Epigenetics-associated protein decomposition technology

Abstract

The purpose of the present invention is to provide molecules that induce, in cells, the decomposition of proteins associated with epigenetic modification, particularly enzymes (e.g. DNA methyltransferase) that catalyze methylation of nucleic acids.　More specifically, the purpose of the present invention is to provide a compound represented by formula (I) or a salt thereof, or a solvate or a hydrate of the same. [In formula (I), A is a double-stranded nucleic acid and includes, in one strand, at least one 5'-CpG-3' sequence in which the 5-position of cytosine (C) has been methylated, the C of the 3'-GpC-5' that is complementary to said 5'-CpG-3' may be a modified base for covalently bonding DNMT1 to A, L is a hydrocarbon that may include oxygen, sulfur, phosphorus, or nitrogen, and B is a ligand molecule of an E3 ubiquitin ligase.]

Description

Epigenetics-related protein degradation technology

[0001] The present invention relates to molecules that induce the degradation of proteins involved in the epigenetic modification of nucleic acids, and more specifically, to molecules that induce the degradation of enzymes that catalyze the methylation of nucleic acids (e.g., DNA methyltransferases) in cells.

[0002] Epigenetics regulates gene expression beyond the genomic DNA sequence information, and is involved in various biological phenomena, from development to viral infection control and carcinogenesis. Cytosine methylation, one of these mechanisms, is known to contribute to cancer progression by silencing tumor suppressor genes (e.g., p16, VHL, and RB) through excessive cytosine methylation at CpG sequences in their promoters (non-patent literature 1). Reversing this hypermethylation state with pharmaceuticals would enable the simultaneous expression of tumor suppressor genes that remain silenced by methylation even after cell proliferation, making it a promising cancer treatment strategy. Inhibition of DNA methyltransferases (DNMTs), enzymes that recognize genomic CpG sequences and catalyze methylation transfer, has been investigated as a potential cancer treatment strategy for reversing this hypermethylation state.

[0003] One inhibitor that has been put to practical use to date is 5-aza-2'-deoxycytidine (AzaC), a deoxycytidine analog (Non-Patent Document 2). AzaC exerts its therapeutic effect by incorporating into CpG sequences in place of natural cytosine during DNA replication and then covalently binding to DNMTs that recognize CpG sequences. However, AzaC's genomic incorporation efficiency is low, making it less effective against solid cancers. Furthermore, AzaC is incorporated into the genomic DNA of normal cells and blood cells, causing severe side effects. This creates a dilemma: despite its low efficacy, it is not possible to increase the dosage to avoid side effects.

[0004] Epigenetic proteins that use DNA as a substrate, such as DNMTs, lack small molecule binding pockets, and no effective ligands have been identified. Therefore, nucleic acid-based therapeutics are considered promising. Recently, several proteolysis-targeting chimeras (PROTACs) have been reported that target transcription factors (TFs) and RNA-binding proteins (RBPs), which lack ligand binding sites like epigenetic proteins (see Non-Patent Document 3). PROTACs utilize the ubiquitin-proteasome system to induce targeted protein degradation. The ubiquitin-proteasome system is a protein degradation system involving E1, E2, and E3 ligases that are widely present in cells. PROTAC molecules are bifunctional molecules that bind to a protein of interest (POI) (POI-binding molecule) and an E3 ligase (E3-binding molecule) via a linker. By binding to both the POI and E3 ligase, they bring the POI and E3 ligase into close proximity, resulting in the transfer of a ubiquitin marker for proteasomal degradation to the POI, inducing its degradation.

[0005] When designing PROTAC molecules, it is believed that the key is how stably they bind (capture) target proteins. Despite several previous studies, the development of clinically applicable PROTAC molecules has been difficult for epigenetics-related proteins due to the difficulty in capturing target proteins (Non-Patent Document 4). One reason for this is that nucleic acid-recognition proteins, including DNA epigenetics-related enzymes, lack pockets for the small molecule compounds that serve as ligands for conventional PROTACs. Furthermore, when nucleic acid molecules are used as ligands, PROTACs can be inactivated by modification of the nucleic acid ligand itself; therefore, it is extremely important to design ligands that can catalytically degrade epigenetics-related proteins.

[0006] Goll and Bestor, Annu. Rev. Biochem., 2005, 74:481-514. Christman, Oncogene, 2002, 21:5483-5495. Shih et al., Pharmaceutics, 2023, 15:765. Yan and Higgins, Biochim Biophys Acta. 2013, 1835:76-85.

[0007] In view of the above circumstances, an object of the present invention is to provide a molecule that induces the degradation of proteins involved in epigenetic modification, particularly enzymes that catalyze the methylation of nucleic acids (e.g., DNA methyltransferases) within cells.

[0008] The present inventors attempted to create a molecule that induces the degradation of the epigenetics-related protein DNMT1 (DNA (cytosine-5)-methyltransferase 1). Introducing the created molecule into cancer cells induced the degradation of DNMT1 present in the cells, resulting in a decrease in the viability of the cancer cells. The structure and mechanism of action of the molecule developed by the inventors are shown in Figure 1. The molecule developed by the inventors consists of a chimeric structure in which an artificial nucleic acid targeting an epigenetics-related protein (e.g., DNMT1) is linked to a ubiquitin ligase ligand molecule via a linker (hereinafter referred to as "EpiTAC (Epigenetics Targeting Chimera)" or "EpiTAC-DNMT1").

[0009] The artificial nucleic acid portion constituting EpiTAC-DNMT1 may be a double-stranded nucleic acid, with one strand containing a methylated cytosine (mC) and the other strand containing a chemically modified base for selective and strong binding of DNMT1. Specifically, the 5'-CpG-3' cytosine (C) in the artificial nucleic acid is methylated at the 5th position, and the complementary cytosine in 3'-GpC-5' is substituted with a modified base (e.g., 5-fluorocytosine, 2-hydroxypyrimidine, 5-azacytosine, etc.) to tightly bind DNMT1 (referred to as "POI" in Figure 1) to the artificial nucleic acid. DNMT1 has high selectivity for methylation of 5'-CpG-3' on hemimethylated DNA, and thus artificial nucleic acids with these characteristics can selectively bind (capture) DNMT1.

[0010] On the other hand, the ubiquitin ligase (E3 ubiquitin ligase) ligand molecule that constitutes EpiTAC-DNMT1 recruits the E3 ubiquitin ligase to the proximal region of DNMT1, promoting DNMT1 ubiquitination. The E3 ubiquitin ligase then recruits the ubiquitin-bound E2 ubiquitin-conjugating enzyme, recognizes the amino acid that serves as the substrate for the target protein to be ubiquitinated, and catalyzes the transfer of ubiquitin from the E2 to that amino acid. Ubiquitinated DNMT1 is degraded in the proteasome. As described above, the EpiTAC-DNMT1 created by the inventors is a groundbreaking molecule that promotes the degradation of overactive DNMT1 in cancer cells and other tissues, promoting the expression of tumor suppressor genes and other genes, thereby suppressing cancer cell proliferation and ultimately enabling the regression of cancer tissue.

[0011] That is, the present invention relates to the following (1) to (11): (1) A compound represented by formula (I) or a salt thereof, or a solvate or hydrate thereof. [In formula (I), A is a double-stranded nucleic acid, one strand of which contains at least one 5'-CpG-3' sequence in which the 5-position of cytosine (C) is methylated, and the C of 3'-GpC-5' complementary to the 5'-CpG-3' may be a modified base for binding DNMT1 to A, L is a hydrocarbon that may contain oxygen, sulfur, phosphorus, or nitrogen, and B is a ligand molecule for E3 ubiquitin ligase.] (2) The compound according to (1) above or a salt thereof, or a solvate or hydrate thereof, wherein the modified base is 5-fluorocytosine or 2-hydroxypyrimidine. (3) The compound or salt thereof, or a solvate or hydrate of any of the above (1) or (2), wherein A is a 16-bp double-stranded DNA consisting of the nucleic acid represented by SEQ ID NO: 1 and the nucleic acid represented by SEQ ID NO: 2, wherein the 9th base from the 5' side in SEQ ID NO: 1 is substituted with C or another modified base, and the 5th position of the 10th C from the 3' side in SEQ ID NO: 2 is methylated. (4) The compound or salt thereof, or a solvate or hydrate of any of the above (1) or (2), wherein A is a 20-bp double-stranded DNA consisting of the nucleic acid represented by SEQ ID NO: 3 and the nucleic acid represented by SEQ ID NO: 4, wherein the 11th base from the 5' side in SEQ ID NO: 3 is substituted with C or another modified base, and the 12th C from the 3' side in SEQ ID NO: 4 is methylated at the 5th position. (5) The compound or salt thereof, or a solvate or hydrate thereof according to (1), wherein B is a ligand molecule of von-Hippel-Lindau (VHL), a ligand molecule of Cereblon (CRBN), a ligand molecule of IAP (inhibitor of apoptosis protein), or a ligand molecule of MDM2. (6) The compound or salt thereof, or a solvate or hydrate thereof according to (5), wherein the ligand molecule of VHL is VH032.(7) The compound or salt thereof according to (1), or a solvate or hydrate thereof, wherein L is a hydrocarbon having 1 to 50 carbon atoms which may contain oxygen, sulfur, phosphorus, or nitrogen. (8) The compound or salt thereof according to (7), or a solvate or hydrate thereof, wherein L is a compound represented by formula (II) or formula (III). [In formula (II), n is an integer of 1 to 20, and in formula (III), n is an integer of 1 to 20.] (9) An agent for inducing intracellular DNMT1 degradation, comprising the compound or salt thereof according to (1), or a solvate or hydrate thereof. (10) The agent for inducing DNMT1 degradation according to (9), wherein the cells are cancer cells. (11) A medicament or pharmaceutical composition for treating cancer, comprising the agent for inducing DNMT1 degradation according to (10) as an active ingredient. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of the symbol.

[0012] The molecules of the present invention can suppress the excessive methylation of CpG sequences present in the promoter sequences of tumor suppressor genes, thereby activating the expression of tumor suppressor genes and inhibiting the oncogenic transformation of cells and the malignant progression of cancer.

[0013] Figure 1 shows an overview of the structure and mechanism of action of the compound (EpiTAC-DNMT1) of the present invention. Figure 2 shows an example of an "EpiTAC-DNMT1" of the present invention. Figure 3 shows an example of the double-stranded DNA sequence of "EpiTAC-DNMT1" of the present invention. Figure 4 shows the results of analyzing the DNMT1 degradation activity of EpiTAC of formula (V). Panel A shows the results of Western blotting using an anti-DNMT1 antibody (DNMT1) with a 16-bp nucleic acid molecule (16) and a 20-bp nucleic acid molecule (20). The ratio (%) of the band density detected by the anti-DNMT1 antibody to the band density detected by the anti-β-actin antibody (bactin) was calculated and used to determine the DNMT1 degradation activity of EpiTAC. Labels such as C2 indicate that an EpiTAC with an X n of 2 in formula (V) was used. Panel B shows a graph of the DNMT1 degradation activity (%) calculated based on the results of Panel A. Figure 5 shows the results of an investigation into the mechanism of DNMT1 degradation by EpiTAC. We examined the ability of EpiTAC to degrade DNMT1 in the presence of the proteasome inhibitor bortezomib. Figure 6 shows the results of an investigation into the function of the nucleic acid portion (decoy) of EpiTAC-DNMT1. Panel A shows the results of an investigation into whether the decoy portion has DNMT1 degradation activity. Panel B shows the results of an investigation into whether the decoy portion has the ability to bind to DNMTs. Figure 7 shows the results of an analysis of the DNMT1 degradation activity of EpiTAC of formula (IV). Panel A shows the results of Western blotting using an anti-DNMT1 antibody (DNMT1) with a 20-bp nucleic acid molecule. The ratio (%) of the band density detected by the anti-DNMT1 antibody to the band density detected by the anti-β-actin antibody (bactin) was calculated and used as the DNMT1 degradation activity of EpiTAC. The symbols C6 and P3 indicate that EpiTACs were used in which X in formula (IV) is an alkyl chain and n is 6, and X in formula (IV) is PEG and n is 3, respectively. "w / o ligand" represents the control results in which a molecule containing no E3 ubiquitin ligand was added. Panel B is a graph of DNMT1 degradation activity (%) calculated based on the results in Panel A.Figure 8 shows the results of an investigation into the effects of EpiTAC of formula (V) on cancer cells. MDA-MB-231 cells were transfected with EpiTAC and cell viability was examined. "Decoy" indicates transfection with only the nucleic acid portion of EpiTAC, and "20bp-C2" indicates transfection with EpiTAC in which the decoy portion is 20bp and X in formula (V) is 2. Figure 9 shows a schematic diagram of the structure and characteristics of modified bases contained in the double-stranded DNA of the decoy portion of EpiTAC. "C-EpiTAC" does not contain modified bases and remains as cytosine. Figure 10 shows the results of an investigation into the effects of F-EpiTAC (F), C-EpiTAC (C), and Z-EpiTAC (Z) shown in Figure 9 on the viability of cancer cells (MDA-MB-231 and MDA-MB-468).

[0014] Hereinafter, embodiments of the present invention will be described. The term "present embodiment" refers to all embodiments described in this specification unless otherwise specified. The first embodiment is a compound represented by the following formula (I), or a salt thereof, or a solvate or hydrate thereof: [In formula (I), A is a double-stranded nucleic acid, one of whose strands contains at least one 5'-CpG-3' sequence in which the 5th position of cytosine (C) is methylated, and the C of 3'-GpC-5' complementary to the 5'-CpG-3' may be a modified base for binding DNMT1 to A, L is a hydrocarbon that may contain oxygen, sulfur, phosphorus, or nitrogen, and B is a ligand molecule for E3 ubiquitin ligase.]

[0015] The compound represented by formula (I) can bind (sometimes referred to as "capture" herein) to DNMT1 via the double-stranded nucleic acid portion A in cells, and B binds to an E3 ubiquitin ligase, bringing the E3 ubiquitin ligase into proximity with DNMT1. The E3 ubiquitin ligase cooperates with an E1 ubiquitin-activating enzyme and an E2 ubiquitin-conjugating enzyme to transfer ubiquitin to DNMT1. The ubiquitinated DNMT1 is then degraded by the proteasome. The compound represented by formula (I) with the above functions, i.e., the aforementioned "EpiTAC-DNMT1," can suppress methylation of the promoter region of tumor suppressor genes, which are known to be hypermethylated in cells, particularly cancer cells, thereby promoting the expression of tumor suppressor gene products and suppressing cancer cell proliferation.

[0016] Figure 2 shows an example of a compound of formula (I) (this is merely an example and is not limited to the compound shown in Figure 2). A corresponds to the artificial nucleic acid in Figure 2. A may have a hairpin structure at the end as shown in Figure 2, or it may be a nucleic acid without a hairpin structure. A must have at least one 5'-CpG-3' sequence in either strand, and multiple sequences may be present. Furthermore, the 5'-CpG-3' sequence is methylated at the 5th position of the cytosine (C) (denoted as "mC" in Figure 2), and the C in the 3'-GpC-5' sequence complementary to the 5'-CpG-3' sequence in the other strand may remain as C or may be substituted with a modified base (denoted as "Y" in Figure 2) that allows DNMT1 to bind to A. The binding between DNMT1 and A may be irreversible or reversible, with reversible being preferable. Furthermore, the modified base is not particularly limited, but examples include 5-azacytosine, 5-fluorocytosine, 2-hydroxypyrimidine, and 5,6-dihydro-5-azacytosine, as shown in Figure 2. From the viewpoint of stability, 2-hydroxypyrimidine or 5-fluorocytosine is preferred. In particular, modified bases such as 2-hydroxypyrimidine are preferred because they do not undergo methylation and reversibly bind to DNMT1, allowing them to be reused after DNMT1 degradation. The use of such modified bases allows for the reuse of EpiTAC, enabling catalytic induction of target protein degradation. This allows for high efficacy with low dosages and frequent administration, which is expected to lead to reduced side effects and reduced medical costs.

[0017] The length of A is not particularly limited, but may be, for example, 12 bp or more, and the upper limit of the length is also not particularly limited, but may be, for example, 100 bp or less. Those skilled in the art can appropriately determine the length of A depending on the intended use, for example, by referring to Berkyurek et al., J Biol Chem 289 379-386 2014.

[0018] As used herein, "nucleic acid" includes not only DNA and RNA, but also nucleic acid analogs in which DNA or RNA has been chemically modified. The sugars contained in nucleic acids may be ribose, deoxyribose, or chemically modified versions of these. The bases may be adenine, guanine, thymine, cytosine, uracil, or inosine, and may further be chemically modified versions of these, such as 7-deazaadenine, 7-deazaguanine, and hypoxanthine. Nucleic acid analogs are well known in the art and may be selected by those skilled in the art. Examples of such analogs include phosphorothioates, in which one oxygen atom in the phosphodiester bond of a nucleic acid is replaced with sulfur; locked nucleic acids (LNAs), in which the oxygen atom at the 2' position of RNA is bridged with a methylene between the oxygen atom at the 4' position; and monophosphorylated nucleic acid analogs.

[0019] More specific examples of the sequence of A include a 16-bp double-stranded DNA consisting of the sequences shown in SEQ ID NOs: 1 and 2 in Figure 3 (Example 1: in SEQ ID NO: 1, the 9th base from the 5' side is substituted with C or another modified base, and in SEQ ID NO: 2, the 5th position of the C at the 10th position from the 3' side is methylated), and a 20-bp double-stranded DNA consisting of the sequences shown in SEQ ID NOs: 3 and 4 (Example 2: in SEQ ID NO: 3, the 11th base from the 5' side is substituted with C or another modified base, and in SEQ ID NO: 4, the 5th position of the C at the 12th position from the 3' side is methylated). However, these are merely examples and are not intended to be limiting. The double-stranded nucleic acid of A can be easily prepared using a nucleic acid synthesizer and a nucleic acid synthesis method well known to those skilled in the art (e.g., the phosphoramidite method). The bond between A and L may be at either the 3' or 5' end of A, and an element (e.g., an organic molecule) necessary for the bond with L may be included. Such elements may include azide groups, hydroxyl groups, maleimide groups, carboxyl groups, cyclic alkynes, amino groups, nitrile groups, thiol groups, and the like.

[0020] In formula (I), B is a ligand molecule for E3 ubiquitin ligase, i.e., a molecule that binds to E3 ubiquitin ligase. In Figure 2, it corresponds to "VH032 (CAS number: 1448189-80-7)." The E3 ubiquitin ligase in this embodiment is not particularly limited and includes all E3 ubiquitin ligases known to those skilled in the art. For example, in addition to von-Hippel-Lindau (VHL), which is the target of VH032, E3 ubiquitin ligases that are targeted in the prior art PROTAC can be used (see Non-Patent Document 3), such as Cereblon (CRBN), IAP (inhibitor of apoptosis protein), and MDM2.Ligand molecules that bind to these E3 ubiquitin ligases include the aforementioned VH032 (CAS number: 1448189-80-7) and its derivatives (VHL ligands), as well as VH-298 (CAS number: 2097381-85-4) and its derivatives (VHL ligands), (S,R,S)-AHPC (monohydrochloride) (CAS number: 1448189-80-7) and its derivatives (VHL ligands), pomalidomide (CAS number: 19171-19-8) and its derivatives ( Cereblon ligand), Thalidomide-5-OH (CAS number: 64567-60-8) and its derivatives (Cereblon ligand), Iberdomide (CAS number: 1323403-33-3) and its derivatives (Cereblon ligand), CC-885 (CAS number: 1010100-07-8) and its derivatives (Cereblon ligand), Lenalidomide (CAS number: 191732-72-6) and its derivatives (Cereblon ligand), cIAP1 Examples include ligand 1 (CAS No.: 2095244-42-9) and its derivatives (IAP ligands), E3 ligase ligand 9 (CAS No.: 87304-15-2) and its derivatives (IAP ligands), E3 ligase ligand 13 (CAS No.: 2701565-75-3) and its derivatives (IAP ligands), and nutlin carboxylic acid (CAS No.: 2249750-27-2) and its derivatives (MDM2 ligands). B may contain an element (e.g., an organic molecule) necessary for binding to L. Such an element may include an azide group, a hydroxyl group, a maleimide group, a carboxyl group, a cyclic alkyne, an amino group, a nitrile group, a thiol group, etc.

[0021] In formula (I), L is a hydrocarbon that may contain oxygen, sulfur, phosphorus, or nitrogen, and is preferably water-soluble and biocompatible. L plays the role of a linker that connects A and B. There is no particular limitation on the number of carbon atoms contained in L, but it is, for example, 1 to 50, preferably about 5 to 20. L may contain an element (such as an organic molecule) necessary for bonding with A and B. Such an element may include an azide group, a hydroxyl group, a maleimide group, a carboxyl group, a cyclic alkyne, an amino group, a nitrile group, a thiol group, etc.

[0022] L may contain, as a main component, for example, a hydrocarbon chain represented by the following formula (II) or polyethylene glycol (PEG) represented by the following formula (III). The length of L is not particularly limited, and those skilled in the art can determine the optimal length through appropriate preliminary experiments depending on the structure of the artificial nucleic acid and the type of E3 ligase ligand molecule. When the main component of L is an alkyl chain of formula (II), for example, n in formula (II) is an integer of 1 to 20, preferably an integer of 5 to 15. When the main component of L is a PEG chain of formula (III), for example, n in formula (III) is an integer of 1 to 20, preferably an integer of 2 to 10.

[0023] In formula (I), the method for linking the A moiety, the L moiety, and the B moiety can be easily selected by a person skilled in the art, and for example, a click reaction, a phosphoramidite method, or the like can be used as appropriate.

[0024] The second embodiment is an intracellular DNMT1 degradation inducer (also referred to as the "DNMT1 degradation inducer of this embodiment") comprising a compound represented by the following formula (I) or a salt thereof, or a solvate or hydrate thereof: [In formula (I), A is a double-stranded nucleic acid, one strand of which contains at least one 5'-CpG-3' sequence in which the 5th position of cytosine (C) is methylated; the C in the 3'-GpC-5' complementary to the 5'-CpG-3' may be a modified base for covalently linking DNMT1 to A; L is a hydrocarbon that may contain oxygen, sulfur, phosphorus, or nitrogen; and B is a ligand molecule for E3 ubiquitin ligase.] The DNMT1 degradation inducer according to this embodiment contains the compound according to the first embodiment as an active ingredient and functions particularly effectively in cancer cells. Therefore, the "cell" in the second embodiment is preferably a cancer cell. For implementing the second embodiment, see also the description of the first embodiment.

[0025] The third embodiment is a medicament and pharmaceutical composition for cancer treatment that contains a DNMT1 degradation inducer according to this embodiment as an active ingredient, i.e., a medicament and pharmaceutical composition for cancer treatment that contains a compound of formula (I) as an active ingredient (hereinafter also referred to as "medicament, etc. according to this embodiment"). As described above, the compound represented by formula (I) induces the degradation of DNMT1 present in cancer cells and suppresses hypermethylation in tumor suppressor gene promoter regions, thereby suppressing cancer cell proliferation. Therefore, a medicament, etc. containing a compound represented by formula (I) as an active ingredient is thought to induce the regression of cancer tissue and can be used for cancer treatment. The medicament, etc. according to this embodiment may be administered as the active ingredient (compound represented by formula (I)) itself, but generally may be administered in the form of a composition that contains one or more active ingredients as well as one or more formulation additives.

[0026] Dosage forms of pharmaceuticals and the like according to this embodiment include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, eye drops, and the like. These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents at the time of use. Tablets and granules may also be coated by known methods. Injections are prepared by dissolving the active ingredient in water, but may also be dissolved in physiological saline or glucose solution as needed, and buffers and preservatives may also be added.

[0027] Preparations for oral or parenteral administration are provided in any dosage form. Examples of dosage forms include oral pharmaceuticals in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or solutions, and parenteral pharmaceuticals in the form of injections for intravenous, intramuscular, or subcutaneous administration, drip infusions, transdermal absorbents, transmucosal absorbents, nasal drops, inhalants, suppositories, eye drops, ointments, creams, and the like. Injections and drip infusions can also be prepared in a powdered dosage form, such as a lyophilized form, and dissolved in an appropriate aqueous medium, such as physiological saline, before use.

[0028] Those skilled in the art can appropriately select the type of formulation additives used in the production of the pharmaceuticals, etc. according to this embodiment, the ratio of the formulation additives to the active ingredient, the production method, etc. As the formulation additives, inorganic or organic substances, or solid or liquid substances can be used, and they can generally be blended in an amount of 1 to 90% by weight relative to the weight of the active ingredient. Specific examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0029] To prepare solid formulations for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, and anhydrous silicic acid to form a powder, or, if necessary, with binders such as sucrose, hydroxypropyl cellulose, and polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose and calcium carboxymethylcellulose, followed by wet or dry granulation to form granules. To prepare tablets, these powders and granules may be compressed as is, or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets may be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate and methacrylic acid-methyl methacrylate polymer to form enteric-coated formulations, or coated with ethylcellulose, carnauba wax, and hydrogenated oil to form sustained-release formulations. To prepare capsules, the powder or granules may be filled into hard capsules, or the active ingredient may be dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like and then coated with a gelatin membrane to form soft capsules.

[0030] To prepare parenteral preparations for injection, the active ingredient can be dissolved in distilled water for injection, optionally with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, or an isotonic agent such as sodium chloride or glucose, and then sterile filtered and filled into ampoules. Alternatively, mannitol, dextrin, cyclodextrin, gelatin, or the like can be added, followed by vacuum freeze-drying to produce an injectable preparation that can be dissolved immediately. Alternatively, the active ingredient can be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, or the like to produce an emulsion for injection.

[0031] Ointments and creams can be produced by kneading and mixing the active ingredient with a base and additives. Oil-based ointments can be produced, for example, by warming and melting an oil-based base such as oils, waxes, or hydrocarbons such as paraffin, adding the active ingredient, mixing to dissolve or disperse the active ingredient, and kneading until the entire mixture is homogeneous. Water-soluble ointments can be produced, for example, by warming and melting a water-soluble base such as macrogol, adding the active ingredient, and kneading until the entire mixture is homogeneous. Creams can be produced, for example, by adding the active ingredient to an oil phase containing petrolatum, higher alcohol, or the like, either as is or with the addition of an emulsifier or other additive, or to an aqueous phase containing purified water or with the addition of an emulsifier or other additive, heating each phase, and emulsifying the oil and aqueous phases by stirring until the entire mixture is homogeneous.

[0032] To prepare a parenteral rectal preparation, the active ingredient may be dissolved by wetting together with a suppository base such as cacao butter, tri-, di-, or monoglycerides of fatty acids, or polyethylene glycol, and then poured into a mold and cooled; alternatively, the active ingredient may be dissolved in polyethylene glycol, soybean oil, or the like, and then coated with a gelatin film.

[0033] The dosage and frequency of administration of the pharmaceuticals and the like according to this embodiment are not particularly limited and can be appropriately selected at the discretion of a physician depending on conditions such as the progression or worsening of the cancer being treated, the purpose of treatment, the type of disease, and the patient's weight and age. Generally, the daily oral dose for adults is approximately 0.01 to 1000 mg (weight of active ingredient), and can be administered once a day, in divided doses, or every few days. When used as an injection, it is desirable to administer a daily dose of 0.001 to 100 mg (weight of active ingredient) to adults, either continuously or intermittently.

[0034] The pharmaceuticals of this embodiment may be formulated as sustained-release formulations, such as implants and microencapsulated delivery systems, using carriers that can prevent immediate elimination from the body. Such carriers include biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Liposomes can be prepared as lipid compositions containing, but not limited to, phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanol (PEG-PE), filtered through a filter with an appropriate pore size to obtain a suitable size, and then purified by reverse-phase evaporation.

[0035] The pharmaceuticals and compositions according to this embodiment may be provided in the form of a kit together with instructions for administration, etc. The pharmaceuticals included in the kit are supplied in containers made of materials that maintain the activity of the components of the therapeutic agent for a long period of time, do not adsorb to the inside of the container, and do not alter the components. For example, a sealed glass ampoule may contain a buffer sealed in the presence of a neutral, non-reactive gas such as nitrogen gas. The instructions for use included with the kit may be printed on paper or stored on an electromagnetically readable medium and provided to the user, or may be an electronic file available via the Internet, etc.

[0036] The fourth embodiment is a method for treating cancer, comprising administering a medicament, etc. according to this embodiment (i.e., a medicament, etc. containing the compound of formula (I) above as an active ingredient) to a subject (a subject for preventing or treating a disease). The subject to which the medicament, etc. according to this embodiment is administered may be any animal classified as a mammal, and is not particularly limited, and examples thereof include, in addition to humans, animals belonging to primates, pet animals such as dogs, cats, rabbits, and ferrets, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred subject to administer is humans.

[0037] When this specification is translated into English and includes the singular words "a," "an," and "the," it is intended to include not only the singular but also the plural, unless the context clearly indicates otherwise. Furthermore, in this specification, "about" or "approximately" refers to a numerical range of ±10%. The present invention will be further explained below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention.

[0038] I. Synthesis of Compounds and Experimental Methods I-1. Synthesis of EpiTAC (1) The synthesis of EpiTAC of the following formula (IV) is explained below. The case where X is an alkyl chain with n=12 and PEG with n=4 is explained. I-1-1. Synthesis of Nucleic Acids (A- in Formula (I)) All oligonucleotides used in this example were synthesized using an automated DNA / RNA synthesizer. The synthesized DNA sequences are shown in Table 1. The synthesized oligonucleotides are hairpin-type double-stranded DNAs in which the "TTT" portion forms a loop structure (see also Formula (IV)). In the sequence of SEQ ID NO: 5, the 9th C (underlined) from the 5' side is methylated at the 5th position, and the 26th C (underlined) is substituted with 5-fluorocytosine. In the sequence of SEQ ID NO: 6, the 11th C (underlined) from the 5' side is methylated at the 5th position, and the 33rd C (underlined) is substituted with 5-fluorocytosine.

[0039] I-1-2. Synthesis of E3 ubiquitin ligase ligand molecules containing a linker (the -LB portion in Formula (I)). VH-032 (compound 1; a ligand for E3 ubiquitin ligase (VHL)) was synthesized according to the procedure previously described (Galdeano, C. et al. J. Med. Chem. 2014, 57, 8657-8663). The VH032 phosphoramidite used in the synthesis of EpiTAC was synthesized according to the procedure previously described (Jing, L. et al. J. Am. Chem. Soc. 2021, 143, 8902-8910; Jingwei, S. et al. Adv. Sci. 2021, 8, 2102555).

[0040] (1) Synthesis of Compound 3 when X is an alkyl chain Compound 1 (1.0 equiv.) was dissolved in DCM / DMF (1:1), and compound 2 (1.0 equiv.), TEA (3.0 equiv.), and HATU (1.1 equiv.) were added. The mixture was stirred at room temperature overnight. The reaction solution was diluted with DCM and washed with saturated aqueous NaHCO3. The organic phase was concentrated and purified by flash chromatography to give compound 3.

[0041] Synthesis of compound 4 Compound 3 (1.0 equiv.) was dissolved in DCM and cooled to 0 °C, followed by the addition of TEA (1.5 equiv.) and DMAP (0.01 equiv.). The mixture was stirred, and AcO (1.5 equiv.) was added slowly. The reaction was stirred at 0 °C for 1 h. The reaction solution was washed with water, and the organic phase was dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography to give compound 4.

[0042] Synthesis of compound 5 Compound 4 (1.0 equiv.) was dissolved in THF and TBAF (1 M in THF, 2.0 equiv.) was added. The mixture was stirred at room temperature overnight. The solvent was removed, and the residue was purified by flash chromatography to give compound 5.

[0043] Synthesis of compound 6 Compound 5 (1.0 equivalent) was dissolved in anhydrous DCM, and DIPEA (2.0 equivalents) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.5 equivalents) were added. The mixture was stirred at room temperature for 1 hour. The solvent was removed, and the residue was purified by flash chromatography to give compound 6. The NMR data for compound 6 are shown in the table below.

[0044] (2) Synthesis of compound 8 when X is PEG Compound 1 (1.0 equiv.) was dissolved in DCM and DMF (1:1), and compound 7 (1.0 equiv.), TEA (3.0 equiv.), and HATU (1.1 equiv.) were added. The mixture was stirred at room temperature overnight. The reaction solution was diluted with DCM and washed with saturated aqueous NaHCO3. The organic phase was concentrated and purified by flash chromatography to give compound 8.

[0045] Synthesis of compound 9 Compound 8 (1.0 equiv.) was dissolved in DCM and cooled to 0 °C. TEA (1.5 equiv.) and DMAP (0.01 equiv.) were then added. The mixture was stirred, and AcO (1.5 equiv.) was slowly added. The reaction was stirred at 0 °C for 1 h. The reaction solution was washed with water, and the organic phase was dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography to give compound 9.

[0046] Synthesis of compound 10 Compound 9 (1.0 equiv.) was dissolved in THF and TBAF (1 M in THF, 2.0 equiv.) was added. The mixture was stirred at room temperature overnight. The solvent was removed, and the residue was purified by flash chromatography to give compound 10.

[0047] Synthesis of compound 11 Compound 10 (1.0 equiv.) was dissolved in anhydrous DCM, and DIPEA (2.0 equiv.) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.5 equiv.) were added. The mixture was stirred at room temperature for 1 hour. The solvent was removed, and the residue was purified by flash chromatography to give compound 11. The NMR data for compound 11 are shown in the table below.

[0048] I-1-3. Synthesis of EpiTAC of Formula (IV) (ALB in Formula (I)) DNA was synthesized using a DNA / RNA synthesizer as described above and loaded onto a column. A 0.1 M solution of compound 6 or compound 11 in ACN and 0.1 M 5-ethylthio-1H-tetrazole was passed through the column and allowed to react for 1 hour. Then, 0.02 M iodine in THF / pyridine / water was passed through the column. After the reaction was complete, the DNA was deprotected using 28% aqueous ammonia, and the target product (EpiTAC of Formula (IV)) was purified by HPLC.

[0049] I-2. Synthesis of EpiTAC (2) The synthesis of EpiTAC of the following formula (V) is described below. Here, we explain the case where X is an alkyl chain (n=2), but the synthesis can be performed in the same way when X is PEG.

[0050] [Correction based on Rule 91 05.03.2025] I-2-1. Synthesis of Nucleic Acids (Part A- in Formula (I)) Similar to I-1-1 above, synthesis was performed using an automated DNA / RNA synthesizer. The synthesized DNA sequences are shown in Table 2. However, in the sequence of SEQ ID NO: 5, the 9th C (underlined) from the 5' side is methylated at the 5th position, and the 26th C (underlined) is either left as cytosine or substituted with 5-fluorocytosine or 2-hydroxypyrimidine. In addition, in the sequence of SEQ ID NO: 6, the 11th C (underlined) from the 5' side is methylated at the 5th position, and the 33rd C (underlined) is either left as cytosine or substituted with 5-fluorocytosine or 2-hydroxypyrimidine.

[0051] I-2-2. Synthesis of E3 ubiquitin ligase ligand molecules containing a linker (the -LB portion in Formula (I)). VH-032 (compound 1; a ligand for E3 ubiquitin ligase (VHL)) was synthesized according to the procedure previously described (Galdeano, C. et al. J. Med. Chem. 2014, 57, 8657-8663). The VH032 phosphoramidite used in the synthesis of EpiTAC was synthesized according to the procedure previously described (Jing, L. et al. J. Am. Chem. Soc. 2021, 143, 8902-8910; Jingwei, S. et al. Adv. Sci. 2021, 8, 2102555).

[0052] Synthesis of compound 12 Compound 1 (1.0 equiv.) was dissolved in DCM and DMF (1:1), and N-(tert-butoxycarbonyl)-β-alanine (1.0 equiv.), TEA (3.0 equiv.), and EDCI (1.1 equiv.) were added. The mixture was stirred at room temperature for 16 h. The reaction solution was diluted with DCM and washed with HO. The organic phase was concentrated and purified by flash chromatography to give compound 12.

[0053] Synthesis of compound 13 Compound 12 was dissolved in ethyl acetate and HCl (1M in ethyl acetate) was added. The mixture was stirred at room temperature for 3 hours and filtered to give compound 13.

[0054] Synthesis of compound 15 Compound 13 (1.0 equiv.) was dissolved in DMF, and compound 14 (1.0 equiv.) and TEA (1.0 equiv.) were added. The mixture was stirred at room temperature for 30 minutes. The resulting mixture was purified by preparative HPLC (10%-100% acetonitrile / 0.08% NH4HCO3 in H2O). All product fractions were collected and extracted with ethyl acetate (3 × 20 mL). The organic layers were combined, washed with water (20 mL) and brine (20 mL), dried over Na2SO4, and concentrated to give compound 15 (ESI-MS m / z = 700.3 [M + Na] + ).

[0055] I-2-3. Synthesis of EpiTAC of Formula (V) (ALB in Formula (I)) DNA synthesized using a DNA / RNA synthesizer was reacted with NHS-azide (20 equivalents) at room temperature for 4 hours and purified by HPLC. After purification, the azide-containing DNA was conjugated with compound 15 (1.5 equivalents) in PBS buffer for 15 hours, and the target product (EpiTAC of Formula (V)) was subsequently purified by HPLC.

[0056] I-3. Western Blotting. EpiTAC was transfected into cells (MDA-MB-231 or MDA-MB-468, both cell lines derived from human breast adenocarcinoma. MDA-MB-468 is a cell line that highly expresses DNMT1) cultured in DMEM medium. After 12 hours, the medium was removed and the cells were washed three times with PBS. RIPA buffer was added to the cells, and the cells were detached using a scraper on ice. The collected cells were incubated on ice for 15 minutes and centrifuged at 15,000 × g for 10 minutes to collect the supernatant. The total protein content in the supernatant was measured by BCA assay, and 10 μg of the lysate was loaded onto an 8% SDS-PAGE gel. Proteins separated by SDS-PAGE were transferred to a PVDF membrane using the iBlot system (Thermo Scientific Fisher). DNMT1 on the membrane was detected and quantified using the iBind system (Thermo Scientific Fisher).

[0057] I-4. Cell viability assay Cells (MDA-MB-231) were seeded into a 48-well plate (200 μL per well, 2 × 10 per well). 4Cells were cultured until they reached approximately 80% confluence within 24 hours. The medium was changed from DMEM to Opti-MEM, and cells were transfected with 0.5 mL of EpiTAC (0.5 μM) using Xtremegene HP (Roche). After 6 hours, the medium was changed to standard growth medium (DMEM), and the cells were incubated for an additional 18 hours. Cell viability was measured using PrestoBlue Cell Viability Reagent (Invitrogen). Fluorescence in each well was measured using a multiwell Cytation 5 plate reader (BioTek Instruments) with excitation at 560 nm and emission at 590 nm.

[0058] II. Results II-1. Degradation of DNMT1 by EpiTAC of Formula (V) We investigated the effect of EpiTAC on DNMT1 degradation in cells. EpiTAC of Formula (V) was used, with the nucleic acid moiety being 16 or 20 bp long, the modified base being 5-fluorocytosine, X being an alkyl chain, and n = 2, 4, 6, 8, or 10. MDA-MB-231 cells were transfected with EpiTAC (1 μM) and cultured for 6 hours. The medium was replaced with DMEM medium (10% FBS) and then cultured for another 6 hours. Cell lysates were then subjected to Western blotting using an anti-DNMT1 antibody, and the amount of detected DNMT1 was quantified. Figure 4A shows the results of Western blotting, and Figure 4B shows the ratio of the DNMT1 band intensity to the β-actin band intensity. We confirmed that EpiTAC of Formula (V) had DNMT1 degradation activity regardless of whether the nucleic acid moiety was 16 or 20 bp long. Regarding linker length, there appears to be an optimal value depending on the type of linker. In this experiment, when the nucleic acid length was 16 bp, n was 4 to 10, and when the length was 20 bp, n was 2 to 6, resulting in the highest degradation efficiency of DNMT1.

[0059] Next, we investigated the mechanism of DNMT1 degradation by EpiTAC. MDA-MB-231 cells were transfected with EpiTAC (20 bp nucleic acid fragment, 5-fluorocytosine modified base, X alkyl chain; n = 2) (1 μM) in the presence or absence of the proteasome inhibitor bortezomib (1 μM). DNMT1 levels were quantified by Western blotting as described above (Figure 5). In the absence of bortezomib, EpiTAC reduced DNMT1 levels by 58%. However, when proteasome function was inhibited by bortezomib, EpiTAC reduced DNMT1 levels by 92%, similar to those in the absence of bortezomib and EpiTAC. These results suggest that the proteasome is involved in EpiTAC-induced DNMT1 degradation.

[0060] We further investigated the effect of the nucleic acid portion of EpiTAC alone (hereafter referred to as "Decoy") on DNMT1. MDA-MB-231 cells were transfected with Decoy (1 μM) and cultured for 12 hours. The amount of DNMT1 in the cells was quantified. The amount of DNMT1 remained unchanged regardless of the presence or absence of Decoy, indicating that the nucleic acid portion of EpiTAC does not have the ability to degrade DNMT1 (Figure 6A). Next, we investigated whether the nucleic acid portion of EpiTAC could bind (capture) DNMT1. MDA-MB-231 cells were transfected with a biotin-conjugated Decoy ("Biotin-Decoy") and cultured for 12 hours. A pull-down assay using streptavidin beads was performed. In the presence of Biotin-Decoy, DNMT1 was recovered in the streptavidin bead fraction, confirming the binding of the Decoy, the nucleic acid portion of EpiTAC, to DNMT1 (Figure 6B).

[0061] II-2. Degradation of DNMT1 by EpiTAC of Formula (IV). MDA-MB-231 cells were transfected with EpiTAC of Formula (IV) (n = 6, 8, 10, 12, or 14 when X is an alkyl chain; n = 3, 4, or 5 when X is PEG) and cultured for 6 hours. The medium was replaced with DMEM (10% FBS) and then cultured for another 6 hours. Western blotting of cell lysates was then performed using an anti-DNMT1 antibody to quantify the amount of detected DNMT1. Figure 7A shows the results of Western blotting, and Figure 7B shows the ratio of the DNMT1 band intensity to the β-actin band intensity. EpiTAC of Formula (IV), whether the linker moiety was an alkyl chain or PEG, was confirmed to degrade DNMT1 in cells, similar to EpiTAC of Formula (V). There appeared to be an optimal linker length depending on the type of linker. In this experiment, the decomposition effect of DNMT1 was high when n=12 in the case of alkyl chains and when n=4 in the case of PEG.

[0062] II-3. Effect of EpiTAC on Cancer Cells We investigated the effect of EpiTAC on cancer cell viability by degrading DNMT1 in cancer cells. Cancer cells (MDA-MB-231) were transfected with EpiTAC of formula (V) (20 bp nucleic acid portion, 5-fluorocytosine modified base, X alkyl chain, n = 2). After 2 hours, the medium was changed. After 22 hours of culture, cell viability was measured. Cell viability was found to be reduced to approximately 50% compared to that without EpiTAC (Figure 8; 20bp-C2). Although viability was slightly reduced when Decoy (nucleic acid portion only) was added, the addition of EpiTAC significantly reduced cell viability compared to that without Decoy (Figure 8). These results demonstrate that EpiTAC (V), a compound of formula (I), induces the degradation of DNMT1 in cancer cells and reduces cancer cell viability.

[0063] Next, we investigated the effects of each EpiTAC on DNMT1 degradation and cancer cell viability using EpiTACs of formula (V), whose nucleic acid moiety is the sequence shown in SEQ ID NO: 6, where the 33rd C (underlined) is 5-fluorocytosine ("F-EpiTAC" in Figure 9), cytosine (no substitution, "C-EpiTAC" in Figure 9), or 2-hydroxypyrimidine ("Z-EpiTAC" in Figure 9). When 5-fluorocytosine or cytosine is included in the nucleic acid moiety, DNMT1 binds to the nucleic acid moiety and methylates the 5-position of the 5-fluorocytosine or cytosine. Since the 5-position remains methylated even after DNMT1 degradation, the F-EpiTAC and C-EpiTAC used to induce DNMT1 degradation are not reused. On the other hand, when the nucleic acid portion contains 2-hydroxypyrimidine, even if DNMT1 binds to the nucleic acid portion, the 5-position of the 2-hydroxypyrimidine is not methylated. Therefore, after DNMT1 is degraded, Z-EpiTAC is recycled and is thought to be able to catalytically induce the degradation of DNMT1.

[0064] Therefore, we investigated the ability of F-EpiTAC, C-EpiTAC, and Z-EpiTAC to induce DNMT1 degradation and their effects on cancer cell viability. Although F-EpiTAC, C-EpiTAC, and Z-EpiTAC all had the ability to induce DNMT1 degradation, Z-EpiTAC exhibited the highest activity (Figure 10A). These results suggest that Z-EpiTAC indeed catalytically induced DNMT1 degradation.

[0065] Next, we examined the effects of EpiTAC on the cancer cell lines MDA-MB-231 and MDA-MB-468 cells. Both MDA-MB-231 and MDA-MB-468 cells are derived from human breast adenocarcinoma, but MDA-MB-468 cells highly express DNMT1. F-EpiTAC, C-EpiTAC, and Z-EpiTAC all reduced cell viability, but Z-EpiTAC had the highest cancer cell killing ability, as well as the ability to degrade DNMT1 (Figure 10B and C). Furthermore, when comparing the cell killing ability of each EpiTAC on MDA-MB-231 and MDA-MB-468 cells, the addition of 0.25 μM of each EpiTAC had a more pronounced effect on MDA-MB-468 cells, which highly express DNMT1. As mentioned above, MDA-MB-468 cells highly express DNMT1 and are therefore thought to have stronger characteristics as cancer cells. Therefore, the EpiTAC of the present invention, which exerts a higher killing ability on such cells, is suggested to have a higher cell-killing ability against cancer cells than normal cells.

[0066] The compounds of the present invention induce the degradation of DNMT1 in cells and can suppress hypermethylation of the promoter region of tumor suppressor genes in cancer cells. Therefore, the present invention is expected to be useful in the medical field, such as cancer treatment.

Claims

1. A compound represented by the following formula (I) or a salt thereof, or a solvate or hydrate thereof: [In formula (I), A is a double-stranded nucleic acid, one of whose strands contains at least one 5'-CpG-3' sequence in which the 5th position of cytosine (C) is methylated, and the C of 3'-GpC-5' complementary to the 5'-CpG-3' may be a modified base for binding DNMT1 to A, L is a hydrocarbon that may contain oxygen, sulfur, phosphorus, or nitrogen, and B is a ligand molecule for E3 ubiquitin ligase.] 2. The compound according to claim 1, or a salt thereof, or a solvate or hydrate thereof, wherein the modified base is 5-fluorocytosine or 2-hydroxypyrimidine.

3. The compound or salt thereof, or a solvate or hydrate thereof according to claim 1 or 2, wherein A is a 16-bp double-stranded DNA consisting of a nucleic acid represented by SEQ ID NO: 1 and a nucleic acid represented by SEQ ID NO: 2, in which the 9th base from the 5' side in SEQ ID NO: 1 is substituted with C or another modified base, and the 5th position of the 10th C from the 3' side in SEQ ID NO: 2 is methylated.

4. The compound or salt thereof, or a solvate or hydrate thereof according to claim 1 or 2, wherein A is a 20 bp double-stranded DNA consisting of a nucleic acid represented by SEQ ID NO: 3 and a nucleic acid represented by SEQ ID NO: 4, in which the 11th base from the 5' side in SEQ ID NO: 3 is substituted with C or another modified base, and the 5th position of the 12th C from the 3' side in SEQ ID NO: 4 is methylated.

5. The compound or salt thereof, or a solvate or hydrate thereof according to claim 1, wherein B is a ligand molecule of von-Hippel-Lindau (VHL), a ligand molecule of Cereblon (CRBN), a ligand molecule of IAP (inhibitor of apoptosis protein), or a ligand molecule of MDM2.

6. The compound according to claim 5, or a salt thereof, or a solvate or hydrate thereof, wherein the VHL ligand molecule is VH032.

7. The compound according to claim 1, or a salt thereof, or a solvate or hydrate thereof, wherein L is a hydrocarbon having 1 to 50 carbon atoms which may contain oxygen, sulfur, phosphorus or nitrogen.

8. The compound according to claim 7, wherein L is a compound represented by the following formula (II) or formula (III), or a salt thereof, or a solvate or hydrate thereof: [In formula (II), n is an integer of 1 to 20] [In formula (III), n is an integer of 1 to 20] 9. An agent for inducing intracellular DNMT1 degradation, comprising the compound according to claim 1 or a salt thereof, or a solvate or hydrate thereof.

10. The DNMT1 degradation inducer described in claim 9, wherein the cells are cancer cells.

11. A medicine and pharmaceutical composition for treating cancer, comprising the DNMT1 degradation inducer described in claim 10 as an active ingredient.

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