-1 cells with drug conjugate 1 and 2 (Cy5.5) in Experimental Example 8.

[0127] Figure 22 shows a confocal microscope image observed after fluorescent staining following treatment of PC-3 cells with drug conjugate 1 and 2 (Cy5.5) in Experimental Example 8.

[0128] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0129]

[0130] Examples

[0131]

[0132] [Preparation Example 1] Synthesis of HD(RGD dimer)-ADIBO

[0133] HD(RGD dimer)-ADIBO was synthesized from ADIBO-amine according to Reaction Scheme 1 below.

[0134] [Reaction Equation 1]

[0135]

[0136] 1. Synthesis of Compound A-1: ​​Fmoc-Asp(OtBu)-OH (13.58 g, 33 mmol), HBTU (17.07 g, 1.35 eq), HOBt (6.08 g, 1.35 eq), DIPEA (10.45 mL, 1.82 eq), and DMF (300 mL) were placed in a reaction flask and stirred at room temperature for 10 minutes. DBCO-amine (8.29 g, 30 mmol) was added and stirred at room temperature for 3 more hours. After extraction with ethyl acetate / water, silica gel flash column chromatography (ethyl acetate / hexane=80:20) was performed to obtain a pale yellow target product (16.63 g, 79.2%). The theoretical molecular weight was 669.28, the measured molecular weight was 670, and the mass spectrum results were consistent with the target.

[0137] 1 H NMR (500 MHz, Methanol-d4) δ 7.80 (dd,J= 7.4, 5.0 Hz, 2H), 7.71 - 7.17 (m, 14H), 5.01 (dd,J= 28.2, 14.0 Hz, 1H), 4.48 - 4.28 (m, 3H), 4.22 (t,J= 6.9 Hz, 1H), 3.61 (dd,J= 16.0, 13.9 Hz, 1H), 3.28 - 3.09 (m, 2H), 2.63 (ddd,J= 16.0, 8.8, 5.4 Hz, 1H), 2.58 - 2.35 (m, 2H), 1.94 (dt,J= 16.8, 6.0 Hz, 1H), 1.41 (d,J= 5.2 Hz, 9H)

[0138] 2. Synthesis of Compound A-2: Compound A-1 (18.68 g, 27.9 mmol) was placed in a reaction flask, 20% TFA / DCM (380 mL) was added, and the mixture was stirred at room temperature for 2 to 3 hours. After evaporating the solvent under reduced pressure, silica gel flash column chromatography (methanol / dichloromethane = 5 / 95) was performed to obtain a pale yellow solid (8.68 g, yield 50.7%). The theoretical molecular weight was 613.22, the measured molecular weight was 613, and the mass spectrum results were consistent with the target product.

[0139] 1H NMR (500 MHz, DMSO-d6) δ 7.91 - 7.87 (m, 2H), 7.71 (td,J= 8.3, 3.7 Hz, 2H), 7.65 - 7.52 (m, 3H), 7.50 - 7.45 (m, 1H), 7.44 - 7.35 (m, 4H), 7.35 - 7.27 (m, 4H), 5.31 (d,J= 3.9 Hz, 1H), 4.98 (dd, J = 19.6, 14.1 Hz, 1H), 4.29 - 4.15 (m, 4H), 3.64 - 3.55 (m, 1H), 3.29 (t,J=6.8 Hz, 1H), 3.10 (dd,J= 14.5, 8.7 Hz, 1H), 2.96 (dtd,J= 16.6, 7.8, 5.3 Hz, 1H), 2.47 - 2.34 (m, 2H), 1.80 (ddd,J= 16.2, 8.3, 5.5 Hz, 1H)

[0140] 3. Synthesis of Compound A-3: Compound A-2 (8.68 g, 14.1 mmol) was placed in a reaction flask, and dichloromethane / dimethylformamide (300 mL / 100 mL) was added. DCC (3.78 g, 1.3 eq) and NHS (2.11 g, 1.3 eq) were added, and after stirring at room temperature for 4 hours, the solvent was evaporated under reduced pressure, and the precipitated solid was removed by vacuum filtration. HD-[(cRGDfK)]2 (14.69 g, 0.8 eq), which had been pre-dissolved in dimethylformamide and DIEA (3.94 mL, 1.6 eq), was added to a flask containing ADIBO-NHFmoc-NHS (10.02 g, 14.1 mmol) and stirred at room temperature for 3 hours. After confirming that the reaction was complete by HPLC, some of the dimethylformamide was removed, and ether (2 L) was added to induce precipitation. After filtration under reduced pressure, an ivory solid (16.90 g obtained, yield 78.7%) was obtained. The theoretical molecular weight was 1898.85, the measured molecular weight was 949, and the mass spectrum results were consistent with the target product.

[0141] 4. Synthesis of HD(RGD dimer)-ADIBO: Compound A-3 (16.51 g, 8.69 mmol) was placed in a reaction flask, 20% piperidine / DMF (434.5 mL) was added, and the mixture was stirred at room temperature for 30 minutes. Ether (2 L) was added to induce precipitation, and after filtration under reduced pressure, the substance was purified by C18 silica gel flash column chromatography. A white solid (2.22 g, 15.2%) was obtained by freeze-drying. The theoretical molecular weight was 1676.78, and the measured molecular weights were 838 and 1676, respectively; the mass spectrum results were consistent with the target substance. Figure 1 shows the results of HPLC analysis of the synthesized HD(RGD dimer)-ADIBO under the following conditions.

[0142] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0143] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 50% over 10 minutes, maintain for 2 minutes)

[0144] Rt = 6.12 min (Flow rate: 1 mL / min)

[0145]

[0146] [Preparation Example 2] Synthesis of Peptide Boc-VK(Fmoc)G-OH

[0147] The peptide Boc-VK(Fmoc)G-OH shown in Chemical Formula 6 below was synthesized by the solid phase peptide synthesis method. The theoretical molecular weight was 624.32, the measured molecular weight was 625.2, and the mass spectrum results were consistent with the target product.

[0148] Device: SHIMADZU SCL-40

[0149] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0150] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 95% over 20 minutes, hold for 2 minutes)

[0151] Rt = 13.5 min (Flow rate: 1.0 mL / min)

[0152] [Chemical Formula 6]

[0153]

[0154]

[0155] [Example 1] Preparation of drug conjugate VKG_01

[0156] The drug conjugate VKG_01 was synthesized from the peptide Boc-VK(Fmoc)G-OH according to reaction scheme 2 below.

[0157] [Reaction Equation 2]

[0158]

[0159] 1. Synthesis of Compound B-1: Peptide Boc-VK(Fmoc)G-OH (1.93 g, 3.09 mmol), p-aminobenzyl alcohol (762 mg, 2 eq), EEDQ (1.5 g, 2 eq), and DMF (15 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. 200 mL of ether was added to induce precipitation, after which the solid was filtered and vacuum dried for 1 hour. Silica gel flash column chromatography (methanol / dichloromethane=5 / 95) was performed to obtain a pale yellow solid (880 mg, yield 39%). The obtained solid (880 mg, 1.21 mmol), Bis-NPC (920 mg, 2.5 eq), and DMF (60 mL) were placed in a reaction flask and stirred at room temperature for 5 hours. Afterward, water (100 mL) was added to induce precipitation, the solid was filtered, and vacuum dried for 6 hours. A pale yellow target product (1.03 g, 95.4%) was obtained. The theoretical molecular weight is 894.96, and the measured molecular weight is 917.2 (M+Na +) and the mass spectrum results matched the target.

[0160] 2. Synthesis of Compound B-2: Compound B-1 (1.03 g, 1.15 mmol), exatecan mesylate (642 mg, 1.05 eq), DIEA (600 μL, 3 eq), and DMF (28 mL) were placed in a reaction flask and stirred at room temperature for 16 hours, then evaporated under reduced pressure. Ether (300 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A slightly gray target product was obtained without purification. The theoretical molecular weight was 1190.51, the measured molecular weight was 1191.4, and the mass spectrum results were consistent with the target product.

[0161] 3. Synthesis of Compound B-3: Compound B-2 (1.55 g, 1.30 mmol) was placed in a reaction flask and 10% TFA / DCM (130 mL) was added, followed by stirring at room temperature for 1 hour. After evaporation drying under reduced pressure, silica gel flash column chromatography (methanol / dichloromethane = 5:95) was performed to obtain a white target product (672 mg, 47.4%). The theoretical molecular weight was 1090.46, the measured molecular weight was 1091.4, and the mass spectrum results were consistent with the target product.

[0162] 4. Synthesis of Compound B-4: Compound B-3 (200 mg, 0.183 mmol), succinic anhydride (22 mg, 1.2 eq), DIEA (64 μL, 2 eq), and DMF (18 mL) were placed in a reaction flask and stirred at room temperature for 1 hour, then evaporated under reduced pressure. Ether (2–30 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. The theoretical molecular weight was 1190.48, the measured molecular weight was 1191.3, and the mass spectrum results were consistent with the target product. The obtained solid (203.2 mg, 0.171 mmol), EDCI (65.6 mg, 2 eq), NHS (39.4 mg, 2 eq), and DMF (17 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. After adding ether (2–30 mL) to induce precipitation, the solid was filtered and vacuum dried for 6 hours. A white target product (215.4 mg, 97.8%) was obtained. The theoretical molecular weight was 1287.49, and the measured molecular weights were 1288.2 and 1310.2, and the mass spectrum results were consistent with the target product.

[0163] 5. Synthesis of VKG-01: Compound B-4 (100 mg, 0.078 mmol), HD(RGD dimer)-ADIBO (130.2 mg, 1 eq), DIEA (27 uL, 2 eq), and DMF (8 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. Ether (2–30 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (120.9 mg, 54.7%) was obtained without further purification. The obtained solid (114.1 mg, 0.04 mmol) was placed in a reaction flask, 4% piperidine / DMF (4 mL) was added, and after stirring at room temperature for 40 minutes, evaporated under reduced pressure. Ether (2–30 mL) was added to induce precipitation, after which the solid was filtered under reduced pressure. After performing C18 silica gel flash column chromatography (acetonitrile / 0.1% formic acid in water), freeze-drying was performed to obtain a white target product (12.8 mg, 12.2%). Figure 2 shows the results of HPLC analysis performed on VKG-01 under the following conditions, and Figure 3 shows the mass spectrum. The theoretical molecular weight of VKG-01 is 2627.18, and the measured molecular weight is half that value, 1313.9, and the mass spectrum results were consistent with the target product.

[0164] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0165] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 50% over 10 minutes, maintain for 2 minutes)

[0166] Rt = 8.03 min (Flow rate: 1 mL / min)

[0167]

[0168] [Example 2] Preparation of drug conjugate VKG-02

[0169] The drug conjugate VKG-02 was synthesized from compound B-3 according to the following reaction scheme 3.

[0170] [Reaction Equation 3]

[0171]

[0172] 1. Synthesis of Compound B-5: Compound B-3 (672 mg, 0.616 mmol), HO-PEG4-NHS ester (362 mg, 1.5 eq), DIEA (214 uL, 2 eq), and DMF (30 mL) were placed in a reaction flask and stirred at room temperature for 4 hours, then evaporated under reduced pressure. After adding ether (300 mL) to induce precipitation, the mixture was filtered under reduced pressure, followed by silica gel flash column chromatography (methanol / dichloromethane = 5:95) to obtain a white target product (563 mg, 66.8%). The theoretical molecular weight was 1366.58, the measured molecular weight was 1365.2, and the mass spectrum results were consistent with the target product. The obtained solid (563 mg, 0.412 mmol), EDCI (158 mg, 2 eq), NHS (95 mg, 2 eq), and DMF (29 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. Ether (300 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (452 ​​mg, 68%) was obtained. The theoretical molecular weight was 1463.60, the measured molecular weight was 732.5, and the mass spectrum results were consistent with the target product.

[0173] 2. Synthesis of VKG-02: Compound B-5 (339 mg, 0.232 mmol), HD(RGD dimer)-ADIBO (323 mg, 0.9 eq), DIEA (67 uL, 2 eq), and DMF (19 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. Ether (4–50 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (418 mg, 71.6%) was obtained without further purification. The obtained solid (418 mg, 0.138 mmol) was placed in a reaction flask and 4% piperidine / DMF (7 mL) was added. After stirring at room temperature for 1 hour, the mixture was evaporated under reduced pressure. Ether (4–50 mL) was added to induce precipitation, after which the solid was filtered under reduced pressure. After performing C18 silica gel flash column chromatography (acetonitrile / 0.1% formic acid in water), freeze-drying was performed to obtain a white target product (139 mg, 36%). Figure 4 shows the results of HPLC analysis performed on VKG-02 under the following conditions, and Figure 5 shows the mass spectrum. The theoretical molecular weight of VKG-02 is 2803.29, and the measured molecular weight is half that value, 1402.1, and the mass spectrum results were consistent with the target product.

[0174] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0175] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 50% over 10 minutes, maintain for 2 minutes)

[0176] Rt = 8.27 min (Flow rate: 1 mL / min)

[0177]

[0178] [Example 3] Preparation of drug conjugate EVCit-01

[0179] The drug conjugate EVCit-01 was synthesized according to the following reaction scheme 4.

[0180] [Reaction Equation 4]

[0181]

[0182] 1. Synthesis of Compound C-1: The peptide Fmoc-VCit-PAB-OH (2 g, 3.3 mmol) was placed in a reaction flask and 20% piperidine / DMF (20 mL) was added. After stirring at room temperature for 30 minutes, the mixture was evaporated and dried under reduced pressure. After extraction with ethyl acetate and water, silica gel flash column chromatography (methanol / dichloromethane = 20:80) was performed to obtain a white target product (1.02 g, 81.3%). The theoretical molecular weight was 379.22, and the measured molecular weights were 379.9 and 401.9, and the mass spectrum results were consistent with the target product.

[0183] 2. Synthesis of Compound C-2: Fmoc-Glu(OtBu)-OH (725.8 mg, 1.706 mmol), HBTU (970.5 mg, 1.5 eq), HOBt (492 mg, 1.5 eq), and DMF (6 mL) were placed in a reaction flask, and DIEA (0.6 mL, 2 eq) was added. The mixture was stirred at room temperature for 10 minutes. Compound C-1 (1.02 g, 1.5 eq) was dissolved in DMF (3 mL) and added to the reaction mixture, followed by stirring at room temperature for 3 hours. After extraction with ethyl acetate and water, silica gel flash chromatography (methanol / dichloromethane = 5:95) was performed to obtain a white target product (938 mg, 70%). The theoretical molecular weight is 786.4, and the measured molecular weight is 787.7. It was 809.5, and the mass spectrum results matched the target.

[0184] 3. Synthesis of Compound C-3: Compound C-2 (1.5 g, 1.91 mmol), DMAP (279 mg, 1.2 eq), and DMF (27 mL) were placed in a reaction flask and stirred. Bis-NPC (870 mg, 1.5 eq) was added to the reaction solution and stirred at room temperature for 2–3 hours. Then, water (5–60 mL) was added under an ice bath to induce precipitation. The solid was washed with water and ether, filtered under reduced pressure, and vacuum dried for 6 hours. A slightly yellow target product (1.37 g, 75.3%) was obtained. The theoretical molecular weight was 951.4, the measured molecular weight was 952.2, and the mass spectrum results were consistent with the target product.

[0185] 4. Synthesis of Compound C-4: Compound C-3 (1.031 g, 1.083 mmol), exatecan mesylate (633 mg, 1.1 eq), DIEA (943 μL, 5 eq), and DMF (30 mL) were placed in a reaction flask and stirred at room temperature for 12 hours, then evaporated under reduced pressure. Ether (100 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A slightly gray target product (1.074 g, 79.4%) was obtained. The theoretical molecular weight was 1247.53, and the measured molecular weights were 1248.2 and 1270.3, and the mass spectrum results were consistent with the target product.

[0186] 5. Synthesis of Compound C-5: Compound C-4 (1.07 g, 0.86 mmol) was placed in a reaction flask and 20% Piperidine / DMF (30 mL) was added. After stirring at room temperature for 30 minutes, the mixture was evaporated and dried under reduced pressure. Ether (100 mL) was added to induce precipitation, the solid was filtered, and silica gel flash column chromatography (methanol / dichloromethane = 10:90 followed by the addition of 1% triethylamine) was performed to obtain a slightly brown target product (0.49 g, 55.5%). The theoretical molecular weight was 1025.47, the measured molecular weight was 1026.3, and the mass spectrum results were consistent with the target product.

[0187] 6. Synthesis of Compound C-6: Compound C-5 (245 mg, 0.239 mmol), succinic anhydride (28.7 mg, 1.2 eq), DIEA (83 μL, 2 eq), and DMF (20 mL) were placed in a reaction flask and stirred at room temperature for 1 hour, then evaporated under reduced pressure. Ether (2–30 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (234.8 mg, 87.2%) was obtained, and the following reaction was carried out without further purification. The theoretical molecular weight was 1125.48, the measured molecular weight was 1126.2, and the mass spectrum results were consistent with the target product. The obtained solid (234.8 mg, 0.21 mmol), EDCI (80.5 mg, 2 eq), NHS (48.3 mg, 2 eq), and DMF (10.5 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. Ether (100 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (204.1 mg, 79.4%) was obtained. The theoretical molecular weight was 1222.50, the measured molecular weight was 1223.2, and the mass spectrum results were consistent with the target product.

[0188] 7. Synthesis of EVCit-01: Compound C-6 (204.1 mg, 0.167 mmol), HD(RGD dimer)-ADIBO (285.2 mg, 1 eq), DIEA (59.2 μL, 2 eq), and DMF (17 mL) were placed in a reaction flask and stirred at room temperature for 3 days, then evaporated under reduced pressure. Ether (2–30 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (136.6 mg, 28.8%) was obtained without further purification. The obtained solid (114.1 mg, 0.04 mmol) was placed in a reaction flask, 20% TFA / DCM (10 mL) was added, and after stirring for 2 hours at 0 °C to room temperature, ether (100 mL) was added to induce precipitation. After HPLC column purification (acetonitrile / 0.1% formic acid in water) and freeze-drying, a white target product (12.8 mg, 12.2%) was obtained. Figure 6 shows the results of HPLC analysis performed on EVCit-01 as described below, and Figure 7 shows the mass spectrum. The theoretical molecular weight of EVCit-01 is 2728.19, and the measured molecular weight is half that value, 1365.5; the mass spectrum results were consistent with the target product.

[0189] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0190] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 50% over 10 minutes, maintain for 2 minutes)

[0191] Rt = 8.82 min (Flow rate: 1 mL / min)

[0192]

[0193] [Example 4] Preparation of drug conjugate EVCit-02

[0194] The drug conjugate EVCit-02 was synthesized from compound C-5 according to the following reaction scheme 5.

[0195] [Reaction Equation 5]

[0196]

[0197] 1. Synthesis of Compound C-7: Compound C-5 (245 mg, 0.239 mmol), HO-PEG4-NHS ester (140.3 mg, 1.5 eq), DIEA (83.3 μL, 2 eq), and DMF (20 mL) were placed in a reaction flask and stirred at room temperature for 1 hour, then evaporated under reduced pressure. After adding ether (2–30 mL) to induce precipitation, the solid was filtered, and silica gel flash column chromatography (methanol / dichloromethane = 15:85 followed by the addition of 1% acetic acid) was performed to obtain a white target product (320.2 g, 100%). The theoretical molecular weight was 1301.59, the measured molecular weight was 1302.3, and the mass spectrum results were consistent with the target product. The obtained solid (274 mg, 0.21 mmol), EDCI (120.8 mg, 3 eq), NHS (72.5 mg, 3 eq), and DMF (10.5 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. Ether (100 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (181 mg, 61.6%) was obtained. The theoretical molecular weight is 1398.48, and the measured molecular weight is 1421.2 (M+Na + ) and the mass spectrum results matched the target.

[0198] 2. Synthesis of EVCit-02: Compound C-7 (181 mg, 0.129 mmol), HD(RGD dimer)-ADIBO (195 mg, 0.9 eq), DIEA (45 μL, 2 eq), and DMF (6 mL) were placed in a reaction flask and stirred at room temperature for 24 hours, then evaporated under reduced pressure. Ether (5–60 mL) was added to induce precipitation, after which the solid was filtered and vacuum dried for 6 hours. A white target product (175 mg, 45.7%) was obtained without further purification. The obtained solid (172 mg, 0.059 mmol) was placed in a reaction flask, 10% TFA / DCM (6 mL) was added, and after stirring at room temperature for 2 hours, ether (2–30 mL) was added to induce precipitation. The solid was then filtered and subjected to C18 silica gel flash column chromatography (acetonitrile / 0.1% formic acid in water). After freeze-drying, a white target product (13 mg, 7.7%) was obtained. Figure 8 shows the results of HPLC analysis performed on EVCit-02 under the following conditions, and Figure 9 shows the mass spectrum. The theoretical molecular weight of EVCit-02 is 2904.3, and the measured molecular weight is half that value, 1453.9; the mass spectrum results were consistent with the target product.

[0199] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0200] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 50% over 10 minutes, maintain for 2 minutes)

[0201] Rt = 8.95 min (Flow rate: 1 mL / min)

[0202]

[0203] [Example 5] Preparation of a 2-drug conjugate

[0204] A second drug conjugate was synthesized from compound HD-[(cRGDfK)]2 according to the following reaction scheme 6.

[0205] [Reaction Equation 6]

[0206]

[0207] HD-[(cRGDfK)]2 (2 g, 1.53 mmO) and DMF (60 mL) were placed in a reaction flask, and 1 M NaHCO3 aqueous solution was added to adjust the pH to 8. Azidobutyric acid NHS ester (380.7 mg, 1.1 eq) was dissolved in DMF (40 mL), added to the starting material reaction solution, stirred at room temperature for 16 hours, and then evaporated under reduced pressure. Ether (300 mL) was added to induce precipitation, the solid was filtered, and C18 silica gel flash column chromatography (acetonitrile / 0.1% Formic acid in water) was performed. After freeze-drying, a white target product (1.83 g, 84.7%) was obtained. Figure 10 shows the results of HPLC analysis performed on the second component under the following conditions, and Figure 11 shows the mass spectrum.

[0208] Column: Agilent Eclips Plus C18, 3.5 µm, 4.6 mm X 100 mm

[0209] HPLC Analysis Conditions: Acetonitrile / water (0.1% formic acid added) gradient (increase from 5% to 50% over 10 minutes, maintain for 2 minutes)

[0210] Rt = 5.72 min (Flow rate: 1 mL / min)

[0211]

[0212] [Example 6] Preparation of Drug Conjugate 2-Cy5.5 (AZIDO-Flamma675-cRGDfK dimer)

[0213] In the following experiments, a compound of Chemical Formula 7, in which Cy5,5 is attached to a drug conjugate 2, was used for detection.

[0214] [Chemical Formula 7]

[0215]

[0216]

[0217] [Experimental Example 1] Evaluation of Integrin Binding Ability of Drug Conjugates

[0218] The drug conjugates prepared in Examples 1 to 4 above are integrin α expressed in cancer cells v β3 and α v The following experiment was performed to evaluate whether it binds to β5 and competitively inhibits the extracellular matrix, vitronectin. Specifically, 100 μL of a coating solution (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) containing vitronectin at concentrations of 2 μg / mL or 1 μg / mL was applied to each 96-well ELISA plate and coated at 4°C for 16 hours. The coated plates were washed with 200 μL of phosphate buffer (137 nM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.46 mM KH2PO4, 0.05% tween-20, pH 7.4) and treated with 150 μL of blocking buffer (25 mM Tris, 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.6, 1% BSA) at room temperature for 1 hour to prevent non-specific binding. After washing three times with 200 μL of phosphate buffer, 50 μL of drug conjugates (Examples 1 to 4) serially diluted from 1000 nM to 0.32 nM in blocking buffer were applied to wells B to G. Well A was treated with 100 μL of blocking buffer and used as a blank, and well H was treated with 50 μL of blocking buffer. 50 μL of 2 μg / mL integrin α v β3 or α v After adding β5 to wells B through H, the reaction was carried out at room temperature for 1 hour. After washing 3 times with 200 μL of phosphate buffer, the primary antibody (α v β3: mouse anti-human CD51 / 61; α vAfter adding 100 μL of β5: anti-αv mouse anti-human MAB1978, the reaction was carried out at room temperature for 1 hour. After washing three times with 200 μL of phosphate buffer, 100 μL of the secondary antibody was added and the reaction was carried out at room temperature for 1 hour. After washing three times with 200 μL of phosphate buffer, the reaction was carried out with 50 μL of TMB substrate solution. The reaction was stopped by adding 50 μL of 2N sulfuric acid to the colored ELISA plate, and the absorbance was measured at a wavelength of 450 nm using a microplate spectrometer. IC 50 The values ​​were repeated twice for each drug conjugate and calculated using GraphPad Prism 8 software.

[0219] α v β3(nM)α v β5(nM)c(RGDfK)2 dimer 1.12 ± 0.30 2.57 ± 0.21 VKG_02 1st agent 3.32 ± 1.36 9.32 ± 2.07 VKG_02 1st agent + 2nd agent 0.59 ± 0.09 1.57 ± 0.09

[0220] As shown in Table 1 above, integrin α v β3 or α v Compared to the case where drug conjugate VKG_02 Agent 1 was administered alone to β5, when drug conjugates VKG_02 Agent 1 and Agent 2 were administered together, integrin α v β3 and α v It was observed that the binding ability to β5 increased by 5.6 times and 5.9 times.

[0221]

[0222] [Experimental Example 2] Evaluation of Glioblastoma Proliferation Inhibitory Ability

[0223] To evaluate the proliferation inhibitory ability of the complex drug conjugate according to the present invention against glioblastoma cells, U-87 MG and U251 cell lines were prepared as glioblastoma cell lines. On the day before the experiment, U87 MG and U251 cells were cultured in a 96-well plate for 24 hours. U87 MG and U251 cells were cultured at 37°C under a humidified atmosphere of 5% CO2 in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin). After culture, the medium was removed, and drug conjugate 1 (Example 1 or 2) and drug conjugate 2 (Example 5) were added to the DMEM medium to make a total volume of 100 μL. At this time, U87 MG cells were treated with each drug conjugate diluted threefold from 1000 nM to 0.15 nM, and U251 cells were treated with each conjugate diluted threefold from 3000 nM to 0.45 nM. U87 MG cells and U251 cells were cultured at 37 ℃ for 144 hours and 96 hours, respectively, under a humidified atmosphere of 5% CO2. After adding 10 μL of WST-8 reagent to the cell-cultured plates and allowing the reaction to occur in the incubator, changes in cell viability were measured by determining the absorbance at a wavelength of 450 nm using a microplate spectrometer. GI 50 The values ​​were repeated 3 times for each drug and calculated using a statistical analysis program (GraphPad Prism 8).

[0224] Classification GI 50 (nM)U87 MGU251VKG_01394.111265.43VKG_0293.84278.04

[0225] As a result, as shown in Fig. 12, compared to the case where U87 MG and U251 cells were treated with Drug Conjugate 2 alone, cell viability was significantly reduced when Drug Conjugate 1 (Example 1 or 2) and Drug Conjugate 2 were treated together; furthermore, when treated at a concentration of approximately 1000 nM, cell viability was observed to decrease to 30% or less. Both combinations of the two drug delivery systems exhibited excellent glioblastoma proliferation inhibitory activity, but the combination of VKG_02 1 and 2 in Example 2 was particularly observed to have a very superior glioblastoma proliferation inhibitory effect. Additionally, while the aforementioned complex drug delivery systems were confirmed to have excellent growth inhibitory effects in both types of glioblastoma cells, the degree of proliferation inhibition was particularly high in U87 MG cells. This is because integrin α in U87 MG cells compared to U251 cells v It is expected to be due to the high expression level of β3 (Fig. 19).

[0226]

[0227] [Experimental Example 3] Evaluation of cell permeability of drug conjugates according to integrin αVβ3 expression levels

[0228] On the day before the experiment, U87 MG and U251 cells were cultured in 4-well cell culture slides (SPL) for 24 hours. U87 MG and U251 cells were cultured at 37°C under a humidified atmosphere of 5% CO2 in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin / Streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin). U87 MG and U251 cells were treated with Drug Conjugate 1 (Example 1) and Drug Conjugate 2 (Example 6) at 0.5 μM each for 1 hour. The treatment interval between Drug 1 and Drug 2 was 10 minutes. To observe the cell permeability of the test substance, cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 15 minutes. To directly detect the internalized drug conjugate 1 within the cells, the cells were washed three times with phosphate buffer solution, treated with an antifade reagent containing DAPI, a nuclear fluorescence staining solution, and then covered with a cover glass to dry thoroughly. Additionally, during confocal microscopy, the original shape of the cells was observed using a Nomarski filter, and Cy5.5 fluorescence and DAPI fluorescence were observed using filters corresponding to their respective fluorochromes.

[0229] As shown in Figures 13 and 14, when the cells were treated with drug conjugate 1 (Example 1) and drug conjugate 2 (Example 6), it was confirmed that these drug conjugates entered the cells, and in particular, that they were distributed in the cytoplasm. Furthermore, as examined in the previous experiment, integrin α v It was confirmed that cell permeability was higher in U87 MG cells with high β3 expression (an increase of more than 2 times).

[0230]

[0231] [Experimental Example 4] Evaluation of changes in cell permeability according to the treatment concentration of the drug conjugate

[0232] On the day before the experiment, U87 MG cells were cultured in 4-well cell culture slides (SPL) for 24 hours. U87 MG and U251 cells were cultured at 37°C under a humidified atmosphere of 5% CO2 in DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin / Streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin, Invitrogen). U87 MG cells were treated with VKG_01 Agent 1 of Example 1 and Agent 2 of Example 6 (Cy5.5) at different concentrations (0.1, 0.2, 0.5 μM) for 1 hour. The treatment interval between Agent 1 and Agent 2 was 10 minutes. To observe the cell permeability of the test substance, cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 15 minutes. For the direct detection of the internalized drug conjugate VKG_01 within the cells, the cells were washed three times with phosphate buffer solution, treated with an antifade reagent containing DAPI, a nuclear fluorescence staining solution, and then covered with a cover glass to dry thoroughly. Additionally, during confocal microscopy, the original shape of the cells was observed using a Nomarski filter, and Cy5.5 fluorescence and DAPI fluorescence were observed using filters corresponding to their respective fluorochromes.

[0233] As shown in Figure 15, it was confirmed that as the treatment concentration of the complex drug conjugate increased, the amount of drug internalized within the cell increased, and the expressed fluorescence intensity also increased.

[0234]

[0235] [Experimental Example 5] Evaluation of In-Vertically Blood-Brain Barrier (BBB) ​​Permeability of Drug Conjugates

[0236] To determine the permeability of substances by mimicking the blood-brain barrier (BBB) ​​environment in vitro, hCMEC / d3 endothelial cells were cultured on a transwell membrane for 72 hours to verify the permeability. The culture medium for hCMEC / d3 cells was EBM-2 endothelial basal medium supplemented with 5% fetal bovine serum (FBS), 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin), chemically defined lipid concentrates, 5 μg ascorbic acid, 1 ng hBFGF (h basal fibroblast growth factor), 10 mM HEPES, and 1.4 μM hydrocortisone. Cells were placed in a transwell (1.0 μm pore size, PET membrane, 12-well) and incubated at 37 °C for 72 hours under a humidified atmosphere of 5% CO2, with the medium being replaced every 24 hours. Subsequently, all medium was removed, the remaining medium was washed with phosphate buffer, and the samples were treated using phenol red-free EBM medium. The samples were treated with 2 μM Lucifer yellow, 0.5 μM of the second agent of Example 5, and a mixture of the first agent of Example 1 VKG_01 and the second agent of Example 6 (0.5 μM) for 6 hours. The solutions from the inside of the transwell and the bottom of the chamber were transferred to a 96-well plate, and fluorescence intensity was measured using a SpectraMax iD3-V7. Each sample was observed using a filter appropriate for its respective fluorochrome. To confirm the integrity of the cell tissue used in the experiment, the transwell after the reaction was washed twice with phosphate buffer and then treated with 2 μM FITC-dextran.After reacting for 2 hours, the solution inside the transwell and at the bottom of the chamber was transferred to a 96-well plate and the fluorescence intensity was measured.

[0237] As a result, as shown in Figure 16, despite the low BBB permeability of the polymer compound according to the present invention, when the composite drug conjugate was treated, it showed about 40% BBB permeability, indicating that the BBB permeability is high.

[0238]

[0239] [Experimental Example 6] Evaluation of in vivo brain tumor suppression ability of a complex drug conjugate (1)

[0240] To evaluate the brain tumor suppression ability of the complex drug conjugate according to the present invention in vivo, the following experiment was performed. Specifically, 5 x 10⁶ U-87 mg cells were injected into congenital thymic defect mice (5 weeks old, balb / c nude mice, OrientBio, Female). 6 Cells were suspended in serum-free DMEM medium and subcutaneously injected into the right flank (upper thigh) of a mouse; after a growth period of approximately 3 weeks, the cells reached about 100 mm 3 ~150 mm 3 A tumorigenetic model of a certain size was selected and used. The drug conjugate VKG_01 of Example 1 was administered to the prepared nude mouse tumor model at a dose of 1.25 mg / kg, and 15 minutes later, the drug conjugate of Example 6 was administered at an equal dose (n = 5). As a control, Exatecan was administered at doses of 3.125 mg / kg and 6.25 mg / kg (n = 5 each). Changes in tumor size were observed after administering the drugs intravenously a total of 4 times at 4-day intervals.

[0241] As shown in Figure 17, even though the drug conjugate VKG_01 containing exatecan was administered at a low dose of 1.25 mg / kg, a tumor suppression effect similar to that observed when exatecan was administered at a dose of 3.125 mg / kg or more was confirmed.

[0242]

[0243] [Experimental Example 7] Evaluation of in vivo brain tumor suppression ability of complex drug conjugates (2)

[0244] In a glioblastoma xenograft mouse model prepared in the same manner as Experimental Example 6, drug conjugate 1 VKG_01 (Example 1) or VKG_02 (Example 2) was administered such that the dose of exatecan contained therein was 2.5 mg / kg, and 15 minutes later, drug conjugate 2 (Example 6) was administered in the same amount as drug 1 (n = 5). As a control, exatecan was administered at a dose of 2.5 mg / kg (n = 5). Changes in tumor size were observed after administering the mixture intravenously a total of 4 times at 4-day intervals.

[0245] As shown in Figure 18, it was confirmed that the tumor suppression effect was more than twice as superior when VKG_01 and VKG_02, which are drug conjugates containing exatecan, were administered at a dose of 2.5 mg / kg of exatecan included therein, and when the second drug was administered together with them, compared to when exatecan alone was administered at 2.5 mg / kg.

[0246]

[0247] [Experimental Example 8] Evaluation of Cell Permeability of Drug Conjugates Against Integrin Overexpressing Carcinomas

[0248] Through the above experiment, the tumor-inhibiting effect of the complex drug conjugate according to the present invention was confirmed in brain cancer among integrin-overexpressing carcinomas. Accordingly, to confirm the anti-tumor effect on other integrin-overexpressing carcinomas other than brain cancer, breast cancer cell line MDA-MB-231, pancreatic cancer cell line Panc-1, and prostate cancer cell line PC-3 cells were prepared the day before the experiment and cultured for 24 hours in 4-well cell culture slides (SPL). MDA-MB-231 and PC-3 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (10,000 units penicillin and 10,000 μg / mL streptomycin), and Panc-1 cells were cultured in DMEM medium at 37°C under a humidified atmosphere of 5% CO2. MDA-MB-231, Panc-1, and PC-3 cells were treated with Drug Conjugate 1 (Example 1) and Drug Conjugate 2 (Example 6) at a concentration of 0.5 μM each for 1 hour. The treatment interval between Drug Conjugate 1 and Drug Conjugate 2 was set to 10 minutes. To observe the cell permeability of the test substances, the cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 15 minutes. To directly detect the internalized Drug Conjugate 1 within the cells, the cells were washed three times with phosphate buffer solution, treated with an antifade reagent containing DAPI (a nuclear fluorescence staining solution), and then covered with a cover glass to allow them to dry thoroughly. Additionally, during confocal microscopy, the original shape of the cells was observed using a Nomarski filter, and Cy5.5 fluorescence and DAPI fluorescence were observed using filters corresponding to their respective fluorochromes.

[0249] As shown in FIGS. 20 to 22, integrin α vWhen drug conjugate 1 (Example 1) and drug conjugate 2 (Example 6) were treated with MDA-MB-231, a breast cancer cell line with high β3 expression, Panc-1, a pancreatic cancer cell line, and PC-3, a prostate cancer cell line, it was confirmed that these drug conjugates were introduced into the cells.

[0250] Through this, the indications of the composite drug conjugate according to the present invention include integrin α in addition to glioblastoma. v It was found that it can be applied to various cancers with high expression levels of β3.

[0251]

[0252] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0253] The present invention provides a method for effectively preventing, improving, or treating cancers that overexpress integrin, such as brain cancer, breast cancer, prostate cancer, and pancreatic cancer, using a novel complex drug conjugate.

Claims

1. A first compound selected from a compound represented by any one of the following chemical formulas 1 to 4, a pharmaceutically acceptable salt, optical isomer, hydrate, and solvate thereof; and A pharmaceutical composition for the prevention or treatment of integrin-overexpressing cancer comprising, as an active ingredient: a second compound selected from a compound represented by the following chemical formula 5, and pharmaceutically acceptable salts, optical isomers, hydrates, and solvates thereof; [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] In the above chemical formulas 2 and 4, n is a natural number from 1 to 30.

2. In Paragraph 1, A pharmaceutical composition in which the first compound and the second compound exist separately outside the body.

3. In Paragraph 1 or 2, A pharmaceutical composition in which the first compound and the second compound combine in the body via a click reaction.

4. In any one of paragraphs 1 through 3, A pharmaceutical composition in which the first compound and the second compound are administered individually.

5. In any one of paragraphs 1 through 4, A pharmaceutical composition in which the first compound and the second compound are administered simultaneously or sequentially.

6. In any one of paragraphs 1 through 5, A pharmaceutical composition wherein the first compound is administered first, and the second compound is administered.

7. In any one of paragraphs 1 through 6, A pharmaceutical composition wherein the first compound and the second compound are administered sequentially, and the administration interval is within one hour.

8. In any one of paragraphs 1 through 7, A pharmaceutical composition wherein the above-mentioned integrin-overexpressing cancer is brain cancer, head and neck cancer, colorectal cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, malignant melanoma, pancreatic cancer, or prostate cancer.

9. In any one of paragraphs 1 through 8, The above-mentioned first and second compounds are pharmaceutical compositions that pass through the blood-brain barrier (BBB).

10. A method for the prevention or treatment of integrin-overexpressing cancer comprising the step of administering a pharmaceutical composition containing a first compound and a second compound as active ingredients to a subject, A method for the prevention or treatment of integrin-overexpressing cancer, wherein the first compound is selected from a compound represented by any one of the following chemical formulas 1 to 4, and its pharmaceutically acceptable salts, optical isomers, hydrates, and solvates, and the second compound is selected from a compound represented by the following chemical formula 5, and its pharmaceutically acceptable salts, optical isomers, hydrates, and solvates: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] In the above chemical formulas 2 and 4, n is a natural number from 1 to 30.

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