Pharmaceutical composition for enhancing the anticancer effect of anticancer agents

Abstract

The present invention relates to a compound for enhancing the anticancer effect of currently used targeted anticancer agents and chemical anticancer agents, more specifically, to an analogue compound of oligopeptide AQTGTGKT that has no side effects and shows excellent anticancer effect, and a composition containing the same. When the compound according to the present invention is mixed with an anticancer agent and treated in combination, it can provide a significant synergistic effect in the cancer growth inhibition effect compared to a control group and each of the groups treated alone. Furthermore, since it shows an excellent combined anticancer effect even with a low concentration of an anticancer agent, it can minimize side effects such as functional and activity damage in normal tissues, bone marrow function reduction, gastrointestinal disorders, alopecia, and anticancer drug resistance. In addition, since the molecular weight of the oligopeptide is smaller than that of an antibody, it has the advantage of being less likely to cause an immune reaction and being easily penetrated into tissues, and it is expected to be used in combination with anticancer agents for various carcinomas.

Description

[Technical Field]

[0001] The present invention relates to a compound that can exhibit a synergistic effect when administered in combination with an anticancer agent such as a targeted anticancer agent or a chemical anticancer agent in cancer treatment, and a pharmaceutical composition containing the compound in combination with the anticancer agent.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2021-0034947 filed on March 17, 2021, and Korean Patent Application No. 10-2022-0032699 filed on March 16, 2022, and the contents disclosed in the specifications and drawings of said applications are incorporated herein by reference. [Background technology]

[0003] Despite the ongoing development of early cancer diagnosis methods and new anti-cancer therapies that have led to improved treatment outcomes, cancer remains a serious disease, competing for the top two causes of death in Japan. Many of the anti-cancer drugs currently used are chemotherapy-based, and their diverse pharmacological effects and toxic side effects vary depending on the type of cancer, posing significant challenges for cancer treatment. Therefore, to overcome the limitations of chemotherapy, there is a continuing need to develop targeted therapeutic agents with clear anti-cancer mechanisms.

[0004] Conventional anticancer drugs penetrate not only cancer cells but also normal cells, damaging their functions and activity, which can lead to side effects such as decreased bone marrow function, gastrointestinal disorders, and alopecia, and can also cause multidrug resistance to anticancer drugs through long-term chemotherapy, presenting major problems in cancer treatment. Therefore, as a solution to these serious problems of conventional anticancer drugs, active development is being conducted into combination drugs that can maximize the efficacy of anticancer drugs while minimizing side effects by administering them in combination with anticancer drugs.

[0005] In particular, combination therapies for cancer treatment are widely used due to their advantage of attacking cancer cells through multiple means. Korean Patent Publication No. 10-2014-0097607 previously disclosed a combination pharmaceutical composition for cancer treatment. Combination therapies can enhance the efficacy of anticancer drugs while reducing the amount of anticancer drug administered, minimizing the toxicity and side effects of the anticancer drug and are also useful when resistance to the anticancer drug has emerged. Even though many effective combination therapies have been identified over the past few decades, the development of effective combination therapies is crucial given the increasing number of people dying from cancer each year. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention was devised to solve the problems of the prior art as described above, and it was found that when the anticancer peptide AQTGTGKT (alanine-glutamine-threonine-glycine-threonine-glycine-lysine-threonine) and its amidated analogue compounds are administered in combination with conventional anticancer drugs, a significant synergistic effect in anticancer activity is observed, leading to the completion of the present invention.

[0007] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises a compound according to the present invention and an anticancer agent as active ingredients.

[0008] Another object of the present invention is to provide a kit for preventing or treating cancer, which comprises the compound according to the present invention and an anticancer agent as active ingredients.

[0009] A further object of the present invention is to provide a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent.

[0010] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by a person having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0011] In order to achieve the above-mentioned object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising (i) a compound represented by the following general formula, and (ii) an anticancer agent as active ingredients:

[0012] (general formula) X-AQTGTGKT

[0013] (In the above general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, and K is lysine, X does not exist, or [ka] It is one or more selected from the group consisting of

[0014] In one embodiment of the present invention, X is absent or [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0015] In another embodiment of the present invention, the anticancer agent may be at least one selected from the group consisting of targeted anticancer agents and chemical anticancer agents, but is not limited thereto.

[0016] In yet another embodiment of the present invention, the targeted anticancer drug may be at least one selected from the group consisting of tyrosine kinase inhibitors, PARP inhibitors, angiogenesis inhibitors, CDK4 / 6 inhibitors (cyclin-dependent kinases 4 / 6 inhibitors), hormonal therapy drugs, and antibody-drug conjugates, but is not limited to this.

[0017] In another embodiment of the present invention, the tyrosine kinase inhibitor may be a drug targeting one or more selected from the group consisting of, but not limited to, EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS Proto-Oncogene 1), BRAF (B-Raf Proto-Oncogene), HER2 (human epidermal growth factor receptor 2), RET (Ret Proto-Oncogene), NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1), MET (Mesenchymal-Epithelial Transition factor), and NRG1 (Neuregulin 1).

[0018] In yet another embodiment of the present invention, the tyrosine kinase inhibitor is osimertinib, afatinib, brigatinib, dasatinib, dacomitinib, erlotinib, gefitinib, lapatinib, neratinib. , Vandetanib, Icotinib, Valitinib, Tesevatinib, Canertinib, Naquotinib, Pelitinib, Poziotinib, Rociletinib, Nazartinib, Alitinib itinib (ALS-1306), pyrotinib, tyrphostin, crizotinib, ceritinib, entrectinib, dabrafenib, trametinib, alectinib, lorlatinib, larotrectinib The anti-cancer agent may be one or more selected from the group consisting of, but is not limited to, larotectinib, lasertinib, olmutinib, AG1478, CUDC-101, MTKi-327 (JNJ-26483327), CL-387785 (EKI-785), CNX-2006, PD168393, TAK285, WZ4002, and AV-412 (MP-412).

[0019] In yet another embodiment of the present invention, the PARP inhibitor may be one or more selected from the group consisting of olaparib, rucaparib, talazoparib, veliparib, and niraparib, but is not limited thereto.

[0020] In yet another embodiment of the present invention, the CDK4 / 6 inhibitor may be one or more selected from the group consisting of trilaciclib, palbociclib, ribociclib, and abemaciclib, but is not limited thereto.

[0021] In yet another embodiment of the present invention, the hormone therapeutic agent may be one or more selected from the group consisting of tamoxifen, toremifene, fulvestrant, goserelin, leuprolide, anastrozole, letrozole, and exemestane, but is not limited thereto.

[0022] In yet another embodiment of the present invention, the antibody-drug conjugate may be one or more selected from the group consisting of sacituzumab govitecan and ladiratuzumab, but is not limited to this.

[0023] In yet another embodiment of the present invention, the targeted anticancer drug may be one or more selected from the group consisting of daratumumab, trastuzumab, and rituximab, but is not limited thereto.

[0024] In yet another embodiment of the present invention, the anticancer chemotherapy agent may be one or more selected from the group consisting of Alimta, oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, and doxorubicin, but is not limited to this.

[0025] In yet another embodiment of the present invention, the compound may enhance the anti-cancer effect of the anti-cancer agent and reduce the side effects, but is not limited thereto.

[0026] In yet another embodiment of the present invention, the composition may be in the form of a mixture in which the compound and the anticancer agent are mixed, but is not limited thereto.

[0027] In yet another embodiment of the present invention, the composition may be in a form in which the compound and the anticancer agent are separately formulated and administered simultaneously or sequentially, but is not limited thereto.

[0028] In yet another embodiment of the present invention, the anti-cancer agent may be contained in a concentration of 0.1 to 10 μM based on the total composition, but is not limited thereto.

[0029] In yet another embodiment of the present invention, the compound represented by the general formula may be contained in a concentration of 1 to 50 μM based on the total composition, but is not limited thereto.

[0030] In yet another embodiment of the present invention, the anticancer drug is a targeted anticancer drug, and the compound represented by the general formula is such that X is absent or [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0031] In yet another embodiment of the present invention, the targeted anticancer drug and the compound may be contained in a molar ratio of 1:1 to 500, but is not limited thereto.

[0032] In yet another embodiment of the present invention, the anticancer agent is a chemical anticancer agent, and the compound represented by the general formula is [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0033] In yet another embodiment of the present invention, the anticancer chemotherapeutic agent and the compound may be contained in a molar ratio of 1:1 to 500, but is not limited thereto.

[0034] In yet another embodiment of the present invention, the cancer may be selected from the group consisting of lung cancer, breast cancer, blood cancer, colon cancer, pancreatic cancer, and combinations thereof, but is not limited thereto.

[0035] The present invention also provides a kit for preventing or treating cancer, which comprises (i) a compound represented by the following general formula, and (ii) an anticancer agent as active ingredients:

[0036] (general formula) X-AQTGTGKT (In the above general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, and K is lysine, X does not exist, or [ka] It is one or more selected from the group consisting of

[0037] The present invention also provides a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, which comprises a compound represented by the following general formula as an active ingredient:

[0038] (general formula) X-AQTGTGKT

[0039] (In the above general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, and K is lysine, X does not exist, or [ka] It is one or more selected from the group consisting of

[0040] In one embodiment of the present invention, the composition may be administered simultaneously, separately, or sequentially with an anti-cancer agent, but is not limited thereto.

[0041] In another embodiment of the present invention, the cancer may be selected from the group consisting of lung cancer, breast cancer, blood cancer, colon cancer, pancreatic cancer, and combinations thereof, but is not limited thereto.

[0042] The present invention also provides a method for preventing or treating cancer, comprising administering to a subject in need thereof a composition comprising (i) a compound represented by the above general formula and (ii) an anticancer agent as active ingredients, wherein the compound and the anticancer agent may each be administered in an effective amount, but the amount is not limited thereto.

[0043] The present invention also provides a method for enhancing the anti-cancer effect of an anti-cancer agent, comprising administering to a subject in need thereof a compound represented by the general formula (I), wherein the compound is administered in an effective amount, but is not limited thereto.

[0044] The present invention also provides use of the compound represented by the above general formula for the manufacture of an agent for enhancing the anticancer effect of an anticancer agent.

[0045] The present invention also provides a use of the compound represented by the above general formula for enhancing the anticancer effect of an anticancer agent, including for suppressing side effects of the anticancer agent.

[0046] The present invention also provides use of a composition comprising (i) a compound represented by the above general formula and (ii) an anticancer agent as active ingredients for the manufacture of a medicament for treating cancer.

[0047] The present invention also provides a use of a composition comprising (i) a compound represented by the above general formula and (ii) an anticancer agent as active ingredients for preventing or treating cancer. [Effects of the Invention]

[0048] The present invention relates to pharmaceutical compositions for cancer prevention / treatment and compositions for enhancing the anticancer effects of anticancer drugs, which contain, as active ingredients, oligopeptide AQTGTGKT and its analog compounds based on the autophagy mechanism, which is a tumor-promoting factor involved in resistance to conventional anticancer drugs. The compounds of the present invention selectively bind to protein targets involved in the early stages of autophagy only when cancer is advanced, thereby overactivating autophagy and specifically targeting cancer cells without affecting normal cells. This allows them to maximize the inhibitory effect on cancer cell proliferation while minimizing the side effects of conventional anticancer drug treatment alone in patients with anticancer drug resistance.

[0049] More specifically, when oligopeptide AQTGTGKT and its analog compounds are administered in combination with conventional anticancer drugs, they exhibit enhanced anticancer effects, and even low drug concentrations exhibit significantly superior anticancer effects by suppressing cancer cell proliferation, thereby minimizing side effects such as functional and activity damage in normal tissues, decreased bone marrow function, gastrointestinal disorders, alopecia, and anticancer drug resistance. Furthermore, oligopeptides have the advantages of having a smaller molecular weight than antibodies, which reduces the risk of immune reactions and allows for easy tissue penetration, and they are expected to be used in combination with anticancer drugs for various cancers. [Brief explanation of the drawings]

[0050] [Figure 1a] FIG. 1a shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound 3-PhPh-AQTGTGKT of the present invention and osimertinib in the lung cancer cell line H1975 (*p<0.05, **p<0.01, ***p<0.001; the same applies below). [Figure 1b] FIG. 1b shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound 4-PhPh-AQTGTGKT according to the present invention and osimertinib in the lung cancer cell line H1975. [Figure 1c] FIG. 1c shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound Ac-AQTGTGKT according to the present invention and osimertinib in the lung cancer cell line H1975. [Figure 1d] FIG. 1d shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound of the present invention, 4-MeOPh-AQTGTGKT, and osimertinib in the lung cancer cell line H1975. [Figure 1e] FIG. 1e shows the results of MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound of the present invention, 4-PhPh-AQTGTGKT, and Alimta in lung cancer cell line H1975. [Figure 1f]FIG. 1f shows the results of MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound of the present invention, 3-PhPh-AQTGTGKT, and Alimta in lung cancer cell line H1975 / OR. [Figure 1g] FIG. 1g shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib in the lung cancer cell line H1975 / OR. [Figure 1h] FIG. 1h shows the results of an MTT assay analysis of the cell activity inhibitory effect of triple combined treatment of the compound 3-PhPh-AQTGTGKT according to the present invention, osimertinib, and Alimta in the lung cancer cell line H1975. [Figure 2a] FIG. 2a shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound AQTGTGKT of the present invention and osimertinib in the lung cancer cell line H820. [Figure 2b] FIG. 2b shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib in the lung cancer cell line H820. [Figure 3] FIG. 3 shows the results of an MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib in the lung cancer cell line PC9 / OR. [Figure 4a] FIG. 4a shows the results of MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound AQTGTGKT of the present invention and Olaparib in breast cancer cell line HCC1937. [Figure 4b] FIG. 4b shows the results of MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound of the present invention, 3-PhPh-AQTGTGKT, and olaparib in breast cancer cell line HCC1937. [Figure 4c]FIG. 4c shows the results of MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound of the present invention, 4-MeOph-AQTGTGKT, and cisplatin in breast cancer cell line HCC1937. [Figure 4d] FIG. 4d shows the results of MTT assay analysis of the cell activity inhibitory effect of combined treatment of the compound of the present invention, 3-PhPh-AQTGTGKT, and cisplatin in breast cancer cell line HCC1937. [Figure 5a] FIG. 5a shows the results of a comparative analysis of the cell growth inhibitory effects depending on the time of combination treatment to explore the optimal combination regimen of the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib in cancer cells. [Figure 5b] FIG. 5b shows the results of a comparative analysis of the cell growth inhibitory effects of the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib on the lung cancer cell line PC9 / OR depending on the duration of combined treatment to specifically explore the optimal combination therapy regimen. [Figure 6a] FIG. 6a shows the results of a comparative analysis of the tumor growth inhibitory effects of combined treatment with the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib after tumor formation in nude mice inoculated with lung cancer cell line H820. [Figure 6b] FIG. 6b shows the results of a comparative analysis of the tumor growth inhibitory effects of combined treatment with the compound 3-PhPh-AQTGTGKT according to the present invention and osimertinib after inoculating the lung cancer cell line H1975 into nude mice to form tumors. [Figure 7] FIG. 7 shows the results of comparative analysis of the inhibitory effects of p-Beclin1S15 in tumors after inoculating lung cancer cell line H820 into nude mice to form tumors, and then treating the tumors with the compound 3-PhPh-AQTGTGKT of the present invention in combination with osimertinib, and then isolating the tumor tissues. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present inventors have confirmed that the oligopeptide AQTGTGKT and its amidated analogs exhibit improved anticancer effects when used in combination with conventional anticancer drugs in various cancer cell lines, and have completed the present invention.

[0052] In one example of the present invention, it was confirmed that when a targeted anticancer drug and / or a chemical anticancer drug was treated in combination with the compound of the present invention in a lung cancer cell line, the anticancer effect was significantly increased compared to when each drug was treated alone (see Example 1).

[0053] In another example of the present invention, it was confirmed that when a targeted anticancer drug or a chemical anticancer drug was treated in combination with the compound of the present invention in breast cancer cell lines, the anticancer effect was significantly increased compared to when each drug was treated alone (see Example 2).

[0054] In another example of the present invention, the tumor growth inhibitory effect of combination therapy with osimertinib and 3-PhPh-AQTGTGKT was analyzed, and it was found that treatment with osimertinib and 3-PhPh-AQTGTGKT in combination followed by treatment with osimertinib alone showed a more improved anti-cancer effect than treatment with osimertinib alone followed by the addition of 3-PhPh-AQTGTGKT (see Example 3.1).

[0055] In yet another example of the present invention, the tumor growth inhibitory effect of combination therapy with osimertinib and 3-PhPh-AQTGTGKT was analyzed, and it was found that continued combination treatment with osimertinib and 3-PhPh-AQTGTGKT after combination treatment with osimertinib and 3-PhPh-AQTGTGKT showed a more improved anti-cancer effect than treatment with osimertinib alone (see Example 3.2).

[0056] In yet another example of the present invention, the effect of combined administration of osimertinib and 3-PhPh-AQTGTGKT was examined using xenograft lung cancer animal models prepared using H820 or H1975 lung cancer cell lines. As a result, it was confirmed that combined administration of the compound and the anticancer agent had a more enhanced tumor growth inhibitory effect than administration of either compound alone (see Example 4).

[0057] In yet another example of the present invention, the autophagy effect of co-administration of osimertinib and 3-PhPh-AQTGTGKT was analyzed, and it was confirmed that the expression of p-Beclin1S15 was significantly reduced in the co-administration group compared to the single administration group (see Example 5).

[0058] Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises (i) a compound represented by the following general formula, and (ii) an anticancer agent as active ingredients:

[0059] (general formula) X-AQTGTGKT

[0060] (In the above general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, and K is lysine, X does not exist, or [ka] It is one or more selected from the group consisting of

[0061] Preferably, said X is absent, or [ka] The compound may be one or more selected from the group consisting of, but is not limited to, the compound.

[0062] The present invention also provides a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, which comprises the compound represented by the above general formula as an active ingredient.

[0063] The present invention also provides a pharmaceutical composition for suppressing side effects of anticancer drugs, comprising the compound represented by the above general formula as an active ingredient. The suppression of side effects of anticancer drugs includes suppression of resistance to the anticancer drugs.

[0064] The present invention also provides a pharmaceutical composition for combined administration with an anticancer agent, which comprises the compound represented by the above general formula as an active ingredient.

[0065] The absence of X in the general formula means that the compound represented by the general formula is AQTGTGKT.

[0066] In the present invention, the X is [ka] The X may be [ka] The compound is designated herein as "3-PhPh-AQTGTGKT." Other names of compounds according to the present invention are listed in Table 1.

[0067] As used herein, the term "oligopeptide" refers to a linear molecule formed by the linking of amino acid residues to each other through peptide bonds. The amidated oligopeptides of the present invention may be prepared by chemical synthesis methods known in the art (e.g., solid-phase synthesis techniques) as well as molecular and biological methods (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)).

[0068] The scope of the compounds according to the present invention may also include pharmaceutically acceptable salts thereof. As used herein, the term "pharmaceutically acceptable" means a compound that is suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic reaction, or other problem or complication, at a reasonable benefit / risk ratio, and within the scope of sound medical judgment. The pharmaceutically acceptable salts include, for example, acid addition salts formed with pharmaceutically acceptable free acids and pharmaceutically acceptable metal salts.

[0069] Specific examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Acid addition salts may be prepared by a conventional method, for example, by dissolving a compound in an excess amount of aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, acid addition salts may be prepared by heating equal molar amounts of a compound and an acid or alcohol in water, followed by evaporating the mixture to dryness or filtering the precipitated salt under suction.

[0070] Salts derived from appropriate bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are particularly suitable for pharmaceutical use, and the corresponding silver salts can be obtained by reacting the alkali metal or alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

[0071] The scope of the compounds of the present invention may include not only pharmaceutically acceptable salts but also all isomers, hydrates and solvates which can be prepared by conventional methods.

[0072] The compounds may contain non-aromatic double bonds and one or more asymmetric centers. Thus, they may occur as racemates and racemic mixtures, single enantiomers, individual partial stereoisomers, partial stereoisomeric mixtures, and cis or trans isomers. All such isomeric forms are contemplated.

[0073] The scope of the compounds of the present invention may also include biologically functional equivalents having amino acid sequence variations that exhibit biological activity equivalent to that of the compounds of the present invention. Such amino acid sequence variations may be based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side chain substitutions reveals that alanine and glycine have similar sizes, lysine is a positively charged residue, and glutamine and threonine are uncharged. Therefore, based on these considerations, alanine and glycine, and glutamine and threonine can be considered biologically functional equivalents.

[0074] When introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydropathic index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tylosin (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0075] The hydrophobic amino acid index is very important for imparting interactive biological functions to proteins. It is a well-known fact that similar biological activity cannot be maintained unless an amino acid is substituted with an amino acid having a similar hydrophobic index. When introducing a mutation based on the hydrophobic index, substitution is preferably performed with amino acids showing a difference in hydrophobic index within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0076] On the other hand, it is also well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0), lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tylosin (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0077] When introducing a mutation based on the hydrophilicity value, substitution is preferably performed with an amino acid that exhibits a difference in hydrophilicity value within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0078] Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0079] Considering the above-mentioned biologically equivalent variants, the amino acid sequence (AQTGTGKT) of the compound of the present invention represented by the general formula is also interpreted as including sequences that show substantial identity thereto. The term "substantial identity" refers to a sequence that shows at least 62.5% homology, more preferably 75% or more homology, and most preferably 87.5% or more homology when the sequence of the present invention is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art.

[0080] As used herein, the term "combined administration" refers to the simultaneous, sequential, or separate administration of individual components of a therapeutic regimen. The combined therapeutic effect is achieved by administering two or more drugs simultaneously, sequentially, or alternately at regular or irregular intervals. Combination therapy can be defined as a regimen that provides a synergistic effect, whereby efficacy, measured, for example, but not limited to, the degree of response, the rate of response, the time to disease progression, or the duration of survival, is therapeutically superior to the efficacy achieved by administering one or the other of the components of the combination therapy at a standard dose.

[0081] The term "anticancer agent" as used herein is used as a general term for substances used to treat malignant tumors. Most anticancer agents are drugs that inhibit nucleic acid synthesis or exhibit anticancer activity by intervening in various metabolic pathways in cancer cells. Anticancer agents currently used in cancer treatment are classified into six categories based on their biochemical mechanism of action: alkylating agents, antimetabolites, antibiotics, vinca alkaloids, hormones, and others. However, the anticancer agent of the present invention may not fall into any of these categories.

[0082] In the present invention, the anticancer agent may be one or more selected from the group consisting of targeted anticancer agents and chemical anticancer agents, but is not limited thereto.

[0083] The present invention may be administered together with a targeted anticancer drug. In the present invention, the term "targeted anticancer drug" refers to a formulation that exhibits anticancer effects by targeting proteins or genes that are specifically altered in cancer cells or cancer tissues and interfering with molecular activity involved in cancer growth and development. The targeted anticancer drug may be, but is not limited to, one or more selected from the group consisting of tyrosine kinase inhibitors (TKIs), PARP inhibitors (poly-ADP ribose polymerase inhibitors), angiogenesis inhibitors, CDK4 / 6 inhibitors (cyclin-dependent kinases 4 / 6 inhibitors), hormonal therapy drugs, and antibody-drug conjugates. Preferably, the targeted anticancer drug according to the present invention may be, but is not limited to, a monoclonal antibody anticancer drug.

[0084] In particular, the tyrosine kinase inhibitor is selected from the group consisting of EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS Proto-Oncogene 1), BRAF (B-Raf Proto-Oncogene), HER2 (human epidermal growth factor receptor 2), RET (Ret Proto-Oncogene), NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1), MET (Mesenchymal-Epithelial Transition The drug may be a targeted drug for one or more selected from the group consisting of NRG1 (Neuregulin1), and non-limiting examples thereof include osimertinib, afatinib, brigatinib, dasatinib, dacomitinib, erlotinib, gefitinib, lapatinib, Neratinib, Vandetanib, Icotinib, Varitinib, Tesevatinib, Canertinib, Naquotinib, Pelitinib, Poziotinib, Rociletinib, Nazartinib, Allitinib;ALS-1306, pyrotinib, tyrphostin, crizotinib, ceritinib, entrectinib, dabrafenib, trametinib, alectinib, lorlatinib ), larotectinib, lasertinib, olmutinib, AG1478, CUDC-101, MTKi-327 (JNJ-26483327), CL-387785 (EKI-785), CNX-2006, PD168393, TAK285, WZ4002 and AV-412 (MP-412).

[0085] In addition, the PARP inhibitor may be one or more selected from the group consisting of olaparib, rucaparib, talazoparib, veliparib, and niraparib, but is not limited thereto.

[0086] In addition, the CDK4 / 6 inhibitor may be one or more selected from the group consisting of trilaciclib, palbociclib, ribociclib, and abemaciclib, but is not limited thereto.

[0087] In addition, the hormone therapeutic agent may be one or more selected from the group consisting of tamoxifen, toremifene, fulvestrant, goserelin, leuprolide, anastrozole, letrozole, and exemestane, but is not limited thereto.

[0088] Furthermore, the targeted anticancer drug according to the present invention may be an antibody-drug conjugate (ADC). The ADC is prepared by covalently conjugating an antibody (antibody) that binds to a specific target antigen on the surface of cancer cells with a drug (drug) that has a strong cell-killing function. By utilizing the target selectivity of the antibody and the strong cell-killing activity of the drug, the drug acts selectively only on cancer cells, thereby enhancing therapeutic efficacy and reducing side effects.

[0089] Preferably, the ADC may be selected from, but is not limited to, Sacituzumab govitecan and Ladiratuzumab.

[0090] In addition, the targeted anti-cancer drug according to the present invention may be selected from, but is not limited to, Daratumumab, Trastuzumab, Rituximab, and the like.

[0091] The present invention may also be administered in combination with a chemotherapeutic agent. As used herein, the term "chemotherapeutic agent" refers to a first-generation anticancer agent, also known as a "cytotoxic anticancer agent" or a "chemical drug anticancer agent." Non-limiting examples of the chemotherapeutic agent include Alimta, oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, and doxorubicin.

[0092] Meanwhile, the compounds according to the present invention can enhance the anticancer effect of anticancer drugs while reducing side effects. This is because appropriate combination therapy can minimize the dosage of anticancer drugs with side effects. Here, "enhancing the anticancer effect" refers to any effect that can ultimately enhance the function of an anticancer drug, and includes not only enhancing the anticancer effects of anticancer drugs, such as inhibiting tumor growth, tumor metastasis, and tumor recurrence, but also enhancing the anticancer effect by suppressing the formation of resistance or tolerance in cancer cells against the anticancer drug. That is, the compounds according to the present invention may be used as compounds for co-administration with known anticancer drugs for the purpose of enhancing the anticancer effect. That is, the compounds according to the present invention can be used in combination with anticancer drugs to enhance the anticancer effect of the anticancer drug.

[0093] In the present invention, the compound or a composition containing the compound may be administered simultaneously, separately, or sequentially with an anticancer drug. When administered sequentially with an anticancer drug, the order of administration is not limited, and the administration regimen may be appropriately adjusted depending on the type of cancer, the type of anticancer drug, the condition of the patient, etc.

[0094] In one embodiment of the present invention, when the compound and osimertinib were co-administered to cancer cells at an early stage, the cancer cell growth inhibitory effect was strongest. Therefore, the compound may be administered before or simultaneously with the anticancer drug. Furthermore, when the compound of the present invention was continuously administered in combination with the anticancer drug, the anticancer effect was even better than when the anticancer drug was administered alone after the combination. Therefore, when the goal is to maximize the anticancer effect, it is preferable to continuously administer the compound of the present invention in combination with the anticancer drug.

[0095] The content of the compound or anticancer agent in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt % or 0.001 to 50 wt % based on the weight of the total composition, but is not limited thereto. The content ratio is a value based on the dry weight after removal of the solvent.

[0096] In the present invention, the anticancer agent may be contained at a concentration of 0.1 to 10 μM, 0.1 to 9 μM, 0.1 to 8 μM, 0.1 to 7 μM, 0.1 to 6 μM, 0.1 to 5 μM, 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, or 0.1 to 1 μM relative to the total composition, but is not limited thereto.

[0097] Furthermore, in the present invention, the compound represented by the general formula may be contained at a concentration of 1 to 50 μM, 1 to 40 μM, 1 to 30 μM, 1 to 20 μM, 1 to 15 μM, 1 to 12 μM, 1 to 10 μM, 1 to 9 μM, 1 to 8 μM, 1 to 7 μM, 1 to 6 μM, 1 to 5 μM, 1 to 4 μM, 1 to 3 μM, 1 to 2 μM, 2 to 12 μM, or 2 to 11 μM relative to the total composition, but is not limited thereto.

[0098] Furthermore, the composition according to the present invention may be in the form of a mixture of the compound and the anticancer drug, and may be in the form for simultaneous administration of the compound and the anticancer drug.

[0099] Furthermore, the composition according to the present invention may be in a form in which the compound and the anticancer agent are separately formulated and administered simultaneously or sequentially. In this case, the composition may be a pharmaceutical composition for combined administration, for simultaneous or sequential administration, comprising a first pharmaceutical composition containing a pharmaceutically effective amount of the compound as an active ingredient and a second pharmaceutical composition containing a pharmaceutically effective amount of the anticancer agent as an active ingredient. In this case, in the case of sequential administration, the order of administration is not limited, and the administration regimen may be appropriately adjusted depending on the condition of the patient, etc.

[0100] That is, when the pharmaceutical composition is a pharmaceutical composition for combined administration for sequential administration, the composition may be one in which the compound ("first component") is administered first, followed by the anticancer drug ("second component"), or the reverse order is also possible.

[0101] In one embodiment of the present invention, the anticancer drug is a targeted anticancer drug, and the compound represented by the general formula is such that X is absent or [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0102] Preferably, the anticancer drug is a tyrosine kinase inhibitor, more preferably osimertinib, and the compound represented by the general formula is such that X is absent or [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0103] Preferably, the anticancer drug is a PARP inhibitor, more preferably Olaparib, and the compound represented by the general formula is such that X is absent or [ka] It may be, but is not limited to this.

[0104] The anticancer drug is a chemical anticancer drug, and the compound represented by the general formula is [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0105] Preferably, the anticancer drug is cisplatin, and the compound represented by the general formula is [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0106] Preferably, the anti-cancer drug is Alimta, and the compound represented by the general formula is [ka] The present invention may be, but is not limited to, one or more selected from the group consisting of:

[0107] In this case, the anticancer agent and the compound may be contained at a molar concentration (molarity) ratio of 1:1 to 500, 1:1 to 400, 1:1 to 300, 1:1 to 200, 1:1 to 180, 1:1 to 150, 1:1 to 130, 11:1 to 120, 1:1 to 110, 1:1 to 100, 1:1 to 90, 1:1 to 80, 1:1 to 70, 1:1 to 60, 1:1 to 50, 1:1 to 40, 1:1 to 30, 1:1 to 20, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, 1:1 to 4, 1:1 to 3, or 1:1 to 2.

[0108] The compounds according to the present invention may be used for the prevention and / or treatment of cancer. As used herein, the term "cancer" refers to uncontrolled cell growth, which can lead to the formation of a cell mass called a tumor, which infiltrates surrounding tissues and, in severe cases, metastasizes to other organs in the body. Academically, it is sometimes called a neoplasm. Cancer is an intractable chronic disease that often cannot be cured fundamentally even with treatments such as surgery, radiation, and chemotherapy, causing pain and ultimately leading to death. There are various causes of cancer, which can be classified as internal or external. While the exact mechanism by which normal cells transform into cancer cells has not been elucidated, it is known that a significant number of cancers develop as a result of external factors, such as environmental factors. Internal factors include genetic factors and immunological factors, while external factors include chemicals, radiation, and viruses. Genes related to the development of cancer include oncogenes and tumor suppressor genes, and cancer develops when the balance between these is disrupted by the internal or external factors mentioned above.

[0109] The cancer may be a solid cancer or a blood cancer, and non-limiting examples thereof may be one or more types selected from the group consisting of squamous cell carcinoma, lung cancer, lung adenocarcinoma, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, cancer near the anus, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, blood cancer, liver cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, etc. More specifically, the cancer may be one or more types selected from the group consisting of lung cancer, breast cancer, blood cancer, colorectal cancer, pancreatic cancer, and combinations thereof.

[0110] In the present invention, the lung cancer may be non-small cell lung carcinoma or lung papillary adenocarcinoma.

[0111] The breast cancer may be, but is not limited to, hormone receptor (HR)-positive breast cancer, or may be triple-negative breast cancer.

[0112] The blood cancer may be leukemia, lymphoma, multiple myeloma, or the like.

[0113] In yet another aspect, the present invention can provide a kit for enhancing the anticancer effect of an anticancer agent, which kit comprises a compound represented by the general formula above.

[0114] In another aspect, the present invention can provide a kit for preventing or treating cancer, which comprises (i) a compound represented by the above general formula, and (ii) an anticancer agent as active ingredients.

[0115] The kit according to the present invention may include, without limitation, other components, compositions, solutions, devices, etc. that are typically required for the prevention or treatment of cancer, in addition to the compound or anticancer agent, and may particularly include instructions for the appropriate use and storage of the compound according to the present invention.

[0116] In the present invention, "prevention" means any action that suppresses or delays the onset of a desired disease, "treatment" means any action that improves or beneficially alters the symptoms of a desired disease and associated metabolic disorders by administering a pharmaceutical composition according to the present invention, and "improvement" means any action that reduces parameters related to a target disease, such as the severity of symptoms, by administering a composition according to the present invention.

[0117] The cancer prevention and / or treatment effect includes not only the effect of suppressing the growth of cancer cells but also the effect of suppressing the progression of cancer due to migration, invasion, metastasis, and the like.

[0118] As used herein, the term "individual" refers to a subject in need of disease prevention or treatment. For example, the individual may be a mammal, including a human or non-human primate, mouse, dog, cat, horse, and cow.

[0119] Meanwhile, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, suitable carriers, excipients and / or diluents that are commonly used for preparing pharmaceutical compositions, and may be formulated and used in the form of oral dosage forms such as acids, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions by a common method.

[0120] The carrier, excipient and diluent that may be contained in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.When formulating the composition, it may be prepared using diluents or excipients that are usually used, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc.

[0121] In the present invention, "administration" means providing a given composition of the present invention to an individual by any suitable method.

[0122] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, a "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently administered drugs, and other factors well known in the medical field. A preferred dosage may be selected depending on the individual's condition and weight, the severity of the disease, drug form, administration route, and duration. Specifically, the pharmaceutical composition may be administered in an amount of 0.001 to 1000 mg / kg, 0.01 to 100 mg / kg, 0.01 to 10 mg / kg, 0.1 to 10 mg / kg, or 0.1 to 1 mg / kg, once or several times daily in divided doses.

[0123] Taking all of the above factors into consideration, it is important to administer an amount that provides the maximum effect at the minimum amount without side effects, and this can be determined by a person skilled in the art. Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, and weight of the patient, the degree of absorption, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and concomitant drugs.

[0124] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are conceivable, including, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, aural administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc. The daily dose may be administered once or several times a day.

[0125] The terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted as meanings and concepts that correspond to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concept of the term in order to explain the invention in the best possible way. [Example]

[0126] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0127] [Example] Experimental materials and methods 1. Synthesis of AQTGTGKT analogue compounds The 13 AQTGTGKT analog compounds of the present invention were synthesized by Sewon Biotechnology. 1 The chemical structure was confirmed by H NMR and UPLC-MS (ultraperformance liquid chromatography-mass spectrometry) techniques.

[0128] 1.1. Reactions in general All reactions were carried out using commercially available materials and reagents without further explanation unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates (Keiselgel 60 F254, Merck) and / or ultra-performance liquid chromatography (UPLC). Visualization of spots on TLC plates was achieved by UV light and by staining the TLC plates with potassium permanganate and / or ninhydrin and charring with a heat gun. All products were 1 Characterization was performed using 1 H NMR and / or UPLC-MS.

[0129] 1.2.Synthesis of Boc / OBn-TG First, TG whose functional groups were protected with benzyl was synthesized according to the following reaction scheme 1. Hereinafter, in each reaction scheme, the compound will be referred to as compound n by the Arabic numeral (n) shown below.

[0130] [Reaction Scheme 1] [ka]

[0131] Specifically, BocThr(OBn)OH (compound 1; 25.0 g, 80.8 mmol, 1.0 equiv.) and NOSu (9.77 g, 84.8 mmol, 1.05 equiv.) were dissolved in dichloromethane (150 mL). The mixture was cooled to 0 °C and placed under an inert atmosphere. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.3 g, 84.8 mmol, 1.05 equiv.) was then added to the mixture. The mixture was warmed to room temperature and stirred for 20 h. The mixture was then washed with NH4Cl (saturated aqueous), and the phases were separated. The organic layer was dried over MgSO4 and concentrated under reduced pressure to give the product, compound 2, as a pale yellow oil (35.7 g, >100% yield, assuming quantitative yield).

[0132] The compound 2, BocThr(OBn)OSu (32.8 g, 80.8 mmol, 1.0 equiv.), was dissolved in 1,4-dioxane (200 mL) and a solution of glycine sodium salt hydrate in distilled water (100 mL) was added in one portion. After stirring at room temperature for 6 h, the mixture was partitioned between ethyl acetate and citric acid (saturated aqueous). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure. The crude material was purified on a C18 (400 g) column using 30–70% acetonitrile (0.1% formic acid) in water (0.1% formic acid) eluent. The desired fractions were combined and partitioned between ethyl acetate and NaHCO (saturated aqueous). The organic layer was dried over MgSO, filtered, and concentrated under reduced pressure to give the product, compound 3, as a pale yellow gum (21.9 g, 74% yield).

[0133] 1.3.Synthesis of CBz / OBn / CO2Bn-KT KT, in which the OH functional group was protected with benzyl, was synthesized according to the following reaction scheme 2.

[0134] [Reaction Scheme 2] [ka]

[0135] Specifically, BocLys(CBz)OH (compound 4; 27.0 g, 70.9 mmol, 1.0 equiv.) and NOSu (9.80 g, 85.1 mmol, 1.2 equiv.) were dissolved in dichloromethane (128 mL). The mixture was cooled to 0 °C and placed under an inert atmosphere. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.3 g, 85.1 mmol, 1.05 equiv.) was then added to the mixture. The mixture was warmed to room temperature and stirred for 20 h. The mixture was then washed with NH4Cl (saturated aqueous), and the phases were separated. The organic layer was dried over MgSO4 and concentrated under reduced pressure to give the product, compound 5, as a pale yellow oil (36.7 g, >100% yield, assuming quantitative yield).

[0136] Next, compound 5 (BocLys(Cbz)OSu; 36.7 g, 70.9 mmol, 1.0 equiv.) and Thr(OBn)OBn.HCl (25.0 g, 74.4 mmol, 1.05 equiv.) were dissolved in 1,4-dioxane (477 mL) at room temperature. To the solution was added a solution of NaHCO3 (6.85 g, 81.5 mmol, 1.15 equiv.) in distilled water (326 mL). The resulting mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate and washed with 10% citric acid (aqueous) and brine. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 6 as a yellow oily solid (55.9 g, >100% yield, assumed quantitative yield).

[0137] Finally, compound 6 (BocLys(Cbz)Thr(OBn)OBn; 55.9 g, 70.9 mmol, 1.00 equiv.) was dissolved in 1,4-dioxane (360 mL) and 4N HCl in 1,4-dioxane (177 mL) was added. The mixture was stirred at room temperature overnight. Then, a saturated aqueous solution of NaHCO3 was added until the pH reached 8. The solution was extracted with ethyl acetate, and the resulting organic solution was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 7 as a yellow oily solid (38.7 g, 97% yield).

[0138] 1.4.Synthesis of CBz / OBn / OBn / CO2Bn-TGKT TG and KT synthesized in 1.2. and 1.3. above were combined according to the following reaction formula 3 to synthesize TGKT.

[0139] [Reaction Scheme 3] [ka]

[0140] More specifically, to a solution of BocThr(OBn)GlyOH (compound 3; 5.61 g, 15.3 mmol, 1.00 equiv.) and compound 8 (Lys(Cbz)Thr(OBn)OBn; 10.0 g, 15.3 mmol, 1.0 equiv.) in dichloromethane (50 mL), N,N-diisopropylethylamine (5.90 mL, 33.7 mmol, 2.2 equiv.) was added. The mixture was stirred at room temperature under an inert atmosphere, and HATU (7.00 g, 18.4 mmol, 1.20 equiv.) was added. The resulting mixture was stirred for 2 h and then washed with NH4Cl (saturated aqueous) and then with NaHCO3 (saturated aqueous). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 9 as a pale orange oily solid (25.0 g, >100% yield, assuming quantitative yield).

[0141] The resulting BocThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 9; 13.9 g, 15.3 mmol, 1.0 equiv. from the previous step) was dissolved in 1,4-dioxane (150 mL) at room temperature under nitrogen. To this solution was added 4N HCl in 1,4-dioxane (20 mL). The mixture was stirred at room temperature for 20 hours. The mixture was concentrated under reduced pressure and purified on a C18 (400 g) column using 20% ​​acetonitrile (0.1% formic acid) in water (0.1% formic acid) eluent. The desired fractions were combined and lyophilized. The resulting powder was dissolved in NaHCO3 (saturated aqueous) and dichloromethane and stirred for 15 minutes. The layers were separated, and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the product, compound 10, as a colorless gum (10.9 g, 88% yield).

[0142] 1.5. Synthesis of CBz / OBn / OBn / OBn / CO2Bn-TGTGKT TG (compound 3) synthesized in 1.2. and 1.4. above and TGKT (compound 10) were combined according to the following reaction formula 4 to synthesize benzyl-protected TGTGKT.

[0143] [Reaction Scheme 4] [ka]

[0144] More specifically, to a solution of BocThr(OBn)GlyOH (compound 3; 5.10 g, 14.0 mmol, 1.05 equiv.) and BocThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 10; 10.8 g, 13.3 mmol, 1.0 equiv.) in dichloromethane (100 mL) was added N,N-diisopropylethylamine (5.10 mL, 29.3 mmol, 2.2 equiv.). The mixture was stirred at room temperature under an inert atmosphere, and HATU (5.60 g, 14.7 mmol, 1.1 equiv.) was added. The resulting mixture was stirred for 2 h and washed with NH4Cl (saturated aqueous) and NaHCO3 (saturated aqueous). The organic layer was concentrated under reduced pressure to give the product, compound 11, as a pale yellow gum (21.4 g, >100% yield, assuming quantitative yield).

[0145] Next, the resulting compound 11 (BocThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn; 15.4 g, 13.3 mmol, 1.00 equiv. from the previous step) was dissolved in 1,4-dioxane (150 mL) at room temperature under nitrogen. To this solution was added 4N HCl in 1,4-dioxane (50 mL), and the mixture was stirred at room temperature for 5 hours. The mixture was concentrated under reduced pressure and purified on a C18 (120 g) column using 20% ​​acetonitrile (0.1% formic acid) in water (0.1% formic acid) eluent. The desired fractions were combined and concentrated to half the volume, then partitioned between NaHCO3 (saturated aqueous) and ethyl acetate. The layers were separated, and the organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the product, compound 12, as an off-white solid (14.6 g, >100% yield, assumed quantitative yield).

[0146] 1.6. Synthesis of CBz / OBn / OBn / OBn / CO2Bn-QTGTGKT Compound 12 (TGTGKT) synthesized in 1.5 above was further bound to Q according to the following reaction formula 5 to synthesize compound 14 (QTGTGKT).

[0147] [Reaction Scheme 5] [ka]

[0148] More specifically, to a solution of Thr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 12; 12.7 g, 12.0 mmol, 1.0 equiv.) and BocGlnOH (3.25 g, 13.2 mmol, 1.1 equiv.) in ethyl acetate (150 mL) and N,N-dimethylformamide (25 mL) was added N,N-diisopropylethylamine (4.60 mL, 26.4 mmol, 2.2 equiv.). The mixture was stirred at room temperature under an inert atmosphere, and HATU (5.47 g, 14.4 mmol, 1.20 equiv.) was added. The resulting mixture was stirred for 1 hour and then washed with NH4Cl (saturated aqueous). The organic layer was further extracted with dichloromethane. The combined organic layers were then concentrated under reduced pressure. The crude material was purified on a 400 g C18 column using a 20-100% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid). The desired fractions were combined and then fractionated between ethyl acetate and NaHCO3 (saturated aqueous) solution. The organic layer was concentrated and residual water was removed by a lyophilization step. The combined fractions gave a total of 12.9 g (89% overall yield) of compound 13.

[0149] The resulting compound 13 (BocGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn; 7.00 g, 5.40 mmol, 1.00 equiv.) was dissolved in 1,4-dioxane (150 mL) at room temperature under nitrogen. To this solution was added 4N HCl in 1,4-dioxane (43.5 mL). The mixture was stirred at room temperature for 20 h. The mixture was concentrated under reduced pressure and lyophilized using a water / acetonitrile (2 / 1) solution. Finally, compound 14 was isolated as a pale yellow powder (6.36 g, 96% yield).

[0150] 1.7. Synthesis of AQTGTGKT Analogues The remaining six analogs, excluding 4-PhPh-AQTGTGKT, were synthesized by combining Compound 14, the final product of Reaction Scheme 5, and Compound 17n, the product of Reaction Scheme 6, according to Reaction Scheme 7.

[0151] [Reaction Scheme 6] [ka]

[0152] [Reaction Scheme 7] [ka]

[0153] Through Reaction Scheme 7, 2.9 mg of 3-PhPh-AQTGTGKT with a purity of 90% or more, 7.0 mg of 4-MeOPh-AQTGTGKT with a purity of 89%, 22.7 mg of 2-PhPh-AQTGTGKT with a purity of 95% or more, 23.2 mg of Ph-AQTGKT with a purity of 90% or more, and 23.2 mg of Naphthyl-AQTGKT with a purity of 85% were finally obtained.

[0154] Each analog synthesis process will be specifically described below.

[0155] Synthesis of 1.7.1.3-PhPh-AQTGTGKT 3-PhPh-AQTGTGKT (compound 19-1), the final target compound, was synthesized by reacting compound 17-1 obtained by the following reaction formula 8 with compound 14 according to reaction formula 9.

[0156] [Reaction Scheme 8] [ka]

[0157] More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv.) was suspended in ethyl acetate (10 mL) and N,N-diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv.) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv.) and [1,1'-biphenyl]-3-carboxylic acid (297 mg, 1.50 mmol, 1 equiv.) were added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and brine. The resulting organics were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified on a 25 g column using a 2-40% ethyl acetate in heptane gradient to give compound 16-1 as a colorless solid (493 mg, 91% yield).

[0158] Next, 10% Pd / C (49 mg) wetted with a minimum amount of water was added to a solution of compound 16-1 (3-PhPh-AlaOBn; 493 mg, 1.37 mmol) in methanol (30 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 2 hours. The mixture was filtered through a Celite pad and washed with methanol and ethyl acetate. The resulting filtrate was concentrated under reduced pressure to give compound 17-1 as a colorless foam (337 mg, 91% yield), which was reacted with compound 14 according to Scheme 9 below.

[0159] [Reaction Scheme 9] [ka]

[0160] More specifically, HATU (106 mg, 0.278 mmol, 1.1 equiv.) was added to a suspension of GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 14; 300 mg, 0.253 mmol, 1 equiv.) and 3-PhPh-AlaOH (compound 17-1; 68.0 mg, 0.253 mmol, 1 equiv.) in N,N-diisopropylethylamine (97.0 μL, 0.556 mmol, 2.2 equiv.) and dichloromethane (20 mL). The mixture was stirred at room temperature for 2 h, and the reaction mixture was washed with NaHCO (saturated aqueous solution). The organics were concentrated under reduced pressure, and the residue was purified on a 60 g C18 column with a 40–100% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) to afford compound 18-1 as a colorless solid (140 mg, 38% yield) after lyophilization.

[0161] Next, 10% Pd / C (85.0 mg) was added to a solution of 3-PhPh-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 18-1; 85.0 mg, 59.1 μmol) in 2 M hydrochloric acid (aqueous, 0.5 mL) and 2-propanol (10 mL). After stirring the mixture under a hydrogen atmosphere (balloon) for 4.5 h, the mixture was filtered through a 0.45 μm syringe filter. The resulting filtrate was concentrated under reduced pressure, and the residue was lyophilized. The material was then purified on a 60 g C18 column using 5–50% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give the desired compound 19-1 as a colorless solid (2.9 mg, 5% yield).

[0162] Compound 19-1 (3-PhPh-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0163] 1H NMR (400MHz; D2O): δ=8.01-7.99(m,1H), 7.86-7.82(m,1H), 7.75-7.66(m,3H), 7.55(t,J7.7Hz,1H), 7. 49(t,J7.5Hz,2H), 7.43-7.38(m,1H), 4.48-4.24(m,5H), 4.23-4.12(m,3H), 4.09(d,J3.8Hz,1H), 4.00- 3.96(m,2H), 3.88(s,2H), 2.90(t,J7.4Hz,2H), 2.35(t,J7.5Hz,2H), 2.15-2.06(m,1H), 2.03-1.91(m,1) H), 1.84-1.72(m,1H), 1.70-1.52(m,3H), 1.45(d,J7.2Hz,3H), 1.40-1.24(m,2H), 1.15-1.05(m,9H), 16 exchangeable protons not visible.

[0164] 1.7.2.4 Synthesis of MeOPh-AQTGTGKT 4-MeOPh-AQTGTGKT (compound 19-2) was synthesized by reacting compound 17-2 obtained by the following reaction formula 10 with compound 14 according to reaction formula 11 to synthesize the final target compound, 4-MeOPh-AQTGTGKT.

[0165] [Reaction Scheme 10] [ka]

[0166] More specifically, H-Ala-OBzl.HCl (425 mg, 1.97 mmol, 1.2 equiv.) was suspended in ethyl acetate (10 mL) and N,N-diisopropylethylamine (715 μL, 4.11 mmol, 2.5 equiv.) was added. After stirring at room temperature for 5 min, HATU (937 mg, 2.46 mmol, 1.5 equiv.) and 4-methoxybenzoic acid (250 mg, 1.64 mmol, 1 equiv.) were added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and brine. The organics were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified on a 25 g column with a 15–50% ethyl acetate in heptane gradient to give compound 16-2 as a colorless solid (360 mg, 70% yield).

[0167] Next, 18 mg of 10% Pd / C wetted with a minimum amount of water was added to a solution of 4-OMePh-AlaOBn (compound 16-2; 180 mg, 0.574 mmol) in methanol (15 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 110 hours. The mixture was then filtered through a pad of Celite and washed with methanol. The resulting filtrate was concentrated under reduced pressure to give compound 17-2 as a colorless oil (128 mg, 100% yield), which was reacted with compound 14 according to Scheme 11 below.

[0168] [Reaction Scheme 11] [ka]

[0169] More specifically, HATU (74.5 mg, 0.196 mmol, 1.2 equiv) was added to a suspension of GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 14; 200 mg, 0.164 mmol, 1 equiv) and 4-OMePh-AlaOH (compound 17-2; 36.6 mg, 0.164 mmol, 1 equiv) in N,N-diisopropylethylamine (63.0 μL, 0.360 mmol, 2.2 equiv) and dichloromethane (20 mL). The mixture was stirred at room temperature for 64 h, and the reaction mixture was diluted with methanol and washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and water. The organics were concentrated under reduced pressure, and the residue was purified on a 60 g C18 column with a 50–95% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) to afford compound 18-2 as a colorless solid (113 mg, 50% yield) after lyophilization.

[0170] Next, 10% Pd / C (100 mg) was added to a solution of 4-OMePh-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 18-2; 108 mg, 77.6 μmol) in 2 M hydrochloric acid (aqueous, 1.0 mL) and 2-propanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 18 h. The mixture was filtered through a 0.45 μm syringe filter. The filtrate was concentrated under reduced pressure, and the residue was dissolved in water and lyophilized. The dried material was purified on a 30 g C18 column using a 5–30% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) and lyophilized to give compound 19-2 as a colorless solid (7.0 mg, 10% yield).

[0171] Compound 19-2 (4-MeOPh-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0172] 1H NMR (400MHz; D2O): δ=7.76-7.71(m,2H), 7.02-6.98(m,2H), 4.42-4.28(m,5H), 4.24-4 .13(m,3H), 4.09(d,J3.9Hz,1H), 4.01-3.96(m,2H), 3.89(s,2H), 3.81(s,3H), 2.91(t, J7.4Hz,2H), 2.34(t,J7.6Hz,2H), 2.14-2.06(m,1H), 2.00-1.91(m,1H), 1.85-1.75(m ,1H), 1.71-1.54(m,3H), 1.42(d,J7.2Hz,3H), 1.40-1.25(m,3H), 1.16-1.07(m,8H), 16 exchangeable protons not visible.

[0173] Synthesis of 1.7.3.2-PhPh-AQTGTGKT 2-PhPh-AQTGTGKT (compound 19-3) was synthesized as the final target compound, 2-PhPh-AQTGTGKT, by reacting compound 17-3 obtained by the following reaction formula 12 with compound 14 according to reaction formula 13.

[0174] [Reaction Scheme 12] [ka]

[0175] More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv.) was suspended in ethyl acetate (10 mL) and N,N-diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv.) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv.) and [1,1'-biphenyl]-2-carboxylic acid (297 mg, 1.50 mmol, 1 equiv.) were added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and brine. The resulting organics were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified on a 25 g column using a 2-40% ethyl acetate in heptane gradient to give compound 16-3 as a colorless oil (416 mg, 77% yield).

[0176] Next, 10% Pd / C (42 mg) wetted with a minimum amount of water was added to a solution of 2-PhPh-AlaOBn (compound 16-3; 416 mg, 1.16 mmol) in methanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 18 hours. The mixture was then filtered through a pad of Celite and washed with methanol. The resulting filtrate was concentrated under reduced pressure to give compound 17-3 as a colorless oil (308 mg, 99% yield), which was reacted with compound 14 according to Scheme 13 below.

[0177] [Reaction Scheme 13] [ka]

[0178] More specifically, HATU (74.5 mg, 0.196 mmol, 1.2 equiv) was added to a suspension of GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 14; 200 mg, 0.164 mmol, 1 equiv) and 2-PhPh-AlaOH (compound 17-3; 44.2 mg, 0.164 mmol, 1 equiv) in N,N-diisopropylethylamine (63.0 μL, 0.360 mmol, 2.2 equiv) and dichloromethane (20 mL). The mixture was stirred at room temperature for 64 h, and the reaction mixture was diluted with methanol, then washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and water. The organic layer was concentrated under reduced pressure, and the residue was purified on a 60 g C18 column with a 50–95% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) to give compound 18-3 as a colorless solid (115 mg, 49% yield) after lyophilization.

[0179] Next, 10% Pd / C (100 mg) was added to a solution of 2-PhPh-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 18-3; 110 mg, 76.5 μmol) in 2 M hydrochloric acid (aqueous, 1.0 mL) and 2-propanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 18 h. The mixture was filtered through a 0.45 μm syringe filter. The filtrate was concentrated under reduced pressure, and the residue was dissolved in water and lyophilized. The dried material was purified on a 30 g C18 column with a gradient of 5–50% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give compound 19-3 as a colorless solid (22.7 mg, 31% yield).

[0180] Compound 19-3 (2-PhPh-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0181] 1H NMR (400MHz; D2O): δ=7.56-7.48(m,2H), 7.44-7.33(m, 7H), 4.38-4.26(m,4H), 4.25 -4.13(m,4H), 4.08(d,J3.9Hz,1H), 4.00-3.96(m,2H), 3.89(s,2H), 2.91(t,J7.5Hz, 2H), 2.25(t,J7.5Hz,2H), 2.09-2.01(m,1H), 1.93-1.77(m,2H), 1.72-1.56(m,3H), 1 .41-1.29(m,2H), 1.18(d,J7.2Hz,3H), 1.12(d,J5.6Hz, 6H), 1.08(d,J6.4Hz,3H), 16 exchangeable protons not visible.

[0182] 1.7.4. Synthesis of Ph-AQTGTGKT Ph-AQTGTGKT (compound 19-4) was synthesized as the final target compound by reacting compound 17-4 obtained by the following reaction formula 14 with compound 14 according to reaction formula 15.

[0183] [Reaction Scheme 14] [ka]

[0184] More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv.) was suspended in ethyl acetate (10 mL) and N,N-diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv.) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv.) and benzoic acid (183 mg, 1.50 mmol, 1 equiv.) were added, and the mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with ethyl acetate and washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and brine. The resulting organics were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified on a 25 g column using a 2–50% ethyl acetate in heptane gradient to give compound 16-4 as a colorless oil (375 mg, 88% yield).

[0185] Next, 10% Pd / C (38 mg) wetted with a minimum amount of water was added to a solution of Ph-Ala-OBn (compound 16-4; 375 mg, 1.32 mmol) in methanol (30 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 4 h. The mixture was then filtered through a pad of Celite and washed with methanol. The resulting filtrate was concentrated under reduced pressure to give compound 17-4 as a colorless glass (glass; 254 mg, 99% yield), which was reacted with compound 14 according to Scheme 15 below.

[0186] [Reaction Scheme 15] [ka]

[0187] More specifically, to a suspension of GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 14; 300 mg, 0.253 mmol, 1 equiv.) and Ph-Ala-OH (compound 17-4; 49.0 mg, 0.253 mmol, 1) in N,N-diisopropylethylamine (97.0 μL, 0.556 mmol, 2.2 equiv.) and dichloromethane (20 mL) was added HATU (106 mg, 0.278 mmol, 1.1 equiv.). The mixture was stirred at room temperature for 2 h, and the reaction mixture was washed with NaHCO (saturated aqueous). The organics were concentrated under reduced pressure, and the resulting residue was purified on a 60 g C18 column with a 40–100% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) to afford compound 18-4 as a colorless solid (200 mg, 58%) after lyophilization.

[0188] Next, 10% Pd / C (110 mg) was added to a solution of Ph-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 18-4; 110 mg, 80.8 μmol) in 2 M hydrochloric acid (aqueous, 1.0 mL) and 2-propanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 18 h. The mixture was filtered through a 0.45 μm syringe filter. The filtrate was concentrated under reduced pressure, and the residue was dissolved in water and lyophilized. The dried material was purified on a 60 g C18 column using a 5–50% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) and lyophilized to give compound 19-4 (23.2 mg, 33% yield), a colorless solid.

[0189] Compound 19-4 (Ph-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0190] 1 H NMR (400MHz; D2O): δ=7.74-7.70(m,2H), 7.58-7.53(m,1H), 7.48-7.43(m,2H), 4.45-4.34(m ,3H), 4.32-4.27(m,2H), 4.25-4.14(m,3H), 4.11(d,J3.8Hz,1H), 4.00-3.96(m,2H), 3.89(s, 2H), 2.91(t,J7.4Hz,2H), 2.34(t,J7.6Hz,2H), 2.14-2.06(m,1H), 2.01-1.91(m,1H), 1.85-1 .76(m,1H), 1.71-1.56(m,3H), 1.43(d,J7.3Hz,3H), 1.40-1.30(m,2H), 1.16-1.07(m,9H), 16 exchangeable protons not visible.

[0191] 1.7.5. Synthesis of Naphthyl-AQTGTGKT Naphthyl-AQTGTGKT (compound 19-5) was synthesized as the final target compound, Naphthyl-AQTGTGKT, by reacting compound 17-5 obtained by the following reaction formula 16 with compound 14 according to reaction formula 17.

[0192] [Reaction Scheme 16] [ka]

[0193] More specifically, H-Ala-OBzl.HCl (388 mg, 1.80 mmol, 1.2 equiv.) was suspended in ethyl acetate (10 mL) and N,N-diisopropylethylamine (653 μL, 3.75 mmol, 2.5 equiv.) was added. After stirring at room temperature for 5 min, HATU (855 mg, 2.25 mmol, 1.5 equiv.) and 2-naphthoic acid (258 mg, 1.50 mmol, 1 equiv.) were added, and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with ethyl acetate and washed with NH4Cl (saturated aqueous), NaHCO3 (saturated aqueous), and brine. The resulting organics were dried (Na2SO4), filtered, and concentrated under reduced pressure. The residue was purified on a 25 g column using a 2–40% ethyl acetate in heptane gradient to give compound 16-5 as a colorless solid (385 mg, 77% yield).

[0194] Next, 10% Pd / C (39 mg) wetted with a minimum amount of water was added to a solution of 2-Naphthyl-AlaOBn (compound 16-5; 385 mg, 1.15 mmol) in methanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 4 hours. The mixture was then filtered through a Celite pad and washed with methanol. The resulting filtrate was concentrated under reduced pressure to give compound 17-5 as a colorless solid (266 mg, 95% yield), which was reacted with compound 14 according to Scheme 17 below.

[0195] [Reaction Scheme 17] [ka]

[0196] More specifically, HATU (106 mg, 0.278 mmol, 1.1 equiv.) was added to a suspension of GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 14; 300 mg, 0.253 mmol, 1 equiv.) and 2-Naphthyl-Ala-OH (compound 17-5; 61.0 mg, 0.253 mmol, 1 equiv.) in N,N-diisopropylethylamine (97.0 μL, 0.556 mmol, 2.2 equiv.) and dichloromethane (20 mL). The mixture was stirred at room temperature for 2 h and then washed with (saturated aqueous) NaHCO. The resulting organic layer was concentrated under reduced pressure, and the residue was purified on a 60 g C18 column with a 40–100% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid) to give compound 18-5 as a colorless solid (230 mg, 64% yield) after lyophilization.

[0197] Next, 10% Pd / C (101 mg) was added to a solution of 2-Naphtyl-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 18-5; 101 mg, 71.5 μmol) in 2 M hydrochloric acid (aqueous, 1.0 mL) and 2-propanol (20 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 3 h. The mixture was filtered through a 0.45 μm syringe filter. The filtrate was concentrated under reduced pressure, and the residue was dissolved in water and lyophilized. The dried material was purified on a 30 g C18 column with a gradient of 5–40% acetonitrile (0.1% formic acid) in water (0.1% formic acid) and lyophilized to give compound 19-5 as a colorless solid (23.2 mg, 33% yield).

[0198] Compound 19-5 (Naphthyl-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0199] 1H NMR (400MHz; D2O): δ=8.32(s,1H), 8.01-7.90(m,3H), 7.79-7.73(m,1H), 7.64-7.55(m,2H), 4.51-3.70(m,13H), 2.96-2.81(m,2H), 2.39-2.30 (m,2H), 2.18-2.04(m,1H), 2.03-1.93(m,1H), 1.85-1.72(m,1H)1.71-1 .53(m,3H), 1.50-1.43(m,3H), 1.39-1.24(m,2H), 1.19-1.04(m,9H), 16 exchangeable protons not visible.

[0200] 1.8.Synthesis of Ac-AQTGTGKT Ac-AQTGTGKT (compound 19-6) was synthesized according to the following reaction scheme 18 to synthesize the final target compound, Ac-AQTGTGKT.

[0201] [Reaction Scheme 18] [ka]

[0202] More specifically, to a suspension of GlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound 14; 300 mg, 0.245 mmol, 1 equiv.) and Ac-Ala-OH (32.1 mg, 0.245 mmol, 1 equiv.) in N,N-diisopropylethylamine (94.0 μL, 0.540 mmol, 2.2 equiv.) and dichloromethane (30 mL) was added HATU (112 mg, 0.294 mmol, 1.2 equiv.). The mixture was stirred at room temperature for 14 h, and the reaction mixture was washed with (saturated aqueous) NH4Cl, NaHCO3, and water. The organic layer was concentrated under reduced pressure, and the residue was purified on a 60 g C18 column using a 50–95% acetonitrile (0.1% formic acid) gradient in water (0.1% formic acid). The desired fractions were combined and lyophilized to give compound 18-6 as a colorless solid (104 mg, 33% yield).

[0203] Next, 10% Pd / C (10 mg) was added to a solution of Ac-AlaGlnThr(OBn)GlyThr(OBn)GlyLys(Cbz)Thr(OBn)OBn (compound) 18-6 (33 mg, 25 μmol) in 2 M hydrochloric acid (aqueous, 0.19 mL) and 2-propanol (5 mL). The mixture was stirred under a hydrogen atmosphere (balloon) for 14 h. The mixture was filtered through a 0.45 μm syringe filter. The resulting filtrate was concentrated under reduced pressure, dissolved in water, and lyophilized. The dried material was purified on a 500 mg SCX-2 cartridge, eluting with 0.5 M ammonia in methanol. The desired fractions were combined, concentrated under reduced pressure, and lyophilized to give compound 19-6 as a colorless solid (7.6 mg, 37% yield).

[0204] Compound 19-6 (Ac-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0205] 1 H NMR (400MHz; D2O): δ=4.40-4.33(m,2H), 4.32-4.27(m,2H), 4.24-4.12(m,4 H), 4.08(d,J4.0Hz,1H), 4.03-3.88(m,4H), 2.92(t,J7.2Hz,2H), 2.32(t,J 7.8Hz,2H), 2.15-2.02(m,1H), 1.99-1.88(m,4H), 1.86-1.76(m,1H), 1.74- 1.55(m,3H), 1.45-1.31(m,2H), 1.29(t,J7.4Hz,2H), 1.20-1.06(m,10H), 16 exchangeable protons visible.

[0206] Synthesis of 1.9.4-PhPh-AQTGTGKT 1.9.1. Synthesis of QTGTGKT intermediate The intermediate QTGTGKT for synthesizing 4-PhPh-AQTGTGKT was synthesized according to the following Reaction Schemes 19 to 23.

[0207] [Reaction Scheme 19] [ka]

[0208] [Reaction Scheme 20] [ka]

[0209] [Reaction Scheme 21] [ka]

[0210] [Reaction Scheme 22] [ka]

[0211] [Reaction Scheme 23] [ka]

[0212] The above-mentioned Reaction Schemes 19 to 23 are almost similar to the above-mentioned Reaction Schemes 1 to 5 except for the presence or absence of a benzyl protecting group, and therefore a redundant explanation will be omitted.

[0213] Synthesis of 1.9.2.4-PhPh-AQTGTGKT Compound 31 obtained in Reaction Scheme 23 was combined with the alanine derivative synthesized in Reaction Scheme 24 below in Reaction Scheme 25 to obtain the final product 4-PhPh-AQTGTGKT.

[0214] [Reaction Scheme 24] [ka]

[0215] [Reaction Scheme 25] [ka]

[0216] The above-mentioned Reaction Schemes 24 and 25 were carried out in a similar manner to the above-mentioned Reaction Schemes, and therefore, redundant explanations will be omitted. Finally, Compound 36 was obtained as a colorless solid (583 mg, 68% yield).

[0217] Compound 36 (4-PhPh-AQTGTGKT) 1 1 H NMR data was measured as follows.

[0218] 1 H NMR (400MHz; D2O): δ=7.85(d,J8.8Hz,2H), 7.77(d,J8.8Hz,2H), 7.70(d,J7.2Hz,2H ), 7.49(t,J7.2Hz,2H), 7.42(t,J7.1Hz,1H), 4.34-4.04(m,6H), 4.07(d,J3.6Hz,1H ), 3.93(d,J2.0Hz,2H), 2.82(t,J7.0Hz,2H), 1.82-1.67(m,1H), 1.66-1.55(m,1H), 1.54-1.44(m,2H), 1.37-1.22(m,2H), 1.18(d,J6.4Hz,3H), 1.06(t,J6.5Hz,3H), 10 exchangeable protons not visible.

[0219] 1.10. Compound Verification The 13 AQTGTGKT analogs obtained by the above-mentioned production method are: 1 The chemical structure was confirmed by H NMR and UPLC-MS (ultraperformance liquid chromatography-mass spectrometry) techniques.

[0220] The structural formulas for the 13 AQTGTGKT analog compounds are shown in Table 1 below.

[0221] [Table 1] JPEG0007805020000043.jpg203170JPEG0007805020000044.jpg238170JPEG0007805020000045.jpg59170

[0222] 2. MTT (tetrazolium) assay The compounds of the present invention were treated in cancer cell lines, and simultaneously or 4 hours later, targeted anticancer drugs (osimertinib or olaparib) or chemo-anticancer drugs (Alimta or cisplatin) were added. The combined effect was measured by the degree of cell proliferation using an MTT assay. Specifically, 2 × 10 cells were added to each well of a 96-well plate. 3 100 μl of cells were dispensed into wells and cultured for 24 hours, after which the compound according to the present invention was introduced. After 4 hours had passed, each cancer cell was treated with an anticancer drug. The treatment concentrations and treatment times of the compound and anticancer drug are specifically described in the Examples. After treatment with the compound and anticancer drug was completed, 10 μl of CellTiter-96 (registered trademark) (Promega Co., USA) reagent was added to each well and allowed to react in an environment of 5% CO2 and 37°C. After 3 hours, the cells were measured using a spectrophotometer (SPECTROstar Nano The absorbance was measured at 490 nm using a chromatograph (BMG).

[0223] 3. Xenograft Lung Cancer Animal Model 5 × 10 H820 cells 6 The cells were mixed with matrigel at a 1:1 ratio at a concentration of 1:2 cells / mouse, and 200 μl of each was subcutaneously injected into the flank of each mouse. After completion of the inoculation, the tumor volume was 70-130 mm. 3 After confirming that the rats reached the target age, they were randomly divided into groups. The test substance was administered via the tail vein and orally seven times at two-day intervals.

[0224] 4. Xenograft Lung Cancer Resistance Animal Model 5 × 10 H1975 cells 6The cells were mixed with matrigel at a 1:1 ratio at a concentration of 1000 cells / mouse, and 200 μl of each was subcutaneously injected into the flank of each mouse. The tumor volume after inoculation ranged from 70 to 130 mm. 3 After confirming that tumor growth had reached 100%, the mice were randomly assigned to groups. After group assignment, osimertinib was administered orally daily. Once tumor growth was suppressed and tumor regrowth was confirmed, administration of the test substance in combination with osimertinib was initiated. The test substance was administered via the tail vein seven times at two-day intervals.

[0225] 5. Immunochemical staining (IHC) Paraffin-embedded tissues were cut into 4 μm thick slides and thoroughly dried before IHC experiments. Each slide was deparaffinized and endogenous enzymes were removed using citrate buffer (pH 6.0). Next, p-Beclin1S15 antibody was diluted 1:100 and incubated at 4°C for 15 hours. After washing four times with TBST washing buffer, rabbit HRP was added for secondary antibody reaction and incubated at room temperature for 30 minutes. After the antibody reaction substrate was added and color development occurred, the slides were washed, mounted, and scanned for the results.

[0226] Example 1: Anticancer effect of the combination of AQTGTGKT or its analogs targeted with chemoanticancer drugs in lung cancer cells 1.1. Combination effects in lung cancer cell line H1975 The anticancer effect of the combined use of AQTGTGKT or its analogues with anticancer drugs was confirmed in the lung cancer cell line H1975 using the MTT assay method described above in "Experimental Materials and Methods."

[0227] First, we investigated the combined effects of 3-PhPh-AQTGTGKT and osimertinib, a targeted anticancer drug. H1975 cells were treated with 25 μM 3-PhPh-AQTGTGKT and then 1.25 μM osimertinib alone or in combination. After 68 hours, we compared the cell proliferation inhibitory effects. As shown in Figure 1a, treatment with 3-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 10% compared to the control (untreated control), and treatment with osimertinib alone reduced cell proliferation by approximately 19% compared to the control. On the other hand, treatment with 3-PhPh-AQTGTGKT and osimertinib combined reduced cell proliferation by approximately 30%.

[0228] Next, H1975 cells were treated with 4-PhPh-AQTGTGKT at a concentration of 2 μM and osimertinib at a concentration of 1 μM, either alone or in combination, and then the cell proliferation inhibitory effects were compared 48 hours later. Treatment with 4-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 12% compared to the control group, and treatment with osimertinib alone reduced cell proliferation by approximately 5% compared to the control group. On the other hand, treatment with 4-PhPh-AQTGTGKT and osimertinib combined inhibited cell proliferation by approximately 24% (Figure 1b).

[0229] Next, H1975 cells were treated with Ac-AQTGTGKT at a concentration of 10 μM and osimertinib at a concentration of 0.5 μM, either alone or in combination, and then the cell proliferation inhibitory effects were compared 48 hours later. Treatment with Ac-AQTGTGKT alone reduced cell proliferation by approximately 14% compared to the control group, and treatment with osimertinib alone reduced cell proliferation by approximately 12% compared to the control group. On the other hand, treatment with Ac-AQTGTGKT and osimertinib combined inhibited cell proliferation by approximately 26% (Figure 1c).

[0230] We also treated H1975 cells with 4-MeOPh-AQTGTGKT at a concentration of 10 μM and the targeted anticancer drug osimertinib at a concentration of 0.5 μM, either alone or in combination, and then compared the cell proliferation inhibitory effects 48 hours later. Treatment with 4-MeOPh-AQTGTGKT alone reduced cell proliferation by approximately 6% compared to the control group, while treatment with osimertinib alone reduced cell proliferation by approximately 21% compared to the control group. On the other hand, treatment with 4-MeOPh-AQTGTGKT and osimertinib combined reduced cell proliferation by approximately 27% (Figure 1d).

[0231] Next, we investigated the combined effect of 4-PhPh-AQTGTGKT with the anticancer drug Alimta (Pemetrexed). Similarly, using the MTT assay described in the "Materials and Methods" section above, the lung cancer cell line H1975 was treated with 4-PhPh-AQTGTGKT at a concentration of 2 μM and the anticancer drug Alimta at a concentration of 0.2 μM. Cell proliferation was then compared 48 hours later. As shown in Figure 1e, treatment with 4-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 9% compared to the control group, and treatment with Alimta alone reduced cell proliferation by approximately 6% compared to the control group. On the other hand, combined treatment with 4-PhPh-AQTGTGKT and Alimta suppressed cell proliferation by approximately 22%.

[0232] In addition, H1975 / OR cells were treated with 3-PhPh-AQTGTGKT at a concentration of 5 μM, followed 4 hours later by treatment with the anticancer drug Alimta at a concentration of 0.1 μM, either alone or in combination. 72 hours later, the cell proliferation inhibitory effects were compared. As shown in Figure 1f, treatment with 3-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 6.5% compared to the control group, and treatment with Alimta alone reduced cell proliferation by approximately 8.3% compared to the control group. On the other hand, combined treatment with 3-PhPh-AQTGTGKT and Alimta suppressed cell proliferation by approximately 20%.

[0233] Next, H1975 / OR cells were treated with 3-PhPh-AQTGTGKT at a concentration of 5 μM, and then treated with the targeted anticancer drug osimertinib at a concentration of 2.5 μM alone or in combination with the drug 4 hours later. The cell proliferation inhibitory effects were compared 68 hours later. As shown in Figure 1g, treatment with 3-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 17% compared to the control group, and treatment with osimertinib alone reduced cell proliferation by approximately 7% compared to the control group. On the other hand, combined treatment with 3-PhPh-AQTGTGKT and osimertinib suppressed cell proliferation by approximately 30%.

[0234] Furthermore, the anticancer effect of triple therapy using the compound of the present invention, a targeted anticancer drug, and a chemotherapeutic drug was confirmed. Specifically, the lung cancer cell killing effect of triple combination therapy using 3-PhPh-AQTGTGKT, osimertinib, and Alimta was confirmed. H1975 cells were treated for 72 hours with 3-PhPh-AQTGTGKT at a concentration of 5 μM, and with osimertinib and Alimta at concentrations of 2.5 μM and 100 nM, respectively (the concentrations used in the experiment to confirm the anticancer effect of the combination with 3-PhPh-AQTGTGKT). The anticancer effect of the triple combination therapy was compared with that of a group treated with 3-PhPh-AQTGTGKT alone and a group treated with the combination of osimertinib and Alimta.

[0235] As a result, the triple combination treatment group of 3-PhPh-AQTGTGKT, osimertinib, and Alimta inhibited cell growth by approximately 35% compared to the control group, demonstrating a far superior cell proliferation inhibitory effect compared to the 3-PhPh-AQTGTGKT monotherapy group and the osimertinib and Alimta combination treatment group (Figure 1h). These results indicate that the combination of the compounds of the present invention with targeted anticancer drugs or chemoanticancer drugs not only provides superior anticancer effects compared to monotherapy, but also significantly enhances the anticancer effects of the triple combination treatment with targeted anticancer drugs and chemoanticancer drugs.

[0236] 1.2.Combination effects in lung cancer cell line H820 The anticancer effect of combined use of AQTGTGKT or its analogues with anticancer drugs was confirmed in the lung cancer cell line H820 using the MTT assay method described above in "Experimental Materials and Methods."

[0237] First, H820 cells were treated with AQTGTGKT at a concentration of 10 μM and simultaneously treated with the targeted anticancer drug osimertinib at a concentration of 5 μM, either alone or in combination. The cell proliferation inhibitory effects were compared 48 hours later. As shown in Figure 2a, treatment with AQTGTGKT alone reduced cell proliferation by approximately 15% compared to the control group, and treatment with osimertinib alone reduced cell proliferation by approximately 28% compared to the control group. On the other hand, combined treatment with AQTGTGKT and osimertinib suppressed cell proliferation by approximately 40%.

[0238] Next, H820 cells were treated with 3-PhPh-AQTGTGKT at a concentration of 40 μM, and then treated with the targeted anticancer drug osimertinib at a concentration of 6 μM alone or in combination with the drug osimertinib 4 hours later. The cell proliferation inhibitory effects were compared 72 hours later. As shown in Figure 2b, treatment with 3-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 8% compared to the control group, and treatment with osimertinib alone reduced cell proliferation by approximately 15% compared to the control group. On the other hand, combined treatment with 3-PhPh-AQTGTGKT and osimertinib suppressed cell proliferation by approximately 22%.

[0239] 1.3.Combination effects in lung cancer cell line PC9 / OR Using the MTT assay method described in the "Experimental Materials and Methods" section above, non-small cell lung cancer cell line PC9 / OR was treated with 3-PhPh-AQTGTGKT at a concentration of 25 μM, and 4 hours later, the targeted anticancer drug osimertinib was treated alone or in combination at a concentration of 4 μM, and cytotoxicity was compared 68 hours later.

[0240] As a result, as shown in Figure 3, treatment with 3-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 0.2% compared to the control group, and treatment with osimertinib alone reduced cell proliferation by approximately 10% compared to the control group. On the other hand, combined treatment with 3-PhPh-AQTGTGKT and osimertinib showed an approximately 40% inhibitory effect on cell proliferation.

[0241] The results of Examples 1.1 to 1.3 above demonstrate that the combined therapy of AQTGTGKT or its analogues according to the present invention and a targeted anticancer drug is far more effective in lung cancer cells than the monotherapy.

[0242] Example 2: Anticancer effects of targeting AQTGTGKT or its analogs in combination with chemoanticancer drugs in breast cancer cells The anticancer effect of combined use of AQTGTGKT or its analogues with anticancer drugs was confirmed in the breast cancer cell line HCC1937 using the MTT assay method described above in "Experimental Materials and Methods."

[0243] First, breast cancer cell line HCC1937 cells were treated with AQTGTGKT at a concentration of 5 μM and simultaneously treated with the targeted anticancer drug olaparib at a concentration of 2.5 μM, either alone or in combination. The cell proliferation inhibitory effects were compared 24 hours later. As shown in Figure 4a, treatment with AQTGTGKT alone did not reduce cell proliferation compared to the control group, while treatment with olaparib alone reduced cell proliferation by approximately 1.2% compared to the control group. On the other hand, combined treatment with AQTGTGKT and olaparib suppressed cell proliferation by approximately 13%.

[0244] Next, HCC1937 cells were treated with 3-PhPh-AQTGTGKT at a concentration of 10 μM and simultaneously treated with the targeted anticancer drug olaparib at a concentration of 2.5 μM, either alone or in combination. After 24 hours, the cell proliferation inhibitory effects were compared. As shown in Figure 4b, treatment with 3-PhPh-AQTGTGKT alone did not reduce cell proliferation compared to the control group, and treatment with olaparib alone did not reduce cell proliferation compared to the control group. On the other hand, combined treatment with 3-PhPh-AQTGTGKT and olaparib suppressed cell proliferation by approximately 8.2%.

[0245] In addition, HCC1937 cells were treated with 4-MeOph-AQTGTGKT at a concentration of 5 μM and simultaneously with the anticancer drug cisplatin at a concentration of 4 μM, either alone or in combination. The cell proliferation inhibitory effects were compared 48 hours later. As shown in Figure 4c, treatment with 4-MeOph-AQTGTGKT alone reduced cell proliferation by approximately 4.6% compared to the control group, and treatment with cisplatin alone reduced cell proliferation by approximately 7.9% compared to the control group. On the other hand, combined treatment with 4-MeOph-AQTGTGKT and cisplatin suppressed cell proliferation by approximately 15%.

[0246] Next, HCC1937 cells were treated with 3-PhPh-AQTGTGKT at a concentration of 10 μM and cisplatin at a concentration of 2 μM, either alone or in combination, and the cell proliferation inhibitory effects were compared 72 hours later. As shown in Figure 4d, treatment with 3-PhPh-AQTGTGKT alone reduced cell proliferation by approximately 3% compared to the control group, and treatment with cisplatin alone reduced cell proliferation by approximately 10% compared to the control group. On the other hand, treatment with 3-PhPh-AQTGTGKT and cisplatin combined inhibited cell proliferation by approximately 20%.

[0247] Example 3: Evaluation of the anti-cancer effect of combination therapy of 3-PhPh-AQTGTGKT and osimertinib 3.1.3 Comparison of anticancer effects depending on the time of combination of PhPh-AQTGTGKT To explore the optimal regimen for the combination of osimertinib and 3-PhPh-AQTGTGKT, we performed experiments in PC9 / OR lung cancer cell lines, dividing patients into two groups: one group treated with osimertinib and 3-PhPh-AQTGTGKT in combination followed by osimertinib alone, and one group treated with osimertinib alone followed by osimertinib and 3-PhPh-AQTGTGKT in combination. Specifically, Combo 1 consisted of 4 μM osimertinib and 25 μM 3-PhPh-AQTGTGKT for 3 days, followed by a medium change and then osimertinib alone for 3 days. Combo 2 consisted of 4 μM osimertinib alone for 3 days, followed by a medium change and then osimertinib and 25 μM 3-PhPh-AQTGTGKT for 3 days. Cells were then stained with crystal violet and growth was measured at 570 nm using a spectrophotometer.

[0248] As a result, as shown in Figure 5a, a more pronounced cell growth inhibitory effect was observed when osimertinib and 3-PhPh-AQTGTGKT were first co-treated followed by osimertinib alone (Combo 1) than when osimertinib alone was treated followed by co-treatment with osimertinib and 3-PhPh-AQTGTGKT (Combo 2).

[0249] 3.2.3 Comparison of anticancer effects depending on the duration of combined use of PhPh-AQTGTGKT To more specifically explore the optimal combination therapy shown in Example 3.1, the PC9 / OR lung cancer cell line was treated with osimertinib and 3-PhPh-AQTGTGKT in combination, followed by osimertinib alone, and then continued with osimertinib and 3-PhPh-AQTGTGKT. Specifically, in Combination 1 (Combo 1), cells were treated with 1 μM osimertinib and 25 μM 3-PhPh-AQTGTGKT for 3 days, followed by a medium change and then osimertinib alone for 3 days. In Combination 2 (Combo 2), cells were treated with 1 μM osimertinib and 25 μM 3-PhPh-AQTGTGKT for 3 days, followed by a medium change and then osimertinib 1 μM and 25 μM 3-PhPh-AQTGTGKT for 3 days. The cells were then stained with crystal violet and cell growth was measured in an absorbance detector at 570 nm.

[0250] As a result, as shown in Figure 5b, when osimertinib and 3-PhPh-AQTGTGKT were first combined, followed by repeated treatment with osimertinib at 1 μM and 3-PhPh-AQTGTGKT at 25 μM for 3 days (Combo 2), a more pronounced cell growth inhibitory effect was observed than when osimertinib was treated alone (Combo 1).

[0251] Example 4: Anti-cancer effect of the combination of 3-PhPh-AQTGTGKT and osimertinib in a lung cancer animal model Nude mice implanted with the H820 lung cancer cell line, an animal model of lung cancer described in the "Experimental Materials and Methods" section above, were treated with 3-PhPh-AQTGTGKT at a dose of 10 mpk seven times at two-day intervals for 14 days, and osimertinib was orally administered daily at 2.5 mpk, and tumor growth was compared.

[0252] As a result, as shown in Figure 6a, when 3-PhPh-AQTGTGKT was treated alone, tumor growth was reduced by approximately 38% compared to the control group (PBS-treated group), while administration of osimertinib alone reduced tumor growth by 46%, and the group treated with 3-PhPh-AQTGTGKT and osimertinib in combination showed a 51% tumor growth inhibitory effect.

[0253] In addition, the efficacy of the combination of the compound of the present invention and osimertinib was evaluated after the development of osimertinib resistance in a lung cancer animal model using the H1975 cell line. The H1975 cell line was inoculated into 4-week-old female Balb / C nude mice, and the presence or absence of tumor formation and growth were observed. The tumor volume was calculated by measuring the short and long diameters. Tumors with a volume of 70-120 mm were considered to be resistant to osimertinib. 3 When the tumor size reached 100 mg / kg, the groups were randomly separated and received either PBS (negative control) or osimertinib 5mpk orally daily.

[0254] The administration started on day 1, and tumor volume was measured twice a week. In the PBS group, the tumor volume reached 1,000 mm on the 20th day of administration. 3 In the osimertinib 5mpk group, the tumor growth suppression began to decrease depending on the number of doses administered from the day of administration, and was statistically significant compared to the PBS group (P<0.001).

[0255] However, tumors began to grow again on day 24 of osimertinib administration. When tumor size in the osimertinib group was compared with the final tumor size on day 28 of administration, the tumor growth rate was over 50%, indicating the patient had acquired resistance to osimertinib. Accordingly, the patients who received osimertinib 5mpk orally daily for 28 days were again divided into two groups and observed up to day 32 of administration. As tumor size gradually increased, the patients were divided into two groups: one group that received osimertinib 5mpk orally daily and PBS administered intravenously every two days (osimertinib + PBS group), and the other group that received osimertinib 5mpk orally daily and 3-PhPh-AQTGTGKT 10mpk administered intravenously every two days (osimertinib + 3-PhPh-AQTGTGKT group), and efficacy evaluation was performed.

[0256] The results are shown in Figure 6b. After the onset of osimertinib resistance, tumor growth in the osimertinib + 3-PhPh-AQTGTGKT group was delayed compared to the osimertinib + PBS group from the second initial administration, and after seven administrations (14 days after administration), a statistically significant difference began to appear between the tumor growth curves of the osimertinib + 3-PhPh-AQTGTGKT group and the osimertinib + PBS group (P<0.01). Tumor size was measured on the final day of administration, with a mean tumor size of 692.6 mm in the osimertinib + PBS group. 3 The tumor size in the osimertinib + 3-PhPh-AQTGTGKT group was measured at 364.7 mm 3 The tumor size was measured and confirmed to be suppressed by approximately 52% in the osimertinib + 3-PhPh-AQTGTGKT group compared to the osimertinib + PBS group. No weight loss or deaths were observed in any individual animals during the experiment.

[0257] These results demonstrate that the combination of 3-PhPh-AQTGTGKT and osimertinib is significantly more effective than monotherapy in a xenograft lung cancer animal model.

[0258] Example 5: Evaluation of the autophagy effect of combined administration Nude mice implanted with the H820 lung cancer cell line, an animal model of lung cancer described in the "Experimental Materials and Methods" section above, were treated with 3-PhPh-AQTGTGKT at a dose of 10 mpk seven times at two-day intervals for 14 days, and osimertinib was orally administered daily at 2.5 mpk. Tumors were then collected and subjected to p-Beclin1S15 immunostaining.

[0259] The results showed that, as shown in Figure 7, in a xenograft lung cancer animal model, when compared with the osimertinib monotherapy group, the expression of p-Beclin1S15 was reduced in the 3-PhPh-AQTGTGKT monotherapy group, and was significantly reduced in the 3-PhPh-AQTGTGKT and osimertinib combination group.

[0260] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. [Industrial Applicability]

[0261] The present invention relates to pharmaceutical compositions for cancer prevention / treatment and compositions for enhancing the anticancer effects of anticancer drugs, which contain, as active ingredients, the oligopeptide AQTGTGKT and its analog compounds, which are based on the autophagy mechanism and are tumor-promoting factors involved in resistance to conventional anticancer drugs. The compounds of the present invention selectively bind to protein targets involved in the upper stages of autophagy only when cancer is advanced, overactivating autophagy and specifically targeting cancer cells without affecting normal cells. This allows them to minimize the side effects of conventional anticancer drug monotherapy in patients with anticancer drug resistance and maximize the inhibitory effect on cancer cell proliferation.

[0262] More specifically, when oligopeptide AQTGTGKT and its analog compounds are administered in combination with conventional anticancer drugs, they exhibit enhanced anticancer effects, and even low drug concentrations exhibit significantly superior anticancer effects by suppressing cancer cell proliferation, thereby minimizing side effects such as functional and activity damage in normal tissues, decreased bone marrow function, gastrointestinal disorders, alopecia, and anticancer drug resistance. Furthermore, oligopeptides have the advantages of having a smaller molecular weight than antibodies, which reduces the risk of immune reactions and allows for easy tissue penetration, and they are expected to be used in combination with anticancer drugs for various types of cancer.

Claims

1. (i) A pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, a compound represented by the following general formula or a pharmaceutically acceptable salt thereof, and which is administered in combination with an anticancer agent: (general formula) X-AQTGTGKT (In the general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, and K is lysine, X is located on the N-terminal side of A in the general formula, and X is represented by any one of the following formulas 2 to 4. (Formula 2) 【Chemistry 1】 (Formula 3) 【Chemistry 2】 (In formula 3, R2 and R4 are each independently hydrogen, a C1 to C5 alkyl group, or a phenyl group. R3 is hydrogen, a C1-C5 alkyl group, a phenyl group, or a C1-C3 alkoxy group. (Formula 4) 【Transformation 3】 (In formula 4, R6 and R7 are each independently hydrogen or a C1-C3 alkyl group.

2. The X is 【Chemistry 4】 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, which is any one selected from the group consisting of:

3. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the anticancer drug is at least one selected from the group consisting of targeted anticancer drugs and chemical anticancer drugs.

4. 4. The pharmaceutical composition for preventing or treating cancer according to claim 3, wherein the targeted anticancer drug is one or more selected from the group consisting of a tyrosine kinase inhibitor, a PARP inhibitor, an angiogenesis inhibitor, a CDK4 / 6 inhibitor, a hormone therapy agent, and an antibody-drug conjugate.

5. The tyrosine kinase inhibitors include EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS Proto-Oncogene 1), BRAF (B-Raf Proto-Oncogene), HER2 (human epidermal growth factor receptor 2), RET (Ret Proto-Oncogene), NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1), MET (Mesenchymal-Epithelial Transition 5. The pharmaceutical composition for preventing or treating cancer according to claim 4, which is a target drug for one or more selected from the group consisting of IL-1 (IL-1), IL-2 (IL-2), IL-3 (IL-4), IL-4 (IL-5), IL-6 (IL-6), IL-7 (IL-8), IL-8 (IL-9), IL-1 (IL-11), IL-1 (IL-12), IL-1 (IL-13), IL

6. The pharmaceutical composition according to claim 4, wherein the tyrosine kinase inhibitor, PARP inhibitor, CDK4 / 6 inhibitor, hormone therapy drug, and antibody drug are each one or more selected from the group consisting of: a) Osimertinib, Afatinib, Brigatinib, Dasatinib, Dacomitinib, Erlotinib, Gefitinib, Lapatinib, Neratinib, Vandetanib tanib), icotinib, varitinib, tesevatinib, canertinib, naquotinib, pelitinib, poziotinib, rociletinib, nazartinib, alitinib (Allitinib; ALS-1306), pyrotinib, tyrphostin, crizotinib, ceritinib, entrectinib, dabrafenib, trametinib, alectinib, loratinib ( lorlatinib), larotectinib, lasertinib, olmutinib, AG1478, CUDC-101, MTKi-327 (JNJ-26483327), CL-387785 (EKI-785), CNX-2006, PD168393, TAK285, WZ4002, and AV-412 (MP-412). b) Olaparib, Rucaparib, Talazoparib, Veliparib, and Niraparib c) Trilaciclib, Palbociclib, Ribociclib, and Abemaciclib d) Tamoxifen, Toremifene, Fulvestrant, Goserelin, Leuprolide, Anastrozole, Letrozole, and Exemestane, and e) Sacituzumab govitecan, and Ladiratuzumab.

7. 5. The pharmaceutical composition for preventing or treating cancer according to claim 4, wherein the targeted anticancer drug is one or more selected from the group consisting of daratumumab, trastuzumab, and rituximab.

8. 4. The pharmaceutical composition for preventing or treating cancer according to claim 3, wherein the anticancer chemotherapeutic agent is at least one selected from the group consisting of Alimta, oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, and doxorubicin.

9. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the compound enhances the anticancer effect of the anticancer drug and reduces the side effects.

10. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the composition is in the form of a mixture of the compound and the anticancer agent.

11. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the compound and the anticancer agent are each formulated and administered simultaneously or sequentially.

12. 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the anticancer agent is contained at a concentration of 0.1 to 10 μM relative to the total composition, and the compound represented by the general formula is contained at a concentration of 1 to 50 μM relative to the total composition.

13. The anticancer drug is a targeted anticancer drug, and the compound represented by the general formula is 【Transformation 5】 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, which is any one selected from the group consisting of:

14. The anticancer drug is a chemical anticancer drug, and the compound represented by the general formula is 【Transformation 6】 2. The pharmaceutical composition for preventing or treating cancer according to claim 1, which is any one selected from the group consisting of:

15. The pharmaceutical composition for preventing or treating cancer according to claim 13 or 14, wherein the anticancer chemotherapeutic agent and the compound are contained in a molar ratio of 1:1 to 500.

16. The pharmaceutical composition for preventing or treating cancer according to claim 1 , wherein the cancer is selected from the group consisting of lung cancer, breast cancer, blood cancer, colorectal cancer, pancreatic cancer, and combinations thereof.

17. (i) A kit for preventing or treating cancer, which comprises, as an active ingredient, a compound represented by the following general formula or a pharmaceutically acceptable salt thereof, and which is administered in combination with an anticancer agent: (general formula) X-AQTGTGKT X is located on the N-terminal side of A in the general formula, and X is represented by any one of the following formulas 2 to 4. (Formula 2) 【Transformation 7】 (Formula 3) 【Transformation 8】 (In formula 3, R2 and R4 are each independently hydrogen, a C1 to C5 alkyl group, or a phenyl group. R3 is hydrogen, a C1-C5 alkyl group, a phenyl group, or a C1-C3 alkoxy group. (Formula 4) 【Chemistry 9】 (In formula 4, R6 and R7 are each independently hydrogen or a C1-C3 alkyl group.

18. A pharmaceutical composition for enhancing the anticancer effect of an anticancer agent, comprising, as an active ingredient, a compound represented by the following general formula or a pharmaceutically acceptable salt thereof: (general formula) X-AQTGTGKT (In the general formula, A is alanine, Q is glutamine, T is threonine, G is glycine, and K is lysine, X is located on the N-terminal side of A in the general formula, and X is represented by any one of the following formulas 2 to 4. (Formula 2) 【Chemistry 10】 (Formula 3) 【Chemistry 11】 (In formula 3, R2 and R4 are each independently hydrogen, a C1 to C5 alkyl group, or a phenyl group. R3 is hydrogen, a C1-C5 alkyl group, a phenyl group, or a C1-C3 alkoxy group. (Formula 4) 【Chemistry 12】 (In formula 4, R6 and R7 are each independently hydrogen or a C1-C3 alkyl group.

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