The complex and its use

Abstract

A complex according to one embodiment of the present invention includes: an acrolein reaction site having a chemical structure represented by formula (1); and a withdrawal site having another chemical structure. The withdrawal site is bound to a reaction site of an acrolein via a linker that can be cleaved by the reaction of the acrolein reaction site and an acrolein.

Description

[Technical Field]

[0001] This invention relates to a composite and its use. [Background technology]

[0002] In cancer targeting therapy, drug delivery systems are being developed to optimize treatment by enhancing drug efficacy while suppressing side effects. These systems selectively deliver drugs to specific tissues (target tissues) that require treatment, while also reducing side effects by minimizing impact on normal tissues.

[0003] For example, research is being conducted on prodrugs that exhibit antitumor activity under conditions characteristic of tumor cells, such as a hypoxic environment, in order to deliver drugs to target tumor cells (Non-Patent Documents 1-2). In addition, research is being conducted on prodrugs that release drugs by reacting with specific trigger compounds (Non-Patent Documents 3-4).

[0004] However, the compounds described in Non-Patent Documents 1-4 are still in the test tube stage, and furthermore, the compounds described in Non-Patent Documents 3-4 require additional compounds as triggers for drug release.

[0005] Acrolein (CH2=CHCHO) is the smallest unsaturated aldehyde molecule and is highly reactive. Acrolein is known to be produced during the combustion of organic matter, and it is also thought to be produced in the body as a metabolite of lipids or polyamines in diseases associated with oxidative stress, such as cancer, Alzheimer's disease, and cerebral infarction.

[0006] Furthermore, recent studies have revealed that acrolein exhibits higher toxicity than hydroxyl radicals, which were previously considered the main cause of oxidative stress. For this reason, it has attracted attention in recent years, particularly for detecting acrolein generated in cells and for elucidating the relationship between acrolein and diseases related to oxidative stress. For example, Non-Patent Document 5 describes a novel compound that selectively reacts with acrolein among molecules present in living organisms. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Calder et.al., Tetrahedron, 2020, Volume 78, Issue 21, Article 131170 [Non-Patent Document 2] Herrlinger et.al., ChemBioChem, 2020, 21(16), p2329-2347 [Non-Patent Document 3] Brakel et.al., Bioconjugate Chem., 2008, 19, 714-718 [Non-Patent Document 4] Matikonda et al., Chem. Sci., 2015, 6, 1212-1218 [Non-Patent Document 5] ACS Sens. 2016, 1, 623-632. [Overview of the project] [Problems that the invention aims to solve]

[0008] However, there has been virtually no research into using compounds that react with acrolein as prodrugs or the like.

[0009] Therefore, the object of the present invention is to provide a novel complex that reacts with acrolein to release an active substance, and its uses. [Means for solving the problem]

[0010] To solve the above problems, the present invention includes the following embodiments. A composite comprising an acrolein reaction site having a chemical structure represented by formula (1), and a release site having another chemical structure, wherein the release site is bonded to the acrolein reaction site via a linker that can be cleaved by the reaction between the acrolein reaction site and acrolein. [ka] (In equation (1), R1 and R2 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom, provided that at least one of R1 and R2 is a C1-C5 alkyl group; R3 and R4 independently refer to a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group (wherein the hydrogen atoms constituting the alkyl group may be substituted by substituents selected from halogen atoms, hydroxyl groups, and optionally substituted amino groups); * indicates the linkage site with the linker. [Effects of the Invention]

[0011] According to one aspect of the present invention, a novel complex that reacts with acrolein to release an active substance, and its uses can be provided. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the fluorescence spectra obtained by fluorescence measurement of a complex (coumarin-ABC) and a control (coumarin) according to one aspect of the present invention. [Figure 2] This figure shows the real-time measurement results for the fluorescence intensity of coumarin-ABC in and without acrolein. [Figure 3] This figure shows the results of measuring the fluorescence intensity of coumarin-ABC and coumarin in MCF10A (normal human mammary cells). [Figure 4] This figure shows the results of measuring the fluorescence intensity of coumarin-ABC and coumarin in A549 (human lung adenocarcinoma cells). [Figure 5] This figure shows the measurement results (concentration-dependent) of the fluorescence intensity of coumarin-ABC and coumarin in HeLa S3 (human cervical cancer cells). [Figure 6] This figure shows the real-time measurement results for the fluorescence intensity of coumarin-ABC (2 μM) in normal cells and cancer cells. [Figure 7] This figure shows the real-time measurement results for the fluorescence intensity of coumarin-ABC (20 μM) in normal cells and cancer cells. [Figure 8] This figure shows the results of HPLC analysis of a mixture of coumarin-ABC and glutathione (GSH). [Figure 9] This figure shows the HPLC analysis results of a mixture of coumarin-ABC and acrolein. [Figure 10] This figure shows the cell viability of MCF10A (normal human mammary cells) in the presence of a complex (MMC-ABC) according to one aspect of the present invention or a control. [Figure 11] This figure shows the cell viability of A549 (human lung adenocarcinoma cells) and HeLa S3 (human cervical cancer cells) in the presence of MMC-ABC or a control. [Figure 12] This graph shows the change in tumor volume in A549 tumor-carrying mice after intratumoral administration of MMC-ABC or a control. [Figure 13] This graph shows the change in body weight in A549 tumor-carrying mice after intratumoral administration of MMC-ABC or a control. [Figure 14] This graph shows the survival rates of A549 tumor-carrying mice after intratumoral administration of MMC-ABC or a control. [Figure 15] These are photographs of mice taken on day 2 after intratumoral administration of MMC-ABC or a control, and on day 4 after the end of administration. [Figure 16] This figure shows the cell viability of MCF10A, A549, or HeLa S3 in the presence of DOX-ABC and DOX. [Figure 17] These are photographs of mice after intratumoral administration of control (compound 9), DOX, or DOX-ABC. [Figure 18] This graph shows the change in tumor volume in A549 tumor-carrying mice after intratumoral administration of control (compound 9), DOX, or DOX-ABC. [Figure 19] This graph shows the survival rates of A549 tumor-carrying mice after intratumoral administration of control (compound 9), DOX, or DOX-ABC. [Figure 20] This graph shows the change in tumor volume in A549 tumor-carrying mice after intratumoral administration of either the control (compound 9) or PUR-ABC. [Modes for carrying out the invention]

[0013] [1. Complex] A complex according to one embodiment of the present invention (hereinafter also simply referred to as "this complex") includes an acrolein reaction site having a chemical structure represented by formula (1), and a release site having another chemical structure, wherein the release site is bonded to the acrolein reaction site via a linker that can be cleaved by a reaction between the acrolein reaction site and acrolein. [ka]

[0014] This complex reacts sensitively with acrolein (2-propenal, CH2=CHCHO) in the presence of acrolein, causing the linker to cleave and the release site to be released. This is because the azide group (N3 group) in the acrolein reaction site of this complex acts as a 1,3-dipole, undergoing a 1,3-dipole cycloaddition reaction (click reaction) with the dipoleophile acrolein to produce an intermediate five-membered ring (1,2,3-triazolin).

[0015] The 1,2,3-triazoline produced by the click reaction (1,3-dipolar cycloaddition reaction) between the acrolein reaction site and acrolein then undergoes an isomerization reaction to form an imine, followed by decarboxylation (removal of carbon dioxide), which cleaves the linker between the acrolein reaction site and the release site, separating the diazo compound derived from the acrolein reaction site from the active substance derived from the release site. [ka]

[0016] The above reaction can proceed under mild conditions, such as in vivo, and exhibits high reaction specificity. Therefore, by administering this complex into the body, the complex reacts with endogenous acrolein, resulting in the release of active substances derived from the release site into the body.

[0017] (Regarding R1 and R2) In formula (1), R1 and R2 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom. However, at least one of R1 and R2 is a C1-C5 alkyl group.

[0018] Examples of halogen atoms include fluorine, chlorine, and bromine atoms. Note that fluorine atoms may also be isotopes.

[0019] C1-C5 alkyl groups may be linear or branched. The alkyl group may have at least one of the above-mentioned halogen atoms substituted for its hydrogen atom, or it may not. Examples of C1-C5 alkyl groups without hydrogen atom substitution include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, and sec-pentyl groups.

[0020] R1 and R2 may be the same or different, but from the viewpoint of improving reactivity with acrolein, it is preferable that both R1 and R2 are alkyl groups having 1 to 5 carbon atoms that are substituted with at least one halogen atom or are not substituted, and it may be more preferable that they are the same alkyl group. The number of carbon atoms constituting the alkyl group may be preferably 1 to 4, and more preferably 2, 3, or 4.

[0021] In other embodiments, it is preferable that one of R1 and R2 is an alkyl group having 1 to 5 carbon atoms that is substituted with or unsubstituted with at least one halogen atom, and the other of R1 and R2 is a halogen atom (particularly fluorine or an isotope). The number of carbon atoms constituting the alkyl group is preferably 1 to 4, and more preferably 2, 3, or 4.

[0022] (Regarding R3 and R4) In formula (1), R3 and R4 are independently a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group. However, the hydrogen atoms constituting the alkyl group may be substituted with substituents selected from halogen atoms, hydroxyl groups, and optionally substituted amino groups.

[0023] The definition and examples of halogen atoms are the same as those described in R1 and R2.

[0024] Examples of amino groups that may have substituents include unsubstituted amino groups, alkylamino groups, and (hetero)arylamino groups. The number of carbon atoms constituting each alkyl group in an alkylamino group is, for example, 1 to 5. A (hetero)arylamino group refers to a group in which at least one hydrogen atom constituting the amino group is substituted with an aryl group that may have a heteroatom (i.e., an aryl group or a heteroaryl group). Examples of (hetero)aryl groups in a (hetero)arylamino group include those in which a ring structure skeleton is formed by 3 to 20 (preferably 4 to 12) atoms. Examples of heteroatoms in a heteroaryl group include sulfur atoms, nitrogen atoms, or oxygen atoms.

[0025] Examples of alkoxy groups having 1 to 5 carbon atoms include methoxy, ethoxy, propoxy, isopropoxy, and butoxy groups.

[0026] Examples of alkylthio groups having 1 to 5 carbon atoms include methylthio, ethylthio, propylthio, butylthio, and pentylthio groups.

[0027] The definition and examples of C1-C5 alkyl groups in which hydrogen atoms are not substituted are the same as those described in R1 and R2. The number of carbon atoms constituting the alkyl group is preferably 1-4, and more preferably 1-3. However, at least one of the hydrogen atoms constituting the alkyl group may be substituted with a substituent selected from a halogen atom, a hydroxyl group, and an optionally substituted amino group. Here, examples of halogen atoms and optionally substituted amino groups are the same as those described in R3 and R4.

[0028] R3 and R4 may be the same or different, but it may be preferable for R3 and R4 to be the same.

[0029] R3 and R4 are preferably, independently of each other, a hydrogen atom, a halogen atom, or a C1-C5 alkyl group which may be substituted with at least one halogen atom. The definition and examples of a C1-C5 alkyl group which may be substituted with at least one halogen atom are the same as those described in R1 and R2.

[0030] (Examples of preferred combinations of R1, R2, R3, and R4 located on the same ring) • Examples of combinations <1> In formula (1), R1, R2, R3, and R4 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom, provided that at least one of R1 and R2 is a C1-C5 alkyl group. • Examples of combinations <2> In formula (1), R3 and R4 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom, and R1 and R2 are C1-C5 alkyl groups. • Examples of combinations <3> In formula (1), R3 and R4 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom, and one of R1 and R2 is a C1-C5 alkyl group, and the other of R1 and R2 is a halogen atom. • Examples of combinations <4> Examples of the above combinations <1> or <2> In this configuration, R3 and R4 are independently a hydrogen atom or a halogen atom, and R1 and R2 are alkyl groups having 1 to 5 carbon atoms. • Examples of combinations <5> Examples of the above combinations <3> In this configuration, R3 and R4 are, independently of each other, a hydrogen atom or a halogen atom.

[0031] (Regarding *) In equation (1), * represents the linking site with the linker for connecting the detachment sites, as described above. This linker can be cleaved by the reaction between the acrolein reaction site and acrolein. More specifically, this linker has a structure in which cleavage is promoted by a 1,3-dipolar cycloaddition reaction between the acrolein reaction site and acrolein to produce a five-membered ring.

[0032] (Linker) An example of a linker that can be cleaved by the reaction between the acrolein reaction site and acrolein is a linker having the chemical structure represented by formula (2). [ka]

[0033] (Regarding R) In formula (2), R is independently a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group. However, the hydrogen atoms constituting the alkyl group may be substituted with substituents selected from halogen atoms, hydroxyl groups, and optionally substituted amino groups.

[0034] The definition and examples of halogen atoms are the same as those described in R1 and R2.

[0035] The definition and examples of an optionally substituted amino group are the same as those described in R3 and R4.

[0036] Examples of alkoxy groups having 1 to 5 carbon atoms include methoxy, ethoxy, propoxy, isopropoxy, and butoxy groups.

[0037] Examples of alkylthio groups having 1 to 5 carbon atoms include methylthio, ethylthio, propylthio, butylthio, and pentylthio groups.

[0038] The definition and examples of C1-C5 alkyl groups in which hydrogen atoms are not substituted are the same as those described in R1 and R2. The number of carbon atoms constituting the alkyl group is preferably 1-4, and more preferably 1-3. However, at least one of the hydrogen atoms constituting the alkyl group may be substituted with a substituent selected from a halogen atom, a hydroxyl group, and an optionally substituted amino group. Here, examples of halogen atoms and optionally substituted amino groups are the same as those described in R3 and R4.

[0039] R atoms bonded to the same carbon atom may be the same or different, but it is sometimes preferable that the R atoms be the same.

[0040] It is preferable that R is independently a hydrogen atom, a halogen atom, or a C1-C5 alkyl group (wherein the hydrogen atom constituting the alkyl group may be substituted with a substituent selected from a halogen atom, a hydroxyl group, and an amino group which may have a substituent), and it is more preferable that R is independently a hydrogen atom, a halogen atom, or a C1-C5 alkyl group which may be substituted with at least one halogen atom. The definition and examples of a C1-C5 alkyl group which may be substituted with at least one halogen atom are the same as those described in R1 and R2.

[0041] (Regarding * and *) In formula (2), ** represents the bonding site with the detachment site. From the viewpoint of rapid linker cleavage, it may be preferable that ** is a bonding site with a nitrogen atom, sulfur atom, or oxygen atom contained in the detachment site (i.e., -N(-)-, -S-, or -O-), and it may be more preferable that it is a bonding site with a nitrogen atom contained in the detachment site.

[0042] In formula (2), * represents the binding site to the acrolein reaction site. Therefore, the complex having a linker with the chemical structure represented by formula (2) has the chemical structure represented by formula (3) below. [ka]

[0043] In equation (3), the definitions and examples of R1, R2, R3, R4, and R are the same as those described in the explanations for equations (1) and (2). Also, in equation (3), ** is the same as that described in the explanation for equation (2).

[0044] A specific example of this complex having the chemical structure represented by formula (3) above is the complex of a release site and 4-azidobenzylcarbamate (hereinafter also simply referred to as "ABC"), represented by formula (4) below (release site-ABC). [ka] In equation (4), ** represents the bonding site with the nitrogen atom contained in the detachment site. Furthermore, when ** represents a bonding site with a nitrogen atom in the release site, the chemical structures shown in formulas (3) and (4) above can also be considered as protecting groups for amino groups in the release site that can be deprotected by acrolein.

[0045] (Mechanism of linker cleavage) As an example of a complex according to one aspect of the present invention, a linker is bonded to a nitrogen atom contained in the release site, and the reaction mechanism by which a linker having the chemical structure represented by formula (2) is cleaved and the release site (active substance) is released will be described below. [ka]

[0046] When this complex approaches acrolein, the azide group of this complex and acrolein undergo a 1,3-dipolar cycloaddition reaction to produce 1,2,3-triazoline as an intermediate. The five-membered ring (triazole ring) of 1,2,3-triazoline opens to form a diazo compound, and intramolecular electron transfer begins. This causes cleavage between the carbonyl carbon in the linker and the nitrogen atom in the release site, and the release site is released as an active substance.

[0047] (Disconnection site) As the release site, any compound that you wish to release in the region where acrolein is present can be used. Examples of such compounds include antitumor compounds, as well as labeling compounds such as dyes and markers.

[0048] The detachment site is preferably a compound having a nitrogen atom, a sulfur atom, or an oxygen atom as a linker bonding site, so that it can be rapidly cleaved from the linker. Furthermore, it may be preferable to have a hydroxyl group, a carboxyl group, a thiol group, or an amino group to facilitate bonding with the linker.

[0049] It is preferable that the bond between the release site and the linker can be rapidly cleaved by the reaction between the acrolein reaction site and acrolein. From this viewpoint, it is preferable that the bond between the release site and the linker is a carbamate bond, a thiocarbamate bond, an ester bond, or a thioester bond.

[0050] (antitumor compound) The inventors have discovered that tumor cells produce significantly more acrolein than normal cells, reaching more than 1000 times the amount found in inflammatory sites of normal cells (Tanaka, K. et al. Adv. Sci. 2019, 6, 1801479; Tanaka, K. et al. Bioorg. Med. Chem. 2019, 27, 2228; Tanaka, K. et al. Adv. Sci. 2020, 1901519). Therefore, this complex, which has an antitumor compound as a release site, can react with acrolein present around tumor cells to release the antitumor compound, making it suitable for use as a tumor treatment agent.

[0051] As the antitumor compound used as the release site, any compound that has activity to kill tumor cells in vivo, such as anticancer drugs, small molecular targeted agents, and radionuclides, can be used without limitation, including all antitumor compounds used in clinical or clinical trials, and antitumor compounds to be developed in the future.

[0052] The molecular weight of the antitumor compound is not particularly limited, but it is preferably low enough to reach tumor cells, for example, 15,000 Da or less, preferably 10,000 Da or less, and more preferably 500 Da or less. The lower limit of the molecular weight of the antitumor compound is not particularly limited, but for example, 100 Da or more, and preferably 300 Da or more.

[0053] It is preferable that the antitumor compound, when bound to the acrolein reaction site via a linker, exhibits reduced antitumor activity (toxicity) compared to its unbound state; in other words, it is in a prodrug state.

[0054] Suitable antitumor compounds for use as release sites include mitomycin C, doxorubicin, dacarbazine, crizotinib, endoxifen, and camptothecin derivatives [for example, the active component of DS-8201 (trastuzumab deruxtecan) (code name: DXd, chemical name: N[(1-S,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl]-2-hydroxyacetamide, Drug Delivery System Vol. 34 No. 1 (2019) pp. 52-58, Yuki Abe, Takashi Nakata, Wagatsuma Examples include, but are not limited to, Toshiki (see "Research and Development of Antibody-Drug Conjugates Carrying Novel DNA Topoisomerase I Inhibitors").

[0055] A specific example of this complex, which has mitomycin C as a release site, is mitomycin C-ABC, which has the following chemical structure. [ka]

[0056] A specific example of this complex, which has doxorubicin as the release site, is doxorubicin-ABC, which has the following chemical structure. [ka]

[0057] A specific example of this complex having dacarbazine as a release site is dacarbazine-ABC, which has the following chemical structure. [ka]

[0058] A specific example of this complex having crizotinib as the withdrawal site is crizotinib-ABC, which has the following chemical structure. [ka]

[0059] A specific example of this complex having endoxifen as a release site is endoxifen-ABC, which has the following chemical structure. [ka]

[0060] A specific example of this complex, which has puromycin as a release site, is puromycin-ABC, which has the following chemical structure. [ka]

[0061] (labeled compound) Examples of labeling compounds used as release sites include, but are not limited to, radioactive isotopes, fluorescent substances, or enzymes.

[0062] in particular, 3 H, 14 C, 125 I, 131 Radioactive isotopes such as I, green fluorescent protein (GFP), fluoroceine isothiocyanate, tetramethylrhodamine isothiocyanate, Eu 3+ Examples of labeling compounds include fluorescent substances such as methylcoumarin compounds, and enzymes such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, and glucose-6-phosphate dehydrogenase.

[0063] This complex, which has a labeled compound as a release site, can react with acrolein present around tumor cells to release the labeled compound, and can therefore be used as a diagnostic agent for detecting the presence or size of tumors and for imaging purposes.

[0064] [2. Method for producing the composite] The method for producing this complex will be explained below using an example of this complex in which a linker having the chemical structure represented by formula (2) is bonded to the nitrogen atom of the release site (i.e., forming a carbamate bond).

[0065] This complex can be produced by any method for synthesizing the carbamate bond. Such methods include reacting an isocyanate with an alcohol, and reacting an amine with a carbonate ester.

[0066] One method for reacting an isocyanate with an alcohol is to obtain an isocyanate by reacting the amino group of the compound used as the release site with triphosgene in the presence of a suitable solvent, and then to produce the complex in which the carbamate bond is formed by reacting the obtained isocyanate with the alcohol shown in formula (5). [ka]

[0067] One method for reacting an amine with a carbonate ester is to react an alcohol represented by formula (5) with a chloroformate ester in the presence of a suitable solvent to obtain a carbonate ester, and then to react the obtained carbonate ester with the amino group of a compound used as the release site to produce the complex in which a carbamate bond is formed.

[0068] Regarding the method for producing this composite, please refer to the description in the examples and to the description in ACS Sens. 2016, 1, 623-632 (Non-Patent Document 5).

[0069] [3. Antitumor treatment agents] A tumor treatment agent according to one embodiment of the present invention (hereinafter also simply referred to as "this treatment agent") contains the complex, as an active ingredient, whose release site is an antitumor compound.

[0070] (Other ingredients) In addition to the complex, this therapeutic agent may also contain a pharmaceutically acceptable carrier, provided that it does not inhibit the activity of the complex. Examples of such carriers include water, electrolyte solutions, and glucose solutions.

[0071] This therapeutic agent may contain adjuvants. Examples of such adjuvants include buffers, pain relievers, stabilizers, preservatives, antioxidants, and colorants.

[0072] Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Examples of pain relievers include propylene glycol, lidocaine hydrochloride, benzyl alcohol, benzalkonium chloride, and procaine hydrochloride. Examples of stabilizers include human serum albumin and polyethylene glycol. Examples of preservatives include benzyl alcohol and phenol. Examples of antioxidants include sulfites and ascorbic acid salts. Suitable examples of colorants include water-soluble colored tar dyes (e.g., food colorants such as Food Red No. 2 and 3, Food Yellow No. 4 and 5, Food Blue No. 1 and 2), insoluble lake dyes (e.g., aluminum salts of the aforementioned water-soluble food colorants), and natural pigments (e.g., β-carotene, chlorophyll, red iron oxide).

[0073] This therapeutic agent may contain additives such as binders, excipients, lubricants, sweeteners, flavoring agents, preservatives, disintegrants, suspending agents, solvents, solubilizers, isotonic agents, and swelling agents.

[0074] Examples of binders include gelatin, corn starch, tragacanth, gum arabic, pregelatinized starch, sucrose, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

[0075] Excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, soft anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, xylitol, sorbitol, and erythritol.

[0076] Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, and polyethylene glycol.

[0077] Examples of sweeteners include sodium saccharin, dipotassium glycyrrhizinate, aspartame, and stevia.

[0078] Flavorings include peppermint, red ginger oil, and cherry.

[0079] Examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0080] Examples of disintegrants include lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, low-substituted hydroxypropylcellulose, soft anhydrous silicic acid, and calcium carbonate.

[0081] Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and polysorbates and polyoxyethylene hydrogenated castor oil.

[0082] Suitable examples of solvents include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, and the like.

[0083] Suitable examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0084] Suitable examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, xylitol, and fructose.

[0085] (Dosage form) This therapeutic agent can be administered as an oral, injectable, or transdermal preparation. Oral preparations include tablets (including sublingual tablets and orally disintegrating agents), capsules (including soft capsules and microcapsules), powders, granules, lozenges, syrups, emulsions, and suspensions. Injectable preparations include intradermal, subcutaneous, intravenous, intramuscular, intrathecal, epidural, and local injections. Transdermal preparations include patches, ointments, and powders. These preparations may also be controlled-release formulations such as immediate-release or sustained-release formulations (e.g., sustained-release microcapsules).

[0086] (Manufacturing method) This therapeutic agent is preferably formulated as an injectable preparation. A sterile composition for injection can be formulated according to standard formulation practices, such as dissolving or suspending the active substance in a vehicle (aqueous solution for injection; such as a naturally occurring vegetable oil like sesame oil or coconut oil). As the aqueous solution for injection, for example, physiological saline, glucose, or an isotonic solution containing other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) can be used, and may be used in combination with a suitable solubilizer, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), or nonionic surfactant (e.g., polysorbate 80™, HCO-50). As the oily solution, for example, sesame oil or soybean oil can be used, and may be used in combination with a solubilizer such as benzyl benzoate or benzyl alcohol. The injectable preparation can be sealed in containers in units of one dose or multiple doses, such as ampoules or vials. Furthermore, the active ingredients and pharmaceutically acceptable carriers can be freeze-dried and stored in a state where they can be dissolved or suspended in a suitable sterile vehicle immediately before use.

[0087] (Content of this complex) The amount of this complex in this therapeutic agent varies depending on the form of the formulation, but is usually about 10 to 25% by mass of the total formulation, preferably about 15% by mass or more, and more preferably about 20% by mass or more.

[0088] (Dosage) The dosage of this therapeutic agent can be appropriately determined considering the type of target, method of administration, type of antitumor agent, type and location of tumor cells, etc. However, when administered intravenously to human solid tumors, the amount of this complex per kg of body weight is preferably 2 to 5 mg, more preferably 3 mg or more, and even more preferably 4 mg or more. These effective doses can be administered in one dose or in several divided doses.

[0089] (Target recipients) This therapeutic agent can be applied to tumors in any mammal. The types of mammals may be non-human mammals or humans. Examples of non-human mammals include rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, livestock such as pigs, cattle, goats, horses, sheep, and mink, pets such as dogs and cats, and primates other than humans such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees.

[0090] The target population for this therapeutic agent is the aforementioned individuals with tumors. While the type of tumor is not particularly limited, examples include lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., cervical cancer, endometrial cancer), gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, gallbladder and bile duct cancer, breast cancer, bladder tumors, osteosarcoma, malignant melanoma, pheochromocytoma, etc.

[0091] [4. Method for detaching the detachment site from the complex or the therapeutic agent] A method for detaching a detachment site from the complex or the therapeutic agent (hereinafter also simply referred to as "the Method") according to one embodiment of the present invention includes a step of reacting the complex or the therapeutic agent with acrolein.

[0092] The acrolein reacted with this complex or this therapeutic agent may be naturally occurring or in vitro.

[0093] When reacting with acrolein present in living organisms, this method can be applied to any mammal. Mammals can be non-human mammals or humans. Examples of non-human mammals include rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, livestock such as pigs, cows, goats, horses, sheep, and mink, pets such as dogs and cats, and primates other than humans, such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees.

[0094] [5. Summary of Embodiments] Embodiments of the present invention include, for example, the following aspects. <1> A composite comprising an acrolein reaction site having a chemical structure represented by formula (1), and a release site having another chemical structure, wherein the release site is bonded to the acrolein reaction site via a linker that can be cleaved by the reaction between the acrolein reaction site and acrolein. [ka] (In equation (1), R1 and R2 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom, provided that at least one of R1 and R2 is a C1-C5 alkyl group; R3 and R4 independently refer to a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group (wherein the hydrogen atoms constituting the alkyl group may be substituted by substituents selected from halogen atoms, hydroxyl groups, and optionally substituted amino groups); * indicates the linkage site with the linker. <2> The linker is a linker having the chemical structure represented by formula (2), <1> The composite described above. [ka] (In equation (2), R independently represents a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group (wherein the hydrogen atoms constituting the alkyl group may be substituted by substituents selected from halogen atoms, hydroxyl groups, and optionally substituted amino groups); * indicates the binding site with the acrolein reaction site. ※※ indicates the connection point with the detachment site. <3> In formula (2), the aforementioned ** is a bonding site with a nitrogen atom, sulfur atom, or oxygen atom contained in the detachment site, as described above. <2> The composite described above. <4> In formula (2), R independently refers to a hydrogen atom, a halogen atom, or a C1-C5 alkyl group (wherein the hydrogen atom constituting the alkyl group may have a substituent selected from a halogen atom, a hydroxyl group, and an amino group which may have a substituent). <2> or <3> The composite described above. <5> In formula (1), R1 and R2 independently refer to C1-C5 alkyl groups which may be substituted with at least one halogen atom. <1> ~ <4> A complex described in any of the following. <6> The aforementioned detachment site is an antitumor compound, <1> ~ <5> A complex described in any of the following. <7> The above-mentioned detachment site detaches upon reaction with acrolein present around the tumor cells. <1> ~ <6> A complex described in any of the following. <8> the above <6> or <7> A tumor treatment agent containing the complex described above. <9> the above <1> ~ <7> A composite as described in any of the above, or the above <8> A method for detaching the detachment site, comprising the step of reacting the therapeutic agent described in [reference] with acrolein.

[0095] The present invention is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Examples]

[0096] [Chemical synthesis] All chemically available reagents were used without further purification. Preparative separation was performed by column chromatography on Merck silica gel 60 (230-400 mesh). 1 H and 1313C NMR spectra were recorded using a JEOL RESONANCE AL400 NMR spectrometer. Unless otherwise specified, CDCl3 was used as the solvent. Chemical shifts were expressed as δ values relative to TMS as the internal standard. High-resolution mass spectrometry (HRMS) was recorded on a micrOTOF-QIII. Fluorescence spectra were measured using a spectrofluorometer (SpectraMax iD3, manufactured by Molecular Devices). Since azide-containing compounds were predicted to be potentially explosive, all operations were carefully carried out in a hood.

[0097] Example 1: Synthesis of 2,6-Diisopropylphenyl Azide (Compound 1)

Chemical Structure

[0098] A mixture of 2,6-diisopropylaniline (846 mg, 4.77 mmol, 1.0 equivalent) and sodium azide (791 mg, 11.9 mmol, 2.5 equivalents) dissolved in acetic acid and distilled water (9:1) (50 mL, [2,6-diisopropylaniline] = 0.1 M) was slowly added with sodium nitrite (814 mg, 11.5 mmol, 2.4 equivalents) at 0 °C. After stirring at 0 °C for 30 minutes, a saturated aqueous NaHCO3 solution was added until the mixture reached pH 7. The mixture was extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane alone) to obtain the desired 2,6-diisopropylphenyl azide (Compound 1) as a yellow oil (912 mg, 94%). 1 1H NMR (400 MHz, CDCl3, 25 °C) δ 7.25 - 7.07 (m, 3H), 3.36 (hept, J = 7.0 Hz, 2H), 1.26 (d, J = 6.8 Hz, 12H); 13C NMR (100 MHz, CDCl3, 25 ℃) δ 143.33, 135.54, 127.01, 124.12, 28.91, 23.59.

[0099] [Production Example 2] Reaction of 2,6-diisopropylphenylazide (compound 1) with acrolein [ka]

[0100] A solution of 2,6-diisopropylphenyl azide (compound 1) (47.3 mg, 0.23 mmol, 1.0 equivalent) in THF (without stabilizers, 310 μL, [1] = 0.7 M) was prepared, to which acrolein (164 μL, 2.33 mmol, 10 equivalents) was added. After stirring at room temperature for 24 hours, the reaction mixture was concentrated to dryness under reduced pressure. The resulting crude product was purified by preparative TLC (n-hexane / ethyl acetate = 3:1) to obtain 4-formyl-1,2,3-triazole (compound 4) (Rf value 0.66, 27%, yellow oily substance) and heterocyclic compound (compound 6) (Rf value 0.42, 53%, red oily substance). 4-Formyl-1,2,3-triazole (compound 4): 1 H NMR (400 MHz, CDCl3, 25℃) δ 10.29 (s, 1H), 8.19 (s, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.8 Hz, 2H), 2.16 (hept, J = 6.9 Hz, 2H), 1.14 (dd, J = 10.4, 6.8 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 185.60, 145.97, 131.65, 128.19, 124.27, 28.63, 24.17, 24.02; ESI-HRMS m / z C 15 H 19 N3NaO ([M+Na] + The calculated value for ) was 280.1420, and the detected value was 280.1419. Heterocycles (compound 6): 1H NMR (400 MHz, CDCl3, 25 ℃) δ 9.38 (s, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 7.22 (d, J = 7.7 Hz, 2H), 7.15 (dd, J = 7.6, 3.1 Hz, 2H), 5.85 (s, 1H), 5.54 (s, 1H), 4.20 (q, J = 26.3 Hz, 2H), 4.11 (d, J = 11.0 Hz, 1H), 3.31 (d, J = 11.0 Hz, 1H), 3.12 (s, 2H), 2.94 (p, J = 6.9 Hz, 1H), 2.83 (p, J = 7.1 Hz, 3H), 1.23 - 1.18 (m, 24H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 189.33, 147.46, 146.89, 143.09, 140.10, 134.52, 129.87, 128.57, 124.37, 124.32, 120.59, 109.18, 92.90, 84.51, 53.37, 51.37, 39.40, 28.76, 28.23, 28.17, 24.78, 24.71, 24.68, 24.48; 33 H 45 N6O2([M+H] + The calculated value for ) is 557.3599, and the detected value is 557.3597.

[0101] [Production Example 3] Synthesis of Coumarin-ABC (Compound 7) (Fluorescent Dye-ABC) The synthesis route for coumarin-ABC (compound 7) synthesized in Production Example 3 is as follows. [ka]

[0102] Each step is described in detail below. (Production Example 3-1) Synthesis of 4-iodo-2,6-diisopropylaniline (compound S1) [ka]

[0103] To a solution (200 mL, [2,6-diisopropylaniline] = 0.2 M) of 2,6-diisopropylaniline (8.8 g, 44.9 mmol, 1.0 equivalent) in saturated aqueous NaHCO3 and Et2O (1:1), iodine (13.7 g, 53.8 mmol, 1.2 equivalents) was added. After stirring at room temperature for 5 hours, Na2S2O3·H2O was added, and the mixture was stirred for 15 minutes. The resulting mixture was extracted with Et2O. The combined organic phase was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure to obtain the desired 4-iodo-2,6-diisopropylaniline (compound S1) as a reddish-black oil (13.4 g, 98%). 1 H NMR (400 MHz, CDCl3, 25 ℃) δ 7.27 (s, 2H), 3.70 (bs, 2H, NH2), 2.81 (hept, J = 6.7 Hz, 2H), 1.21 (d, J = 6.8 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 140.27, 135.08, 131.79, 81.12, 27.82, 22.21; ESI-HRMS m / z C 12 H 19 IN ([M+H] + The calculated value for ) was 304.0557, and the detected value was 304.0558.

[0104] (Production Example 3-2) Synthesis of ethyl 4-amino-3,5-diisopropylbenzoate (compound S2) [ka]

[0105] A mixture of 4-iodo-2,6-diisopropylaniline (compound S1) (6.2 g, 20.5 mmol, 1.0 equivalent) and Pd(PPh3)4 (0.5 g, 0.41 mmol, 0.02 equivalents) in EtOH (17 mL, [S1] = 1.2 M) was mixed with Et3N (4.3 mL, 30.7 mmol, 1.5 equivalents). The resulting solution was stirred under CO pressure (0.5 MPa) at 110°C for 8 hours. The suspension was filtered through Celite, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by gradient elution [n-hexane / ethyl acetate (25:1~15:1)] by column chromatography on silica gel to obtain the desired ethyl 4-amino-3,5-diisopropylbenzoate (compound S2) as a yellow oil (4.4 g, 86%). 1 H NMR (400 MHz, CDCl3, 25 ℃) δ 7.75 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.16 (bs, 2H, NH2), 2.88 (hept, J = 6.9 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H), 1.29 (d, J = 6.8 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 167.64, 145.10, 131.37, 125.04, 119.77, 60.30, 27.97, 22.24, 14.55; 15 H 24 NO2([M+H] + The calculated value for ) was 250.1802, and the detected value was 250.1805.

[0106] (Production Example 3-3) Synthesis of 4-hydroxymethyl-2,6-diisopropylaniline (compound S3) [ka]

[0107] To a 30 mL solution ([S2]=0.1 M) of ethyl 4-amino-3,5-diisopropylbenzoate (compound S2) (764 mg, 3.1 mmol, 1.0 equivalent) in THF at 0°C, DIBAL-H (9 mL, 9.2 mmol, 3.0 equivalents, 1.0 M solution in toluene) was slowly added. The reaction mixture was stirred under a nitrogen atmosphere at 0°C for 10 minutes, then stirred at room temperature for 1 hour. The excess amount of DIBAL-H was carefully quenched at 0°C with MeOH (20 mL), stirred for 30 minutes, and allowed to stand at room temperature. The resulting mixture was filtered through Celite (washed with MeOH), and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by gradient elution [n-hexane / ethyl acetate (15:1~7:1)] using column chromatography on silica gel to obtain the desired 4-hydroxymethyl-2,6-diisopropylaniline (compound S3) as an orange oily substance (627 mg, 99%). 1 H NMR (400 MHz, CDCl3, 25 ℃) δ 7.04 (s, 2H), 4.55 (s, 2H), 3.74 (bs, 2H, NH2), 2. 92 (hept, J = 6.7 Hz, 2H), 1.27 (d, J = 6.8 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 140.06, 132.71, 130.89, 122.46, 66.09, 27.99, 22.44; ESI-HRMS m / z C 13 H 22 NO2([M+H] + The calculated value for ) was 208.1696, and the detected value was 208.1699.

[0108] (Production Example 3-4) Synthesis of (4-azido-3,5-diisopropylphenyl)-methanol (compound S4) [ka]

[0109] A mixture of 4-hydroxymethyl-2,6-diisopropylaniline (compound S3) (763 mg, 3.68 mmol, 1.0 equivalent) and sodium azide (610 mg, 9.20 mmol, 2.5 equivalents) dissolved in acetic acid and distilled water (5:2) (35 mL, [S3] = 0.1 M) was slowly mixed with sodium nitrite (628 mg, 8.83 mmol, 2.4 equivalents) at 0°C. After stirring at 0°C for 4 hours, saturated aqueous solution of NaHCO3 was added to the mixture until the pH reached 7. The mixture was extracted with ethyl acetate. The combined organic phase was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by gradient elution [n-hexane / ethyl acetate (30:1~15:1)] using column chromatography on silica gel to obtain the desired (4-azido-3,5-diisopropylphenyl)-methanol (compound S4) as a yellow oily substance (730 mg, 85%). 1 H NMR (400 MHz, CDCl3, 25 ℃) δ 7.13 (s, 2H), 4.64 (s, 2H), 3.36 (p, J = 6.8 Hz, 2H), 1.27 (d, J = 6.7 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 143.51, 139.31, 134.84, 122.79, 65.27, 28.90, 23.52; ESI-HRMS m / z C 13 H 20 N3O ([M+H] + The calculated value for ) was 234.1601, and the detected value was 234.1605.

[0110] (Production Example 3-5) Synthesis of Coumarin-ABC (Compound 7) [ka]

[0111] To a 0°C mixture (4 mL, [7-amino-4-methylcoumarin] = 0.1 M) of 7-amino-4-methylcoumarin (69 mg, 0.39 mmol, 1.0 equivalent) and DIPEA (250 μL, 1.45 mmol, 3.8 equivalents) dissolved in toluene, triphosgene (140 mg, 0.46 mmol, dissolved in 4 mL of toluene) was slowly added. The reaction mixture was refluxed under a nitrogen atmosphere for 4 hours and then cooled to room temperature. CH2Cl2 (3 mL) was added to the reaction mixture and stirred for 5 minutes until the solution turned dark brown, and compound S4 (117 mg, 0.5 mmol, 1.3 equivalents in 5 mL of CH2Cl2) was added. The reaction mixture was stirred and heated at 55°C for 13 hours and then cooled to room temperature. The resulting solution was concentrated to dryness under reduced pressure while maintaining a temperature of 30°C. The residue was purified by column chromatography (CHCl3 / MeOH 25:1) on silica gel to obtain the desired coumarin-ABC (compound 7) as a pale yellow solid (141 mg, 84%). 1 H NMR (400 MHz, DMSO-d6, 25 ℃) δ 10.28 (bs, 1H, NH), 7.69 (d, J = 8.7 Hz, 1H), 7.55 (d, J = 2.1 Hz, 1H), 7.41 (dd, J = 8.7, 2.0 Hz, 1H), 7.31 (s, 2H), 6.23 (d, J = 1.4 Hz, 1H), 5.15 (s, 2H), 3.29 (p, J = 6.9 Hz, 2H), 2.38 (d, J = 1.3 Hz, 3H), 1. 22 (d, J = 6.9 Hz, 12H); 13 C NMR (100 MHz, DMSO-d6, 25 ℃) δ 160.17, 153.98, 153.33, 153.27, 142.84, 142.81, 134.90, 134.49, 126.17, 124.60, 114.51, 114.35, 112.03, 104.53, 66.29, 28.39, 23.29, 17.99.; ESI-HRMS m / z C 24 H 26 N4NaO4([M+Na] + The calculated value for ) was 457.1846, and the detected value was 457.1840.

[0112] [Manufacturing Example 4] Synthesis of the prodrug MMC-ABC (compound 8) (drug-ABC) The synthesis route for MMC-ABC (compound 8) synthesized in Production Example 4 is as follows. [ka] Each step is described in detail below.

[0113] (Production Example 4-1) Synthesis of 4-azido-3,5-diisopropylbenzyl-(4-nitrophenyl)-carbonate (compound S5) [ka]

[0114] A mixture of compound S4 (297 mg, 1.27 mmol, 1.0 equivalent) and 4-nitrophenyl chloroformate (321 mg, 1.53 mmol, 1.2 equivalents) dissolved in THF (13 mL, [S4] = 0.1 M) at 0°C was prepared by adding pyridine (206 μL, 2.55 mmol, 2.0 equivalents). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. THF was removed using a rotary evaporator, and the resulting crude product was partitioned using siRNA-H2O. The combined organic phase was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane / ethyl acetate 25:1) to obtain the desired 4-azido-3,5-diisopropylbenzyl-(4-nitrophenyl)-carbonate (compound S5) as a yellow oil (424 mg, 84%). 1 H NMR (400 MHz, CDCl3, 25℃) δ 8.27 (d, J = 9.1 Hz, 2H), 7.20 (s, 2H), 5.26 (s, 2H), 3.38 (p, J = 6.9 Hz, 2H), 1.29 (d, J = 6.9 Hz, 12H); 13C NMR (100 MHz, CDCl3, 25 ℃) δ 155.65, 152.50, 145.52, 143.82, 136.11, 132.62, 125.41, 124.63, 121.86, 71.07, 28.94, 23.53.

[0115] (Manufacturing Example 4-2) Synthesis of the prodrug MMC-ABC (Compound 8) [ka]

[0116] A mixture of compound S5 (307 mg, 0.77 mmol, 1.0 equivalent), mitomycin C (170 mg, 0.51 mmol, 1.5 equivalents), and activated powdered molecular sieve (MS4A) dissolved in DMF (8 mL, [S5] = 0.1 M) was mixed with Et3N (130 μL, 0.93 mmol, 1.8 equivalents). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The resulting mixture was filtered through Celite (washed with ethyl acetate), and the filtrate was concentrated to dryness under reduced pressure. The resulting crude product was partitioned using ethyl acetate·H2O. The combined organic phase was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography (CHCl3 / MeOH 30:1) on silica gel to obtain the desired prodrug MMC-ABC (compound 8) as a purple oil (260 mg, 86%). 1H NMR (400 MHz, CDCl3, 25 ℃) δ 7.12 (s, 2H), 5.25 (bs, 2H, NH2), 5.04 (s, 2H), 4.89 (dd, J = 10.8, 4. 7 Hz, 1H), 4.73 (bs, 2H, NH2), 4.44 (d, J = 13.3 Hz, 1H), 4.32 (t, J = 11.0 Hz, 1H), 3.70 (dd, J = 11.0, 4.8 Hz, 1H), 3.48 (dd, J = 13.3, 1.9 Hz, 1H), 3.45 (d, J = 4.6 Hz, 1H), 3.37 - 3.27 (m, 3H), 3.19 (s, 3H), 1.76 (s, 3H), 1.25 (dd, J = 6.9, 4.1 Hz, 12H).; 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 178.53, 176.03, 160.82, 156.46, 154.47, 147.15, 143.52, 135.60, 133.83, 124.67, 110.67, 105.58, 105.31, 68.80, 62.29, 49.90, 48.81, 43.52, 42.01, 40.21, 28.93, 23.55, 8.01; ESI-HRMS m / z C 29 H 36 N7O7([M+H] + The calculated value for ) was 594.2671, and the detected value was 594.2673.

[0117] [Production Example 5] Synthesis of the control compound 2-azido-1,3-diisopropyl-5-methylbenzene (Compound 9) [ka]

[0118] A mixture of 4-methyl-2,6-diisopropylaniline (0.8 g, 4.18 mmol, 1.0 equivalent) and sodium azide (0.7 g, 10.5 mmol, 2.5 equivalents) dissolved in acetic acid and distilled water (5:2) (21 mL, [4-methyl-2,6-diisopropylaniline] = 0.2 M) was slowly added at 0°C with sodium nitrite (0.7 mg, 10.0 mmol, 2.4 equivalents). After stirring at 0°C for 3 hours, a saturated aqueous solution of NaHCO3 was added to the mixture until the pH reached 7. The mixture was extracted with ethyl acetate. The combined organic phase was washed with brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane alone) to obtain the desired 2-azido-1,3-diisopropyl-5-methylbenzene (compound 9) as a colorless oil (0.7 g, 75%). 1 H NMR (400 MHz, CDCl3, 25 ℃) δ 6. 92 (s, 2H), 3.32 (hept, J = 7.1 Hz, 2H), 2.31 (s, 3H), 1.25 (d, J = 7.2 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25 ℃) δ 142.98, 136.42, 124.75, 124.07, 28.88, 23.66, 21.47.

[0119] [Example 1] Release of fluorescent dye in DMEM cell medium Coumarin-ABC (compound 7) and 7-amino-4-methylcoumarin (control compound), synthesized in Production Example 3, were each dissolved in water (0.2% DMSO) at a concentration of 2 μM, and their fluorescence spectra were measured. The results are shown in Figure 1 (dotted line: compound 7, solid line: control compound).

[0120] As shown in Figure 1, it was confirmed that coumarin-ABC (compound 7) does not emit fluorescence.

[0121] Next, coumarin-ABC (compound 7) (20 μM) was incubated in DMEM medium (Dulbecc's modified Eagle medium) (100 μL) on a 96-well plate at room temperature, either in the presence or absence of acrolein (20 mM). Fluorescence intensity was measured in real-time for 30 minutes using a spectrofluorometer (SpectraMax iD3, Molecular Devices). The results are shown in Figure 2 (dotted line: in the presence of acrolein, solid line: in the absence of acrolein).

[0122] As shown in Figure 2, a rapid increase in fluorescence intensity was observed in the presence of acrolein. This indicates that coumarin-ABC (compound 7) reacted with acrolein via an azide-acrolein click reaction, releasing 7-amino-4-methylcoumarin.

[0123] [Example 2] Fluorescence emission (cell-based assay; concentration-dependent) Three cell lines [MCF10A (normal human mammary gland cells), A549 (human lung adenocarcinoma cells), and HeLa S3 (human cervical cancer cells)] were seeded on 96-well plates (2 × 10⁶). 4 Cells were placed in wells and allowed to adhere for 24 hours at 37°C. Then, each cell was subjected to the following three different conditions: (Condition 1) Cells were treated with 100 μL of coumarin-ABC (compound 7) solution [2 μM, 5 μM, 10 μM, 15 μM, 20 μM in medium (1% DMSO)] and incubated at room temperature for 60 minutes. (Condition 2) Cells were treated with 1 mM N-acetylcysteine ​​(NAc-Cys) in culture medium and incubated at 37°C for 2 hours. Then, cells were treated with 100 μL of coumarin-ABC (compound 7) solution [2 μM, 5 μM, 10 μM, 15 μM, 20 μM in culture medium (1% DMSO)] and incubated at room temperature for 60 minutes. (Condition 3) Cells were treated with 100 μL of 7-amino-4-methylcoumarin solution [2 μM, 5 μM, 10 μM, 15 μM, 20 μM in medium (1% DMSO)] and incubated at room temperature for 60 minutes.

[0124] After incubation, fluorescence intensity was measured using a spectrofluorometer (SpectraMax iD3, Molecular Devices). Fluorescence intensity was standardized to 10,000 cells for each cell line. The results are shown in Figures 3-5.

[0125] As shown in Figure 3, in normal cells incubated under condition 3, significant fluorescence emission was observed depending on the compound concentration, while in normal cells incubated under conditions 1 and 2, almost no fluorescence emission was observed. On the other hand, as shown in Figures 4 and 5, in cancer cells incubated under conditions 1 and 3, significant fluorescence emission was observed depending on the compound concentration, while in cancer cells incubated under condition 2, only slight fluorescence emission was observed. These results indicate that because cancer cells overexpress acrolein, in cancer cells, coumarin-ABC (compound 7) reacts with endogenous acrolein via an azide-acrolein click reaction, releasing 7-amino-4-methylcoumarin. In contrast, normal cells produce only a very small amount of acrolein, so in normal cells, most of the coumarin-ABC (compound 7) remains unchanged, resulting in only slight fluorescence emission.

[0126] [Example 3] Fluorescence emission (cell-based assay; time-dependent) Three cell lines (MCF10A, A549, and HeLa S3) were seeded on a 96-well plate (2 × 10⁶). 4 Cells were allowed to adhere to the substrate (cells / well) by leaving them at 37°C for 24 hours. Next, the cells were treated with 100 μL of coumarin-ABC (compound 7) solution [2 μM or 20 μM in medium (1% DMSO)], incubated at room temperature, and fluorescence intensity was measured in real-time for 30 minutes using a spectrofluorometer (SpectraMax iD3, Molecular Devices). Fluorescence intensity was standardized to 10,000 cells for each cell line. The results are shown in Figure 6 (2 μM) and Figure 7 (20 μM).

[0127] As shown in Figures 6 and 7, only slight fluorescence emission was observed in normal cells. In contrast, significant fluorescence emission was observed in cancer cells in a time-dependent manner. These results indicate that, because cancer cells overexpress acrolein, coumarin-ABC (compound 7) reacts with endogenous acrolein via an azide-acrolein click reaction, releasing 7-amino-4-methylcoumarin. On the other hand, normal cells produce only a small amount of acrolein, so in normal cells, most of the coumarin-ABC (compound 7) remains unchanged, resulting in only slight fluorescence emission.

[0128] [Example 4] Emission of fluorescence (HPLC analysis) Coumarin-ABC (compound 7) (2 × 10) -5 Glutathione (GSH) (2 × 10¹⁶) is added to DMSO solution (1 mL, [7] = 20 μM) in an mmol, 1.0 equivalent volume. -5 mmol, 1.0 equivalent) or acrolein (2 × 10⁻¹⁰) -5 mmol (1.0 equivalent) was added. The reaction mixture was stirred, and the reaction mixture was subjected to HPLC analysis at various time intervals (30 minutes, 1 hour, 2 hours, 4 hours, and 12 hours). The reverse-phase HPLC conditions were as follows: Column Cosmosil 5C18-AR300 (Nacalai Tesque Co., Ltd.) 4.6 × 250 mm; Mobile phase A 0.1% TFA in H2O; Mobile phase B 0.1% TFA in CH3CN; Gradient elution 0-4 min at 65%B, 4-14 min at 65-95%B, 14-20 min at 95%B; Flow rate 1 mL / min; Fluorescence detection 360 / 450 nm; Injection volume 6 μL. The results are shown in Figure 8 (GSH) and Figure 9 (Acrolein).

[0129] As shown in Figure 8, coumarin-ABC (compound 7) did not react with glutathione (GSH), and coumarin-ABC (compound 7) remained unchanged even after 12 hours. In contrast, as shown in Figure 9, coumarin-ABC (compound 7) reacted rapidly with acrolein, and the release of 7-amino-4-methylcoumarin was detected in the mixture after only 30 minutes.

[0130] [Example 5] ATP assay (cytotoxicity assay) Three cell lines (MCF10A, A549, and HeLa S3) were seeded on a 96-well plate (2 × 10⁶). 4 Cells were placed in wells and allowed to adhere for 24 hours at 37°C. Then, each cell was subjected to the following three different conditions: (Condition 1) Cells were treated with 100 μL of MMC-ABC (compound 8) solution [0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 50 μM in medium (1% DMSO)] and incubated at 37°C and 5% CO2 for 48 hours. (Condition 2) The mixture was incubated with mitomycin C (MMC) solution in the same manner as in Condition 1. (Condition 3) Cells were treated with 1 mM N-acetylcysteine ​​(NAc-Cys) in culture medium and incubated at 37°C for 2 hours. Then, they were incubated with MMC-ABC (compound 8) solution in the same manner as in Condition 1. (Condition 4) The incubation was carried out in the same manner as in Condition 1 using a solution of 2-azido-1,3-diisopropyl-5-methylbenzene (compound 9).

[0131] After incubation, 100 μL of ATPlite 1-step reagent was added to the cells, and the fluorescence intensity was measured using a spectrofluorometer (SpectraMax iD3, Molecular Devices). The results are shown in Figure 10 [MCF10A (normal human mammary gland cells)] and Figure 11 [A549 (human lung adenocarcinoma cells) and HeLa S3 (human cervical cancer cells)].

[0132] As shown in Figures 10 and 11, the prodrug MMC-ABC (compound 8) released free MMC specifically in cancer cells only when it reacted with endogenous acrolein overexpressed in cancer cells. In contrast, MMC exhibited nonspecific cytotoxicity to both normal and cancer cells.

[0133] [Example 6] Animal experiment In this embodiment, all animal experiments were pre-approved by the Animal Ethics Committee. For all intratumoral injections and tumor weighings, mice were anesthetized with 2% isofran in oxygen at a flow rate of 2.5–3.0 L / min.

[0134] (Cell lines and reagents) A549 cells were obtained from a stock strain preserved in liquid nitrogen. These cells were cultured in DMEM medium (Dulbecco's modified Eagle medium) (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (FBS) (BioWest) and 1% penicillin-streptomycin (Gibco). The cells were then incubated at 37°C in a humidified atmosphere and 5% CO2.

[0135] (A549 tumor-carrying mouse xenograft model) In 6-week-old female nude mice BALB / cAJcl-nu / nu, 2.5–6.0 × 10¹⁴ units were found in 100 μL of 50% Matrigel in cooled, unreconstituted DMEM. 6 A549 xenograft tumors were established by subcutaneous injection of a cell suspension into the left and right shoulders, and tumor growth was observed. Mice were kept in an aerated chamber with controlled temperature, salinity, and sufficient food and water for 12 hours during the day and 12 hours at night. The tumors reached 400-600 mm. 3 After reaching a certain size, A549 tumor-carrying mice were used for cancer treatment.

[0136] (Intratumor (IT) administration) Mice were randomly divided into five groups and treated with either a prodrug or a control. Group 1 received 10 μL of vehicle solution injected into the tumor (n=10). Group 2 received 10 μL of compound 9 at 1.1 mg / kg (n=10). Group 3 received 10 μL of mitomycin C at 1.8 mg / kg (n=10). Group 4 received 10 μL of MMC-ABC (compound 8) at 3.1 mg / kg (n=6). Group 5 received 10 μL of MMC-phenylazide (compound 10) at 2.8 mg / kg (n=6).

[0137] MMC-phenylazide (compound 10) is a control compound prepared in the same manner as MMC-ABC (compound 8) synthesized in Production Example 4, except that 4-azido-methanol is used instead of (4-azido-3,5-diisopropylphenyl)-methanol (compound S4) as the starting material.

[0138] The administration was repeated 12 times, with the same dose given daily. The tumor volume and body weight of the mice were given by the formula: V=W 2 The tumor size was recorded daily using the formula ×L / 2 (where W and L represent the short and long diameters of the tumor, respectively). 3 At the point where the tumor volume was reached, the mice were sacrificed, and the survival rate was calculated using the Kaplan-Meier method. The results are shown in Figure 12 (tumor volume), Figure 13 (body weight), and Figure 14 (survival rate). In addition, the condition of the mice was photographed on the second day after intratumoral administration and on the fourth day after the end of administration. The respective photographs are shown in Figure 15.

[0139] As shown in Figures 12-15, mice treated with the vehicle or compound 9 had larger tumor sizes. On the other hand, in mice treated with mitomycin C and MMC-ABC (compound 8), these administrations were found to be effective in suppressing tumor growth. However, while no side effects were observed with the administration of the prodrug MMC-ABC (compound 8), mice treated with mitomycin C developed systemic inflammation. Furthermore, mice treated with MMC-phenylazide (compound 10) showed a slight reduction in tumor size compared to mice treated with the vehicle or compound 9, but the inhibitory effect of MMC-phenylazide (compound 10) on tumor growth was only slight compared to MMC-ABC (compound 8).

[0140] (Intravenous (IV) administration) Mice were randomly assigned to the following groups: Group 1 received 100 μL of vehicle solution intravenously (n=8). Group 2 received 100 μL of compound 9 at 1.2 mg / kg (n=8). Group 3 received 100 μL of mitomycin C at 1.8 mg / kg (n=9). Group 4 received 100 μL of MMC-ABC (compound 8) at 3.2 mg / kg (n=7). Group 5 received 100 μL of MMC-phenylazide (compound 10) at 2.8 mg / kg (n=8). Administration was performed once every 3 days for a total of 3 injections. Tumor volume and body weight of mice were given using the formula: V=W 2 The tumor size was recorded daily using the formula ×L / 2 (where W and L represent the short and long diameters of the tumor, respectively). 3 When the condition was reached, the mice were sacrificed, and the survival rate was calculated using the Kaplan-Meier method.

[0141] The tumor is 2000 mm 3 Mice treated after reaching a certain stage were sacrificed and perfused intracardiacly with 0.9% physiological saline and then 4% paraformaldehyde. Selected organs (liver, kidney, spleen, heart, lung, and tumor) were removed, immersed overnight in 4% paraformaldehyde, and then treated overnight with 15% and 30% sucrose, respectively, and stored at -80°C until used for hematoxylin and eosin (H&E) analysis.

[0142] (In vivo drug release study) A549 tumor-carrying mouse group (tumors of 1000-1500 mm) 3 Mitomycin C (3.6 mg / kg, n=2), MMC-ABC (compound 8) (6.2 mg / kg, n=2), or MMC-phenylazide (compound 10) (5.6 mg / kg, n=2) were injected intratumorally into mice that had reached a certain tumor size. Two hours after injection of the drug or prodrug, the mice were sacrificed. The tumors, kidneys, and urine of the treated mice were collected, dissolved, and extracted overnight with methanol at 4°C. The extracts were filtered, evaporated, and separated to obtain crude organ extracts, which were then analyzed by HPLC.

[0143] [Production Example 6] Synthesis of the prodrug PUR-ABC (compound S6) To synthesize compound S6, compound S5 was first synthesized using the following synthetic route. [ka]

[0144] (Production Example 6-1) Synthesis of 4-iodo-2,6-diisopropylaniline (compound S1) Iodine (4.3 g, 16.9 mmol, 1.2 equivalents) was added to a saturated aqueous solution of 2,6-diisopropylaniline (2.5 g, 14.1 mmol, 1.0 equivalent) in NaHCO3 and a 1:1 solution of Et2O (140 mL, [2,6-diisopropylaniline] = 0.10 M). After stirring at room temperature for 20 hours, Na2S2O3 was added and the mixture was stirred for a further 30 minutes. The resulting mixture was extracted with Et2O. The organic layer was washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography [n-hexane / siRNA (9:1~7:3)] to obtain the target product, 4-iodo-2,6-diisopropylaniline (compound S1), as a reddish-black oily compound (13.5 g, 96%). 1 H NMR (400 MHz, CDCl3, 25℃) δ7.27 (s, 2H), 3.70 (bs, 2H, NH2), 2.81 (hept, J =6.7 Hz, 2H), 1.21 (d,J = 6.8 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25℃) δ140.27, 135.08, 131.79, 81.12, 27.82, 22.21; ESI-HRMS m / z C 12 H 19 IN ([M+H] + The calculated value for ) was 304.0557, and the detected value was 304.0558.

[0145] (Production Example 6-2) Synthesis of ethyl 4-amino-3,5-diisopropylbenzoate (compound S2) Et3N (1.6 mL, 12 mmol, 2.5 equivalents) was added to an EtOH solution (30 mL, [S1] = 0.16 M) of compound S1, 4-iodo-2,6-diisopropylaniline (1.4 g, 4.7 mmol, 1.0 equivalent), and Pd(PPh3)4 (0.11 g, 0.094 mmol, 0.02 equivalents). The resulting solution was stirred under CO pressure (0.5 MPa) at 110°C for 28 hours. The resulting suspension was filtered by Celite, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified using silica gel column chromatography [n-hexane / siRNA (hexane only ~9:1)] to obtain the target product, 4-amino-3,5-diisopropylbenzoate ethyl (compound S2), as a yellow oily compound (0.51 g, 44%). 1 H MMR (400MHz, CDCl3, 25℃) δ 7.75 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.16 (bs, 2H, NH2), 2.88 (hept, J =6.9 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H), 1.29 (d, J = 6.8 Hz, 12H); 13 C NMR (100MHz, CDCl3, 25℃) δ167.64, 145.10, 131.37, 125.04, 119.77, 60.30, 27.97, 22.24, 14.55; ESI-HRMS m / z C 15 H 24 NO2([M+H] + The calculated value for ) was 250.1802, and the detected value was 250.1805.

[0146] (Production Example 6-3) Synthesis of 4-hydroxymethyl-2,6-diisopropylaniline (compound S3) A THF solution (30 mL, [S2] = 1.0 M) of compound S2 (513 mg, 2.1 mmol, 1.0 equivalent) was cooled to 0°C, and then DIBAL-H (6.2 mL, 6.2 mmol, 3.0 equivalents, 1 M solution in toluene) was added dropwise. The reaction temperature was raised to room temperature and stirred for 30 minutes. The reaction temperature was lowered to 0°C and excess DIBAL-H was quenched with MeOH. The resulting mixture was filtered by Celite while washing with MeOH, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified using silica gel column chromatography [n-hexane / siRNA (9:1~7:3)] to obtain the target product, 4-hydroxymethyl-2,6-diisopropylaniline (compound S3), as an orange oily compound (346 mg, 82%). 1 H NMR (400 MHz, CDCl3, 25℃) δ7.04 (s, 2H), 4.55 (s, 2H), 3.74 (bs, 2H, NH2), 2.92 (hept, J = 6.7 Hz, 2H), 1.27 (d, J = 6.8 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25℃) δ 140.06, 132.71, 130.89, 122.46, 66.09, 27.99, 22.44; ESI-HRMS m / z C 13 H 22 NO ([M+H] + The calculated value for ) was 208.1696, and the detected value was 208.1699.

[0147] (Production Example 6-4) Synthesis of (4-azido-3,5-diisopropylphenyl)-methanol (compound S4) A solution of compound S3 (346 mg, 1.7 mmol, 1.0 equivalent) in acetic acid and distilled water (2:1) (18 mL, [S3] = 0.94 M) was cooled to 0°C, then NaN3 (461 mg, 7.1 mmol, 4.2 equivalents) was added, followed immediately by NaNO2 (477 mg, 6.9 mmol, 4.1 equivalents). After stirring at 0°C for 2 hours, saturated NaHCO3 aqueous solution was added until the pH of the solution reached 7. The mixture was extracted with siRNA. The organic layer was washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified using silica gel column chromatography [n-hexane / siRNA (9:1~7:3)] to obtain the target product, (4-azido-3,5-diisopropylphenyl)-methanol (compound S4), as a yellow oily compound (260 mg, 67%). 1 H NMR (400 MHz, CDCl3, 25℃)δ7.13 (s,2H), 4.64 (s, 2H), 3.36 (p, J = 6.8 Hz, 2H), 1.27 (d J = 6.7 Hz, 12H); 13 C NMR (100MHz, CDCl3, 25℃) δ143.51, 139.31, 134.84, 122.79, 65.27, 28.90, 23.52; ESI-HRMS m / z C 13 H 20 The calculated value for N3O ([M+H]+) was 234.1601, and the detected value was 234.1605.

[0148] (Production Example 6-5) Synthesis of 4-azido-3,5-diisopropylbenzyl-(4-nitrophenyl)-carbonate (compound S5) Pyridine (181 μL, 2.2 mmol, 2 equivalents) was added to a THF solution (12 mL, [S4] = 0.09 M) of compound S4 (260 mg, 1.1 mmol, 1.0 equivalent) and 4-nitrophenyl chloroformate (270 mg, 1.3 mmol, 1.2 equivalents). The reaction mixture was stirred at room temperature under an argon atmosphere for 2 hours. The THF solvent was removed using a rotary evaporator, and the residue was separated using siRNA-H2O. The organic layer was washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was evaporated to dryness under reduced pressure. The residue was purified using silica gel column chromatography [n-hexane / siRNA (n-hexane only ~20:1)] to obtain the target product, 4-azido-3,5-diisopropylbenzyl-(4-nitrophenyl)-carbonate (compound S5) (426 mg, 96%). 1 H NMR (400 MHz, CDCl3, 25℃) δ8.27 (d, J = 9.1 Hz, 2H), 7.20 (s, 2H), 5.26 (s, 2H), 3.38 (p, J = 6.9 Hz), 1.29 (d, J = 6.9, 12H); 13 C NMR (100 MHz, CDCl3, 25℃) δ155.65, 152.50, 145.52, 143.82, 136.11, 132.62, 125.41, 124.63, 121.86, 71.07, 28.94, 23.53.

[0149] (Manufacturing Example 6-6) Synthesis of the prodrug PUR-ABC (compound S6) [ka]

[0150] To a DMF solution (500 μL, [S5] = 0.07 M) of compound S5 (13 mg, 0.033 mmol, 1.0 equiv), DIPEA (52 μL, 0.33 mmol, 10 equiv) was added and stirred well. Then, puromycin dihydrochloride (27 mg, 0.05 mmol, 1.5 equiv) was added. The reaction mixture was stirred at room temperature for 18 h. Toluene was added to the reaction mixture, and volatile substances were removed by a rotary evaporator. The residue was purified by silica gel column chromatography [CHCl3 / MeOH (50:1 - 20:1)] to obtain the target product, PUR-ABC (compound S6), as a yellow oily compound (24 mg, 98%). 1 H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.89 (s, 1H), 7.14 (d, J = 8.1 Hz, 2H), 7.08 (s, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.50 (d, J = 5.0 Hz, 1H), 5.59 (d, J = 7.8 Hz, 2H), 5.42 (d, J = 5.0 Hz, 1H), 5.07 (d, J = 12.0 Hz, 1H), 4.98 (d, J = 11.9 Hz, 1H), 4.76 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 6.8 Hz, 1H), 4.36 (q, J = 5.8 Hz, 1H), 4.04 (dt, J = 4.3, 2.0 Hz, 1H), 3.88 (d, J = 12.6 Hz, 1H), 3.75 (s, 3H), 3.33 (hept, J = 6.6 Hz, 4H), 3.11 (dd, J = 13.9, 6.3 Hz, 1H), 3.02 - 2.82 (m, 2H), 2.14 - 1.99 (m, 3H), 1.69 (s, 1H), 1.24 (d, J = 6.9 Hz, 12H), 0.87 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 172.17, 159.02, 155.09, 151.71, 148.84, 143.73, 137.80, 135.60, 134.54, 130.55, 128.55, 124.22, ESI-MS m / z C 36 H 47 N 10 O7([M+H] + The calculated value for ) was 731.3624, and the detected value was 731.3938.

[0151] [Manufacturing Example 7] Synthesis of the prodrug DOX-ABC (compound S7) [ka]

[0152] Et3N (57 μL, 0.40 mmol, 2.0 equivalents) was added to a DMF solution (2 mL, [S5] = 0.1 M) of compound S5 (80.2 mg, 0.20 mmol, 1.0 equivalent), doxorubicin hydrochloride (105 mg, 0.17 mmol, 0.9 equivalents), and molecular sieves (MS 4A). The reaction mixture was stirred under a nitrogen atmosphere for 20 hours. The resulting mixture was filtered by Celite while washing with ethyl acetate, and the filtrate was concentrated to dryness under reduced pressure. The residue was separated with ethyl acetate-H2O. The organic layer was washed with saturated brine, dried over Na2SO4, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified using silica gel column chromatography CHCl3 / MeOH (25:1) to obtain the target product, DOX-ABC (compound S7), as a red solid (120 mg, 87%). 1H NMR (400 MHz, CDCl3, 25℃) δ7.95 (d, J = 7.7 Hz, 1H), 7.74 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.7 Hz, 1H), 7.05 (s, 2H), 5.49 (bs, 1H, NH), 5.34-5.18 (m, 2H), 4.97 (s, 2H), 4.76 (d, J = 4.6 Hz, 2H), 4.54 (s, 1H), 4.14 (dd, J =14.7, 7.2 Hz, 1H), 4.05 (s, 3H), 3.93-3.82(m, 1H), 3.68 (d, J = 7.8 Hz, 1H), 3.30 (p, J = 6.9 Hz, 2H), 3.18 (d, J = 18.5 Hz, 1H), 3.12 (t, J =6.0 Hz, 1H), 2.85 (d, J =18.8 Hz, 1H), 2.33 (d, J = 14.6 Hz, 2H), 2.15 (dd, J =14.8, 4.1 Hz, 1H), 1.84 (ddd, J =27.1, 13.1, 4.5 Hz, 2H), 1.30 (d, J =6.5 Hz, 3H), 1.22 (d, J = 6.9 Hz, 12H); 13 C NMR (100 MHz, CDCl3, 25℃) δ213.96, 186.91, 186.51, 161.05, 156.22, 155.62, 155.55, 143.42, 135.84, 135.38, 135.25, 134.70, 133.64, 124.01, 120.71, 119.88, 118.57, 111.52, 111.52, 111.36, 100.86, 76.66, 69.81, 69.67, 67.41, 66.85, 65.62, 56.69, 47.15, 35.67, 33.95, 30.26, 28.88, 23.49, 16.92; ESI-HRMS m / z C 41 H 46 N4NaO 13 ([M+H)) + )についての calculated value is 825.2954, and the calculated value is 825.2954.

[0153] [Example 7] ATP assay (cytotoxicity assay) Three cell lines (MCF10A, A549, and HeLa S3) were seeded on a 96-well plate (2 × 10⁶). 4 Cells were placed in wells and allowed to adhere for 24 hours at 37°C. Then, each cell was subjected to the following two different conditions: (Condition 1) Cells were treated with 100 μL of DOX-ABC (compound 7) solution [0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, or 50 μM in medium (1% DMSO)] and incubated at 37°C and 5% CO2 for 48 hours. (Condition 2) The mixture was incubated with doxorubicin (DOX) solution in the same manner as in Condition 1.

[0154] After incubation, 100 μL of ATPlite 1-step reagent was added to the cells, and the fluorescence intensity was measured using a spectrofluorometer (SpectraMax iD3, Molecular Devices). The results are shown in Figure 16.

[0155] As shown in Figure 16, the prodrug DOX-ABC (compound 7) released free DOX specifically in cancer cells only when it reacted with endogenous acrolein, which is overexpressed in cancer cells. In contrast, DOX exhibited nonspecific cytotoxicity to both normal and cancer cells.

[0156] [Example 8] Animal experiment A xenograft model of A549 tumor-carrying mice was prepared in the same manner as in Example 6. The prepared mice were randomly divided into three groups and treated with either a prodrug or a control. Group 1 received 10 μL of compound 9 prepared in Preparation Example 5 as a control, injected into the tumor (n=10). Group 2 received 10 μL of doxorubicin, injected into the tumor (n=10). Group 3 received 10 μL of compound S7, injected into the tumor (n=10).

[0157] The same dose was administered a total of four times on days 0, 4, 7, and 11. The tumor volume of the mice was calculated using the formula: V=W2 It was recorded daily by W×L / 2 (where W and L represent the minor and major diameters of the tumor, respectively). When the tumor reached 2000 mm 3 the mice were sacrificed, and the survival rate was calculated using the Kaplan Meier method.

[0158] Photographs of the mice on the 26th day after intratumoral administration in the first group (control, compound 9), the 11th day after intratumoral administration in the second group (doxorubicin), and the 26th day after intratumoral administration in the third group (compound S7) are shown in Fig. 17.

[0159] Fig. 18 shows a graph indicating changes in tumor volume. Fig. 19 shows a graph indicating changes in the survival rate. The horizontal axis in Figs. 18 and 19 is the number of days since the start of intratumoral administration.

[0160] As shown in Figs. 17 to 19, the mice treated with compound 9 had a large tumor size. On the other hand, in the mice treated with doxorubicin and DOX-ABC (compound S7), it was found that these administrations were effective against tumor growth. However, the survival rate of the mice treated with doxorubicin decreased rapidly after the fourth intratumoral administration. The mice treated with DOX-ABC (compound S7) had a survival rate of 100% even after the fourth intratumoral administration.

[0161] 〔Example 9〕Animal experiment Similar to Example 6, an A549 tumor-bearing mouse xenograft model was prepared. The prepared mice were randomized and divided into two groups, and treated with a prodrug or a control. In the first group, 10 μL of compound 9 was injected intratumorally as a control (n = 10). In the second group, 10 μL of compound S6 was injected intratumorally (n = 10).

[0162] The administration was performed a total of 4 times at 0 day, 4 days, 7 days, and 11 days with the same volume. The tumor volume of the mice was recorded daily by the formula: V = W 2 ×L / 2 (where W and L represent the minor and major diameters of the tumor, respectively).

[0163] Figure 20 shows a graph indicating changes in tumor volume. The horizontal axis in Figure 20 represents the number of days since the start of intratumoral administration.

[0164] As shown in Figure 20, it was found that administration was effective against tumor growth in mice treated with PUR-ABC (Compound S6).

Industrial Applicability

[0165] The present invention can be used, for example, as a therapeutic agent for tumors and a diagnostic agent for the presence or absence of tumors, and can be used in life science research and medical applications and the like.

Claims

1. It includes an acrolein reaction site having the chemical structure represented by formula (1), and a release site having other chemical structures, The detachment site is bonded to the acrolein reaction site via a linker that can be cleaved by the reaction between the acrolein reaction site and acrolein. 【Chemistry 1】 (In equation (1), R1 and R2 independently refer to a C1-C5 alkyl group which may be substituted with a hydrogen atom, a halogen atom, or at least one halogen atom, wherein at least one of R1 and R2 is a C1-C5 alkyl group; R3 and R4 independently refer to a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group (wherein the hydrogen atoms constituting the alkyl group may be substituted by substituents selected from halogen atoms, hydroxyl groups, unsubstituted amino groups, alkylamino groups, and (hetero)arylamino groups); * indicates the linkage site with the linker. The linker is a linker having a chemical structure represented by formula (2), 【Chemistry 2】 (In equation (2), R independently refers to a hydrogen atom, a halogen atom, a hydroxyl group, a thiol group, an optionally substituted amino group, a C1-C5 alkoxy group, a C1-C5 alkylthio group, or a C1-C5 alkyl group (wherein the hydrogen atoms constituting the alkyl group may be substituted by substituents selected from halogen atoms, hydroxyl groups, unsubstituted amino groups, alkylamino groups, and (hetero)arylamino groups); * indicates the binding site with the acrolein reaction site. ※※ indicates the connection site with the detachment site. The aforementioned detachment site is a compound having a nitrogen atom, In formula (2), the aforementioned ** is a composite, which is a bonding site with the nitrogen atom contained in the detachment site.

2. The composite according to claim 1, wherein R in formula (2) independently refers to a hydrogen atom, a halogen atom, or a C1-C5 alkyl group (wherein the hydrogen atom constituting the alkyl group may have substituents selected from a halogen atom, a hydroxyl group, an unsubstituted amino group, an alkylamino group, and a (hetero)arylamino group).

3. The composite according to claim 1 or 2, wherein R1 and R2 in formula (1) independently refer to a C1-C5 alkyl group which may be substituted with at least one halogen atom.

4. The complex according to any one of claims 1 to 3, wherein the release site is an antitumor compound.

5. The complex according to any one of claims 1 to 4, wherein the detachment site detaches upon reaction with acrolein present around tumor cells.

6. A tumor treatment agent comprising the complex described in claim 4 or 5.

7. The complex according to any one of claims 1 to 5, or the therapeutic agent according to claim 6, is delivered to the target tissue requiring treatment. A drug delivery system that reacts acrolein with the complex or the therapeutic agent to detach the detachment site.

Images