Novel autotack chimeric compound and composition containing the same for preventing, ameliorating or treating diseases caused by targeted protein degradation

Abstract

To provide novel AUTOTAC compounds, pharmaceutical or food compositions comprising AUTOTAC compounds as effective ingredients for removing disease-inducing proteins, and biochemical screening methods and techniques targeting and degrading intracellular proteins.SOLUTION: The invention relates to a novel AUTOTAC chimeric compound in which a new p62 ligand and a target-binding ligand are connected by a linker, a stereoisomer, hydrate, solvate or prodrug thereof, and a pharmaceutical or food composition comprising the same as an active ingredient for preventing or treating diseases by degrading the target protein. They can target specific proteins to adjust their concentrations, and can also deliver drugs and other low molecular weight compounds to lysosomes. The AUTOTAC chimeric compound according to the invention can be usefully used as a pharmaceutical composition for preventing, ameliorating or treating various diseases by selectively eliminating specific proteins.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a novel chimeric compound, more specifically, to a chimeric compound in which a p62 ligand and a target-binding ligand are linked by a linker, and to a pharmaceutical or food composition containing the same for preventing or treating diseases caused by targeted protein degradation. [Background technology]

[0002] The N-end rule pathway is a proteolytic system that uses the N-terminus of specific proteins as a degradation signal. N-end rule degradation signals include type 1 N-terminal basic residues such as arginine (Nt-Arg), lysine (Nt-Lys), and histidine (Nt-His), and type 2 hydrophobic residues such as phenylalanine (Nt-Phe), leucine (Nt-Leu), tryptophan (Nt-Trp), tylosin (Nt-Tyr), and isoleucine (Nt-Ile). These N-terminal residues are bound by ligands (referred to as N-ligands) of specific recognition elements (N-recognins). The present inventors were the first to discover or clone the previously known N-recognins, i.e., UBR1, UBR2, UBR4, and UBR5, and previously demonstrated that they contain the UBR box as a substrate recognition domain (Tasaki, T. et al., Mol Cell Biol 25, 7120-36 (2005): Non-Patent Document 1). They also previously demonstrated that the UBR box recognizes substrates by binding type-1 N-end rule ligands (Nt-Arg, Nt-Lys, Nt-His), such as N-terminal Arg residues, and then attaches ubiquitin chains to the substrates. We also found that UBR1 and UBR2 also contain N-domains, which play an important role in binding to type-2 N-end rule ligands (Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, and Nt-Ile) (Sriram, SM, Kim, BY & Kwon, YT, Nat Rev Mol Cell Biol 12, 735-47 (2011): Non-Patent Document 2). The ubiquitinated substrates generated by the binding of N-recognins to N-end rule ligands are delivered to the proteasome and degraded into short peptides.In this process, specific N-terminal residues (Nt-Arg, Nt-His, Nt-Lys, Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, Nt-Leu) are essential determinants for binding because they provide most of the hydrogen bonds required for N-recognin to target N-end rule substrates (Sriram, SM & Kwon, YT, Nat Struct Mol Biol 17, 1164-5 (2010): Non-Patent Document 3).

[0003] Intracellular protein degradation is primarily carried out by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system. Generally, the UPS regulates the intracellular concentration of regulatory factors with normal folding or degrades proteins that have become denatured and no longer function. Substrates are directly or indirectly recognized and ubiquitinated by an estimated 500-1,000 E3 ligases. They then unfold into polypeptide helices and are degraded by the proteasome (Ji and Kwon, Mol Cells 40, 441-449 (2017)). In normal cells, the ubiquitin-proteasome system process is smooth, but disease-related proteins become misfolded proteins, or aggregates formed by the accumulation of misfolded proteins prevent the proteasome from functioning properly, or proteasome function declines with aging, or reprogramming of protein transcription and translation prevents the smooth degradation of disease-related proteins (Ciechanover, A. & Kwon, YT, Exp Mol Med 47, e147 (2015): Non-Patent Document 5). As a representative example, the major pathogenic proteins in proteinopathy (Alzheimer's disease, Huntington's disease, Parkinson's disease, human mad cow disease, Lou Gehrig's disease / amyotrophic lateral sclerosis, α1-antitrypsin deficiency, keratopathy, type 2 diabetes, etc.) are denatured, ubiquitinated, and accumulated, and these excess protein wastes are converted back into clots (Aguzzi and O'Connor, Nat Rev Drug Discov 9, 237-48 (2010): Non-Patent Document 6). Such specific mutant proteins have a strong tendency to be transformed into clots and are therefore not degraded by the proteasome described above. This is because the proteasome has a narrow internal diameter of approximately 13 angstroms, which requires the unfolding of denatured proteins, and once the proteins have coagulated, they cannot be unfolded.As another representative example, cancer cells are known to increase the transcription and translation of oncoproteins while suppressing their degradation through intracellular transcription and translation reprogramming (Xiong et al., J Cell Physiol 234, 14031-14039 (2019): Non-Patent Document 7). Additionally, subunit proteins and transmembrane proteins that form complexes are also subject to degradation by the ubiquitin-proteasome system.

[0004] Autophagy, along with the UPS, is a major intracellular protein degradation system. Autophagy is an essential proteolytic process that maintains cellular homeostasis and genetic stability by degrading aging or dysfunctional organelles, damaged or improperly folded proteins, and their aggregates (Ji and Kwon, Mol Cells 40, 441-449 (2017): Non-Patent Document 4). In particular, when pathogenic proteins and their aggregates accumulate in the cytoplasm, they become cytotoxic and must be degraded by autophagy. Autophagy can be broadly divided into macroautophagy, microautophagy, and chaperone-mediated autophagy. Depending on the purpose of degrading intracellular substrates, autophagy can be further divided into non-selective (bulk) autophagy and selective autophagy (Dikic, I. & Elazar, Z., Nat Rev Mol Cell Biol 19, 349-364 (2018): Non-Patent Document 8). Selective autophagy and chaperone-mediated autophagy selectively degrade dysfunctional intracellular organelles and unnecessary proteins. The development of novel therapeutic strategies for diseases caused by the accumulation of malignant pathogenic proteins and dysfunctional organelles through the induction of selective autophagy is currently building a new paradigm. The p62 / SQSTM1 / Sequestosome-1 protein is important for the initiation and delivery of autophagosome contents, which are mediators of selective autophagy. During this process, the p62 protein binds to pathogenic proteins and their aggregates and delivers them to the autophagosome.When delivering pathogenic proteins to autophagosomes, p62 undergoes self-oligomerization as a key process (Dikic, I. & Elazar, Z., Nat Rev Mol Cell Biol 19, 349-364 (2018): Non-Patent Document 8). At this time, the pathogenic proteins become concentrated together, reducing their volume and facilitating their degradation by autophagy. The PB1 domain mediates p62 self-oligomerization, but its regulatory mechanism is not well understood. The denatured protein-p62 complex delivered to autophagosomes is degraded by lysosomal enzymes when the autophagosomes superimpose on lysosomes.

[0005] Through the aforementioned mechanisms, autophagy is important for maintaining cellular homeostasis through the intracellular fluctuation of damaged proteins and organelles. Impaired autophagy leads to the accumulation and coagulation of misfolded proteins, which can lead to proteinopathy and cancer. Research into activating non-selective autophagy to treat these diseases is actively underway (Ciechanover, A. & Kwon, YT, Exp Mol Med 47, e147 (2015): Non-Patent Document 5). The regulator that normally suppresses non-selective autophagy is mTOR, and the most widely used method of activating autophagy is the use of mTOR inhibitors (Jung, CH, Ro, SH, Cao, J., Otto, NM & Kim, DH, FEBS Lett 584, 1287-95 (2010): Non-Patent Document 9). Specifically, rapamycin was used to remove amyloid beta (Ab) and tau (tau) in an APP-overexpressing animal model of AD while simultaneously improving cognition (Caccamo, A., Majumder, S., Richardson, A., Strong, R. & Oddo, S., J Biol Chem 285, 13107-20 (2010): Non-Patent Document 10), to remove tau in an AD animal model overexpressing tau (Rodriguez-Navarro, JA et al., Neurobiol Dis 39, 423-38 (2010): Non-Patent Document 11), and to remove overexpressed mutant alpha-synuclein protein aggregates in a PD mouse model (Webb, JL, Ravikumar, B., Atkins, J., Skepper, JN & Rubinsztein, DC, J Biol Chem 278, 25009-13: Non-Patent Document 12). It was confirmed that the use of a rapamycin analogue, CCI-779, effectively removed huntingtin aggregates in the HD mice, and also improved animal behavior and cognition (Ravikumar, B., Duden, R. & Rubinsztein, D.C., Hum Mol Genet 11, 1107-17 (2002): Non-Patent Document 13).However, despite the fact that mTOR plays a crucial role in a wide variety of intracellular pathways, including NF-kB, and therefore exhibits excellent activity in removing denatured protein aggregates in proteinopathy, these non-selective autophagy activators that target mTOR have limitations in their use as therapeutic agents. Furthermore, there are no technologies or therapeutic agents that target disease-causing proteins in autophagosomes.

[0006] According to the central dogma, genetic information stored in DNA is transcribed into RNA and translated into proteins to regulate cellular functions. Targeted cleavage of DNA is possible using gene editing technologies such as CRISPR, while targeted degradation of RNA is possible using siRNA. However, targeted degradation techniques for proteins are relatively limited. If disease-causing proteins could be targeted for degradation, they could be used as a platform technology for new drug development in the pharmaceutical industry. PROTAC (PROteolysis Targeting Chimera) is a technology developed for the targeted degradation of intracellular proteins. PROTAC uses a chimeric compound consisting of a ligand that recognizes the target protein and a ligand that recognizes the E3 ubiquitin enzyme. (An and Fu, EBioMedicine 36, 553-562 (2018): Non-Patent Document 14). When a target binder binds to a disease-causing protein and brings it into close proximity with an E3, the E3 recognizes it as a substrate, ubiquitinates it, and induces its degradation by the proteasome. Because the current paradigm for disease treatments relies on protein enzyme inhibition, the development of novel therapeutics for proteins that cannot be targeted by existing enzyme inhibition methods is crucial. From this perspective, Protac offers an attractive new therapeutic development method by enabling the selective degradation of proteins that cannot be targeted by existing enzyme inhibition methods via the ubiquitin-proteasome system. However, because Protac induces proteasome degradation only through the use of ligands that recognize E3 ubiquitin enzymes, degradation of target proteins that are denatured to form aggregates, complexes, or membrane-bound structures is difficult (Bondeson et al., Cell Chem Biol 25, 78-87.e5 (2019): Non-Patent Document 15). Furthermore, Protac is unable to degrade intracellular organelles such as the endoplasmic reticulum and mitochondria, or pathogenic microorganisms such as viruses and bacteria.Therefore, there is a need to develop methods to target and selectively induce autophagy to remove pathogenic proteins, organelles, and aggregates.

[0007] To regulate cellular functions, methods such as editing DNA or degrading RNA to indirectly control protein activity and concentration are widely used. While RNA can be targeted for degradation using siRNA, siRNA has poor cell permeability and must be delivered into cells using transfection reagents, making the process complicated and expensive. Furthermore, if the target protein is stable, it is difficult to reduce protein concentration by degrading RNA. Therefore, there is a need to develop methods and materials that can directly degrade specific proteins, i.e., protein degraders. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Tasaki, T. et al., Mol Cell Biol 25, 7120-36 (2005) [Non-patent document 2] Sriram, SM, Kim, BY& Kwon, YT, Nat Rev Mol Cell Biol 12, 735-47(2011) [Non-patent document 3] Sriram, SM & Kwon, YT, Nat Struct Mol Biol 17, 1164-5(2010) [Non-patent document 4] Ji and Kwon, Mol Cells 40, 441-449(2017) [Non-Patent Document 5] Ciechanover, A. & Kwon, Y.T., Exp Mol Med 47, e147(2015) [Non-patent document 6] Aguzzi and O’Connor, Nat Rev Drug Discov 9, 237-48 (2010)

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

[0009] Traditional drugs, including small molecule synthetic compounds, antibodies, proteins, and peptides, exert their therapeutic effects by binding to the active site of specific proteins and inhibiting the activity of disease-causing proteins. This traditional drug development method has several limitations. First, high drug concentrations are required, which can lead to side effects. The drug-protein bond is not a stable covalent bond and can be dissociated. That is, a drug exerts its therapeutic effect when it binds to a target protein, but loses its efficacy when it dissociates from the target protein. Therefore, to maintain efficacy, high drug concentrations must be maintained throughout the body. However, increasing drug concentrations to achieve efficacy can lead to unexpected binding to other proteins, which can result in side effects. Second, the number of drug target proteins is limited. To date, the U.S. Food and Drug Administration (FDA) has approved approximately 400 drug target proteins. Of these, over 90% are enzymes, transmitter proteins, channel / membrane proteins, etc. These drug target proteins have active and binding sites, making them relatively easy to identify using traditional drug development methods. However, approximately 3,000 disease-related proteins are estimated, and currently approved drugs target only approximately 13% of these. Therefore, a paradigm shift in drug development is necessary. Targeted protein degradation technology offers the following advantages over existing therapeutics. First, it can degrade transcription factors, proteins involved in protein-binding signaling, and proteins that accumulate through aggregation, such as tau, thereby enabling the regulation of undruggable proteins that are difficult to target with existing drugs. Second, it overcomes the phenomenon of reduced drug efficacy due to overexpression or overactivation of target proteins, resistance, or activation of other signaling pathways. Third, targeted protein degraders can be reused after degrading the target protein, allowing for lower doses and reducing side effects.Fourth, since a certain amount of time is required for the target protein to be degraded and reproduced, the administration cycle can be increased, making it more economical.

[0010] To address the above-mentioned problems of existing therapeutic agents, the present invention aims to provide a novel p62 ligand compound that induces p62 protein activation and oligomerization, and a novel AUTOTAC (autophagy-targeting chimera) compound in which a target-binding ligand is linked via a linker (Figure 1).

[0011] Another object of the present invention is to provide a pharmaceutical or food composition that contains the autotack compound as an active ingredient and removes disease-causing proteins.

[0012] Another object of the present invention is to provide biochemical screening methods and techniques for targeting intracellular proteins for degradation. [Means for solving the problem]

[0013] To achieve the above objectives, the present invention provides a novel autotack chimeric compound in which a ligand that binds to the ZZ domain of p62 and a target-binding ligand are linked by a linker.

[0014] The present invention also provides methods using the AutoTack chimeric compounds for (1) inducing p62 self-oligomerization and constitutive activation, (2) increasing the binding of p62 to LC3, (3) delivering p62 and a target protein to an autophagosome, (4) activating autophagosome biogenesis, (5) targeting a target protein for autophagy, (6) deactivating a target protein, and (7) degrading a target protein by lysosomes.

[0015] The present invention also provides a pharmaceutical composition or health functional food containing the AutoTac chimeric compound as an active ingredient for preventing or treating diseases. Preferably, the diseases include not only cancer or degenerative brain diseases, but also other diseases such as rare and intractable diseases that can be treated by targeting and degrading specific proteins.

[0016] The core technology of this invention is to (1) link the target protein to p62 using an AutoTAC agent, (2) induce self-oligomerization of p62, (3) biologically inactivate the target protein by forming a complex with p62, (4) then deliver the target protein-p62 complex to an autophagy membrane such as a phagophore, and (5) degrade the target protein-p62 complex in the lysosome.

[0017] The drug action mechanism and core technology of the present invention are summarized in [Figure 1].

[0018] Specifically, as shown in Figure 1, the AutoTac chimeric compound consists of a target-binding ligand (TBL) and a p62ZZ domain ligand, i.e., an autophagy-targeting ligand (ATL), linked by a linker. The target ligand binds to the target protein, and the autophagy-targeting ligand binds to the p62ZZ domain, an autophagy receptor, and then oligomerizes. p62 then binds to LC3, which protrudes from the autophagosome membrane, targeting it for autophagy, leading to degradation of the target protein in the lysosome. In this study, we aimed to activate p62 using a small molecule ligand that binds to the p62ZZ domain to effectively remove the target protein. While Protac compounds exist that utilize the ubiquitin proteasome system (UPS) for targeted protein degradation, no small molecule compounds have been reported that utilize autophagy to degrade target proteins. Unlike Protac, autophagy can degrade proteins that Protac cannot, such as denatured protein aggregates, membrane-bound proteins, and complex subunits. It can also degrade intracellular structures (inflammasomes, stress granules, etc.), organelles (endoplasmic reticulum, mitochondria, peroxisomes, etc.), and pathogens that have invaded cells (viruses, bacteria, etc.). This targeted degradation can be expected to have preventive and therapeutic effects on various diseases.

[0019] The p62 protein is important in the selective autophagy mechanism, initiating the formation of autophagosomes, a mediator, and delivering their contents, i.e., target proteins. The novel AutoTAC chimeric compound according to the present invention was observed to induce p62 self-oligomerization by activating p62. Furthermore, this self-oligomerization was confirmed to deliver target proteins to autophagosomes, where they are then degraded in lysosomes.

[0020] The AutoTAC chimeric compound according to the present invention can not only induce intracellular autophagy but also induce the autophagosome targeting of target proteins, enabling the selective degradation of proteins that cannot be targeted by existing enzyme inhibition methods through the autophagy mechanism, thereby providing a novel therapeutic agent. [Effects of the Invention]

[0021] The AutoTack chimeric compound of the present invention comprises a target-binding ligand (TBL) and an autophagy-targeting ligand (ATL), which binds to the ZZ domain of p62 protein, linked by a linker. The autophagy-targeting ligand activates autophagy, transporting target proteins to autophagosomes for degradation in lysosomes.

[0022] The present invention is a platform technology that can degrade a desired target protein by linking the ligand of the protein to be degraded to an autophagy targeting ligand-linker, and is therefore applicable as a preventive, ameliorative, and therapeutic agent for various diseases. [Brief explanation of the drawings]

[0023] [Figure 1] This is a schematic diagram showing the structure of the novel AutoTack chimeric compound according to the present invention, and how it binds to p62 protein and simultaneously binds to target proteins, organelles, and aggregates, which are then delivered to autophagosomes, which are intermediates in macroautophagy, via p62 protein, and ultimately degraded by lysosomes. [Figure 2a]Immunoblotting analysis shows that AutoTac compounds induce p62 protein oligomerization and high-molecular-weight aggregation. Treatment with the compounds increases p62 protein oligomerization and high-molecular-weight aggregation. Immunoblotting analysis is shown as a representative graph from at least three independent experiments. [Figure 2b] Immunoblotting analysis shows that AutoTac compounds induce p62 protein oligomerization and high-molecular-weight aggregation. Treatment with the compounds increases p62 protein oligomerization and high-molecular-weight aggregation. Immunoblotting analysis is shown as a representative graph from at least three independent experiments. [Figure 2c] Immunoblotting analysis shows that AutoTac compounds induce p62 protein oligomerization and high-molecular-weight aggregation. Treatment with the compounds increases p62 protein oligomerization and high-molecular-weight aggregation. Immunoblotting analysis is shown as a representative graph from at least three independent experiments. [Figure 3] This shows the effect of the AutoTack chimeric compound of the present invention on the degradation of the corresponding target protein using immunoblotting. It shows that the amount of the target protein is reduced by treatment with the compound. The immunoblotting is shown as a representative graph from at least three independent experiments. [Figure 4a] These immunoblotting results demonstrate that the mechanism by which the AutoTack chimeric compound of the present invention degrades the corresponding target protein is mediated by the autophagy-lysosome pathway. Treatment with the compound reduces the amount of the target protein, and treatment with hydroxychloroquine (HCQ), an inhibitor of the autophagy-lysosome pathway, increases the amount of the target protein again. The immunoblotting results are shown as a representative graph from at least three independent experiments. [Figure 4b]These immunoblotting results demonstrate that the mechanism by which the AutoTack chimeric compound of the present invention degrades the corresponding target protein is mediated by the autophagy-lysosome pathway. Treatment with the compound reduces the amount of the target protein, and treatment with hydroxychloroquine (HCQ), an inhibitor of the autophagy-lysosome pathway, increases the amount of the target protein again. The immunoblotting results are shown as a representative graph from at least three independent experiments. [Figure 5a] These results, using immunoblotting, demonstrate that the target protein degradation efficacy of the AutoTack chimeric compound according to the present invention is superior to that of p62 ligand alone or target-binding ligands. Treatment with the AutoTack chimeric compound shows a greater reduction in target protein levels than treatment with p62 ligand or target protein ligand. Immunoblotting is shown as a representative graph from at least three independent experiments. [Figure 5b] These results, using immunoblotting, demonstrate that the target protein degradation efficacy of the AutoTack chimeric compound according to the present invention is superior to that of p62 ligand alone or target-binding ligands. Treatment with the AutoTack chimeric compound shows a greater reduction in target protein levels than treatment with p62 ligand or target protein ligand. Immunoblotting is shown as a representative graph from at least three independent experiments. [Figure 5c] These results, using immunoblotting, demonstrate that the target protein degradation efficacy of the AutoTack chimeric compound according to the present invention is superior to that of p62 ligand alone or target-binding ligands. Treatment with the AutoTack chimeric compound shows a greater reduction in target protein levels than treatment with p62 ligand or target protein ligand. Immunoblotting is shown as a representative graph from at least three independent experiments. [Figure 6a]Immunofluorescence staining was used to demonstrate the efficacy of the AutoTAC chimeric compound of the present invention in inducing both the target protein and p62 protein into autophagy. After compound treatment, the intracellular puncta and coexistence of the target protein and p62 protein of the AutoTAC compound were gradually increased. [Figure 6b] Immunofluorescence staining was used to demonstrate the efficacy of the AutoTAC chimeric compound of the present invention in inducing both the target protein and p62 protein into autophagy. After compound treatment, the intracellular puncta and coexistence of the target protein and p62 protein of the AutoTAC compound were gradually increased. [Figure 6c] Immunofluorescence staining was used to demonstrate the efficacy of the AutoTAC chimeric compound of the present invention in inducing both the target protein and p62 protein into autophagy. After compound treatment, the intracellular puncta and coexistence of the target protein and p62 protein of the AutoTAC compound were gradually increased. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in detail below.

[0025] The present invention relates to a chimeric compound comprising a p62 ligand (autophagy targeting ligand, ATL), a target binding ligand (TBL), and a linker, and a method for using the chimeric compound to transfer target proteins, organelles, and aggregates to autophagosomes, a mediator of macroautophagy, and for degradation in lysosomes, together with p62. The present invention also relates to a method for preventing, ameliorating, or treating disease-causing proteins, organelles, and aggregates by using the chimeric compound to transfer and degrade disease-causing proteins, organelles, and aggregates through autophagy, and a composition for activating autophagy for disease-related pathogenic proteins, organelles, and aggregates, or for preventing, ameliorating, or treating the disease, which comprises the chimeric compound (hereinafter referred to as the "chimeric compound").

[0026] The inventors have confirmed that by using a p62 ligand, such a p62 ligand can activate autophagy and effectively deliver target proteins, organelles, and aggregates to autophagosomes for removal. When combined with a ligand capable of binding to the pathogenic protein, this compound exhibits excellent pathogenic protein and aggregate removal capabilities. Unlike conventional Protac compounds, it eliminates the need for linker length optimization to form a ternary complex (target protein-linker-E3 ligase ligand) for pathogenic protein folding, E3 ligase selectivity, and effective degradation. The novel chimeric compound developed by the inventors, in which a p62 ligand and a target-binding ligand that binds to a target protein are linked by a linker, has been named AUTOTAC (AUTOphagy TArgeting Chimera).

[0027] A feature of the present invention is a technology that enables the degradation of a desired protein by linking the ligand of the protein to be degraded to a linker linked to a p62 ligand. In other words, a chimeric compound in which a p62 ligand and a target-binding ligand are linked by a linker is a bifunctional small molecule. The target protein to be degraded binds to the targeting ligand near p62, which is involved in protein autophagy, forming a structure that facilitates the degradation of the target protein. The gist of the invention is that by linking various disease-related proteins that one wishes to prevent or treat to p62, one can degrade the target protein in question, thereby achieving the desired therapeutic efficacy.

[0028] In a preferred embodiment, the chimeric compound according to the present invention, comprising a p62 ligand, a target-binding ligand, and a linker, is in the form of a chimeric compound in which the p62 ligand and the target-binding ligand are linked by a linker, and more preferably has the structure of the following Chemical Formula 1: It is possible. [ka]

[0029] In the above formula, A represents a target binding ligand, and B represents a p62 ligand. In the above formula 1, A and B can each be linked to one or more linkers.

[0030] In the present invention, the target-binding ligand is a ligand that binds to a specific target protein in the body, more specifically, to a pathogenic protein, organelle, or aggregate that induces the disease to be targeted. Such target proteins include, but are not limited to, proteins associated with cancer and various proteinopathy disorders. Such target-binding ligands can be used without limitation as long as they bind to proteins associated with the disease to be prevented, ameliorated, or treated, preferably target proteins, organelles, or mutant protein aggregates associated with diseases caused by pathogenic proteins, cancer, proteinopathy, rare intractable diseases, or genetic diseases. Specific embodiments include, but are not limited to, one or more compounds selected from the group consisting of the compounds shown in Table 1 below, or derivative structures derived from these structures.

[0031] The "derivative" structure means that a portion of the structure of the target-binding ligand has been changed by bonding with a linker (for example, a structure in which a carboxyl group in a substituent has been bonded with a linker having an amine group, thereby changing the linking portion between the target-binding ligand and the linker to an amide group).

[0032] [Table 1] TIFF0007733394000003.tif241168TIFF0007733394000004.tif218166TIFF0007733394000005.tif244168TIFF0007733394000006.tif45167

[0033] The linker connecting A and B in Formula 1 can be any linker structurally connecting A and B. For example, such a linker can be -Q-(CH2CH2O) x -(CH2) y -P- or -Q-(CH2CH2CH2O) x -(CH2) y -P- or -Q-(CH2CH2NH)x -(CH2) y -P- or -Q-(CH2CH2CONH) x -(CH2) y It may be, but is not limited to, -P- (wherein Q includes -NH-, -O-, ═N-N(CH3)-, which are moieties modified upon binding with a target-binding ligand; P includes -NH-, -O-, -CH2-, -C(═O)-, which are moieties modified upon binding with a p62 ligand; x is an integer of 0 to 4; and y is an integer of 0 to 3). Preferably, the bond between P and the p62 ligand may be -CONH-, -O-, -NH-, -NHCO-, or -COO-. To form such a bond, the structure of a portion of the p62 ligand, for example, the Rc portion and a portion of the target-binding ligand, may be modified; such modification methods are well known in the art.

[0034] In the present invention, the p62 ligand refers to a substance that binds to p62, more specifically, the ZZ domain of p62. By binding to the p62ZZ domain, such a p62 ligand increases p62 oligomerization and activates autophagy, specifically macroautophagy. In a preferred embodiment, the p62 ligand may have the structure of Chemical Formula 2 below. [ka] W is C6-C10 aryl; L is -(CH2) n1 - or -O-(CH2) n2 -CH(OH)-, provided that the -O-(CH) n2 O in —CH(OH)— is bonded to a benzene ring, and n1 is an integer of 0 to 1; n2 is an integer from 1 to 4; m is an integer from 0 to 2; R a is R1 or -OR1, In this case, R1 is hydrogen or -(CH2) n3 -R'1, R'1 is unsubstituted; or hydroxy, halogen, C 1-4 Alkyl, C 1-4 Alkoxy, nitro, amino, (C 1-4 alkyl)amino, or di(C 1-4 Archi phenyl substituted with amino; n3 is an integer from 1 to 6; R b is -OR2, In this case, R2 is hydrogen or -(CH2) n4 -R'2, R'2 is unsubstituted; or hydroxy, halogen, C 1-4 Alkyl, C 1-4 Alkoxy, nitro, amino, (C 1-4 alkyl)amino, or di(C 1-4 phenyl substituted with alkyl)amino; n4 is an integer from 1 to 6; R c is -(CH2) n5 -OH, -(CH2) n5 -NH-C(=NH)NH2, -C(=NH)NH2, -CH(R3)-COOH, or -CH(COO-R4)-CH2CH2CH2-NH-C(=NH)NH2, -(CH2) n5 -O-(CH2) n5 -OH, -CONH(CH2) n5 -OH, -CO(CH2) n6 -OH, -(CH2) n6 -CH(NH2)-COOH, -(CH2) n6 -CONH2, n5 is an integer from 2 to 4, n6 is an integer from 1 to 4, R3 is hydrogen or C 1-4 is alkyl, R4 is C 1-4 is alkyl, R d is hydrogen, halogen, C 1-4 Alkoxy or C 1-4 It is alkyl. Preferably, W may be phenyl. Preferably, L is —(CH) n1 - or -O-(CH2) n2 -CH(OH)-, provided that the -O-(CH) n2 The O in -CH(OH)- is attached to the benzene ring.

[0035] Preferably, n1 may be an integer of 0-1.

[0036] Preferably, n2 may be an integer of 1 to 2.

[0037] Preferably, R a is hydrogen or -O-(CH2) n3 It may also be -R'1.

[0038] Preferably, R'1 may be phenyl which is unsubstituted; or substituted with hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino, or dimethylamino.

[0039] Preferably, n3 may be an integer of 1 to 4.

[0040] Preferably, R b is hydroxy or -O-(CH2) n4 It may also be -R'2.

[0041] Preferably, R'2 may be phenyl which is unsubstituted; or substituted with hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino, or dimethylamino.

[0042] Preferably, n4 may be an integer of 1 to 4.

[0043] Preferably, R c is -(CH2) n5 -OH, -(CH2) n5 -NH-C(=NH)NH2, -C(=NH)NH2, -(CH2)n5 -O-(CH2) n5 -OH, -CONH(CH2) n5 -OH, -CO(CH2) n6 -OH, -(CH2) n6 -CH(NH2)-COOH, or -(CH2) n6 It may also be -CONH2.

[0044] Preferably, n5 may be an integer of 2 to 3.

[0045] Preferably, n6 may be an integer of 1 to 2.

[0046] Preferably, R d is hydrogen, halogen, C 1-2 Alkoxy or C 1-2 It may also be alkyl.

[0047] Such p62 ligands can be linked to the linker in the form of derivatives in which some groups are modified to facilitate linkage with the linker. This can be modified by those skilled in the art using appropriate known techniques depending on the type of p62 ligand, the type of linker, and the form of binding thereto, and such modified derivative forms are also included in the p62 ligands of the present invention.

[0048] In a specific embodiment, the autotack chimeric compound according to the present invention may be, but is not limited to, the compounds shown in Table 2 below.

[0049] [Table 2] TIFF0007733394000009.tif226168TIFF0007733394000010.tif192169

[0050] Meanwhile, the compounds of the present invention may exist in the form of pharmaceutically acceptable salts. Acid addition salts formed with pharmaceutically acceptable free acids are useful as such salts. The term "pharmaceutically acceptable salt" as used herein refers to any and all organic or inorganic addition salts of the compounds of Formulas 1 to 3 that are relatively non-toxic and harmless to patients and whose side effects do not reduce the beneficial effects of the compounds.

[0051] Acid addition salts are prepared by conventional methods, for example by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent, such as methanol, ethanol, acetone, or acetonitrile. Equal molar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water are heated, and the mixture is then evaporated to dryness. Alternatively, the precipitated salts can be filtered off with suction.

[0052] In this case, the free acid may be an organic acid or an inorganic acid. Examples of inorganic acids that may be used include hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid. Examples of organic acids that may be used include, but are not limited to, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carboxylic acid, vanillic acid, and hydroiodic acid.

[0053] Pharmaceutically acceptable metal salts can also be prepared using bases. Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are particularly suitable for pharmaceutical purposes, but are not limited to these. Corresponding silver salts can also be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (e.g., silver nitrate).

[0054] Pharmaceutically acceptable salts of the compounds of the present invention, unless otherwise specified, include salts of acidic or basic groups that may be present in the compounds of Formula 1. For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p-toluenesulfonate (tosylate) salts, which may be prepared by methods known in the art for preparing salts.

[0055] As the salt of the compound of Chemical Formula 1 of the present invention, any pharmaceutically acceptable salt that exhibits pharmacological activity equivalent to that of the compound of Chemical Formula 1 can be used without limitation.

[0056] The compounds of Formula 1 according to the present invention include, without limitation, not only pharmaceutically acceptable salts thereof, but also solvates such as hydrates that can be prepared therefrom, and all possible stereoisomers. All stereoisomers of the present invention, including enantiomeric and partial stereoisomeric forms (e.g., those that may exist due to asymmetric carbons in various substitutions), are within the scope of the present invention. Individual stereoisomers of the compounds of the present invention may be, for example, substantially free of different isomers (e.g., as pure or substantially pure optical isomers having a specific activity), or may be mixed with all different or different selected stereoisomers, for example, as racemates or in combination with all different or different selected stereoisomers. Chiral centers of the compounds of the present invention may have the S or R configuration as defined by the IUPAC 1974 Recommendations. Racemic forms can be analyzed by physical methods such as separation by chiral column chromatography, separation or crystallization of partial stereoisomeric derivatives, or fractional crystallization. Individual optical isomers can be obtained from the racemate by any suitable method, including, but not limited to, salt formation with an optically active acid followed by crystallization.

[0057] The solvates and stereoisomers of the compound of Formula 1 can be prepared from the compound of Formula 1 using methods known in the art.

[0058] Furthermore, the compound represented by Chemical Formula 1 according to the present invention can be prepared in a crystalline or amorphous form, and if prepared in a crystalline form, it can be optionally hydrated or solvated. The present invention includes not only stoichiometric hydrates of the compound represented by Chemical Formula 1, but also compounds containing various amounts of water. The solvates of the compound represented by Chemical Formula 1 according to the present invention include both stoichiometric and non-stoichiometric solvates.

[0059] The compound of Formula 1 according to the present invention can be prepared by the following exemplary method, and a specific example is shown in the reaction scheme described in the following Examples.

[0060] In the preparation method of the present invention, the reactants used in the above reaction schemes may be purchased as commercially available compounds and used as they are, or may be synthesized by carrying out one or more reactions known in the art, either directly or with appropriate modifications. For example, the compounds may be synthesized by carrying out one or more reactions in a series of steps, taking into consideration the presence, type, and / or position of reactive functional groups and / or heteroatoms contained in the skeletal structure, but are not limited thereto.

[0061] The compound of Chemical Formula 1 according to the present invention acts as a chimeric ligand that binds to the ZZ domain of p62 and simultaneously binds to target proteins, organelles, and aggregates, thereby activating the function of p62 and transmitting target proteins, organelles (endoplasmic reticulum, mitochondria, peroxisomes, etc.), intracellular structures (inflammasomes, stress granules, etc.), pathogens (viruses, bacteria, etc.) that have invaded cells, and aggregates to autophagy together with p62 for degradation.

[0062] Therefore, in another aspect, the present invention provides a pharmaceutical composition for activating autophagy, comprising a compound of Chemical Formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof.

[0063] The compound of Formula 1 according to the present invention can remove pathogenic proteins, organelles, and aggregates of pathogenic protein-related diseases by transferring target proteins, organelles, and aggregates to autophagy and degrading them. At the same time, the compound of Formula 1 binds to the p62ZZ domain as a p62 ligand, activating the PB1 and LIR domains of the p62 protein to induce p62 oligomerization and aggregation, while also enhancing the transfer of target proteins, organelles, and aggregates to autophagosomes along with the p62 protein. Through this process, target proteins, organelles, and aggregates are smoothly removed (see Figure 1). Such target proteins may be major proteins of pathogenic protein-related diseases, and more preferably, prion protein, amyloid precursor protein (APP), alpha-synuclein, superoxide dismutase 1, tau, immunoglobulin, amyloid-A, transthyretin, beta-2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, gelsolin, insulin, lysozyme, fibrinogen, huntingtin, alpha-1-antitrypsin Z, crystallin, c9 open reading frame 72 (c9orf72), glial fibrillary acidic protein (GFAP), and the like. The protein may be one or more selected from the group consisting of cystic fibrosis transmembrane conductance regulator protein, rhodopsin, ataxin, and other proteins having Poly-Q stretches.

[0064] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising an AutoTAC chimeric compound of the following Chemical Formula 1, or a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof. The pharmaceutical composition prevents or treats a disease targeted by a target-binding ligand. Such diseases can be any disease that can be bound to a target-binding ligand, preferably cancer or proteinopathy, and more preferably various diseases that can be treated by targeting and degrading specific proteins, such as rare, intractable diseases or genetic diseases. The pharmaceutical composition according to the present invention is characterized by directly removing the causative protein that induces the disease.

[0065] In another aspect, the present invention provides a pharmaceutical composition for transducing or degrading disease-causing pathogenic proteins and denatured proteins through autophagy, comprising the compound of Chemical Formula 1 or a pharmaceutically acceptable salt, stereoisomer, solvate, hydrate, or prodrug thereof.

[0066] The term "proteinopathy" as used herein includes diseases characterized by altered protein aggregates, such as, but not limited to, neurodegenerative diseases, α1-antitrypsin deficiency, keratopathy, pigmentary retinal salts, type 2 diabetes, and cystic fibrosis.

[0067] According to the present invention, the term "aggregation" refers to the formation of oligomeric or multimeric complexes of typically one or more proteins, accompanied by the incorporation of additional biomolecules, such as carbohydrates, nucleic acids, and lipids, into the complex. Such aggregated proteins can form deposits in specific tissues, more preferably in neural or brain tissue. The degree of aggregation depends on the disease in question.

[0068] The neurodegenerative diseases include Lyme disease, fatal familial insomnia, Creutzfeldt-Jakob disease (CJD), multiple sclerosis (MS), dementia, Alzheimer's disease, epilepsy, Parkinson's disease, stroke, Huntington's disease, Pick's disease, amyotrophic lateral sclerosis (ALS), and cerebrovascular accidents. The preferred therapeutic agent is selected from the group consisting of amyloidosis (ALS), spinocerebellar degeneration, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), injection-localized amyloidosis, β-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, Alexander disease, and Finnish hereditary systemic amyloidosis. The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. An effective dosage is typically about 1 ng to 10 mg / day, particularly about 1 g to 1 mg / day, for an adult (60 kg). It is obvious to those skilled in the art that the dosage may vary depending on various conditions, and therefore, the dosage may be adjusted accordingly. Therefore, the dosage does not limit the scope of the present invention in any way. The number of administrations may be once a day or in divided doses within a desired range, and the administration period is not particularly limited.

[0069] The term "treatment" as used herein refers to any action in which the administration of a pharmaceutical composition of the present invention can improve or favorably alter the various diseases associated with protein coagulation and the disease by targeted protein degradation.

[0070] As described above, the compounds of the present invention (1) induce p62 oligomerization and constitutive activation. This leads to (2) increasing the binding of p62 to LC3, (3) increasing the delivery of p62 to autophagosomes, (4) activating autophagy, and finally (5) exhibiting the effect of removing the target protein. Therefore, pharmaceutical compositions containing this as an active ingredient can be used for the prevention, amelioration, or treatment of various target diseases, preferably cancer or proteinopathy.

[0071] For example, the compositions of the present invention may additionally contain a pharmaceutically acceptable carrier, diluent, or excipient, and may be formulated into various forms, such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or sterile injection solutions, by conventional methods to suit the intended purpose, and may be administered orally or via various routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. Examples of suitable carriers, excipients, or diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition of the present invention may also additionally contain fillers, anti-agglomerating agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like.

[0072] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are formulated by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can be used.

[0073] Examples of oral liquid preparations include suspensions, oral liquids, oils, syrups, etc., which may contain various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin.

[0074] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, oil solutions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin. Injectable solutions may contain conventional additives such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0075] Said dosage forms can be prepared by conventional mixing, granulating or coating methods and contain the active ingredient in an amount effective for medical therapy, specifically for the prevention, amelioration or treatment of diseases associated with protein aggregation.

[0076] In this case, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, without causing side effects. The effective dose level can be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method, time, route and excretion rate of administration, duration of treatment, coadministered or concomitant drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, in single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum efficacy at the minimum dose without side effects, which can be easily determined by one skilled in the art.

[0077] For example, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, body weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

[0078] The preferred dose of the compound of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the administration route and period, but can be appropriately selected by those skilled in the art.

[0079] In yet another aspect, the present invention provides a method for preventing, ameliorating, or treating a target disease, preferably cancer, a pathogenic protein-related disease, or a protein aggregation-related disease, comprising the step of administering the pharmaceutical composition of the present invention to an individual in need thereof.

[0080] In still another aspect, the present invention provides (i) a method for increasing the degradation of a target protein, (ii) a method for increasing the degradation of organelles and structures, (iii) a method for increasing the degradation of viruses and bacteria that have invaded cells, (iv) a method for delivering drugs or small molecule compounds to autophagy and lysosomes, (v) a method for activating the self-oligomerization of p62 and autophagy, and (vi) a method for increasing lysosomal degradation by linking a specific intracellular protein to p62 and delivering it to autophagy, which comprises the step of treating a cell or p62 protein with the compound of Chemical Formula 1 or a pharmaceutically acceptable salt, stereoisomer, solvate, hydrate, or prodrug thereof.

[0081] In another aspect, the present invention provides a method for delivering a drug to a lysosome via an endosome using a therapeutic antibody that specifically binds to a protein exposed on the cell membrane, which is a chimeric compound according to the present invention. The therapeutic antibody can be any antibody that exhibits pharmacological activity against a disease requiring treatment.

[0082] The term "individual" as used herein refers to any animal, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs, suffering from the target disease to be prevented, ameliorated, or treated. Administration of the pharmaceutical composition of the present invention to an individual can effectively prevent, ameliorate, or treat the target disease, preferably cancer, a disease associated with a pathogenic protein, or a disease associated with the aggregation of denatured proteins. Furthermore, the pharmaceutical composition of the present invention acts on a p62 ligand to activate autophagy, which removes denatured protein aggregates through such autophagy activation, thereby exhibiting a preventive or therapeutic effect against diseases associated with such aggregated proteins. Therefore, administration of the pharmaceutical composition in conjunction with an existing therapeutic agent can provide a synergistic effect.

[0083] The term "administration" as used herein means providing a predetermined substance to a patient by any suitable method. The administration route of the compositions of the present invention can be any common route as long as it can reach the target tissue. Administration routes include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, and rectal administration. Furthermore, the pharmaceutical compositions of the present invention can be administered by any device that can deliver the active substance to target cells. Preferred administration methods and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and infusion injections. Injectable preparations can be prepared using aqueous solvents such as physiological saline and Ringer's solution, or non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and can contain pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and preservatives to prevent microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0084] In another aspect, the present invention provides a food composition for preventing or ameliorating proteinopathy, comprising an AutoTAC chimeric compound of Formula 1 or a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof. The food composition may be formulated and used as a functional health food, or may be incorporated into other functional health foods as an additive. Functional health foods are foods that have bioregulatory functions, such as disease prevention or amelioration, biodefense, immunity, post-illness recovery, and anti-aging, and should be harmless to the human body when taken over a long period of time. The amount of active ingredient can be determined appropriately depending on the intended use (prevention, health, or therapeutic treatment).

[0085] There are no particular limitations on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, health drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the usual sense.

[0086] The food composition of the present invention can contain conventional ingredients used in the production of food or food additives, specifically, flavoring agents; natural carbohydrates such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame; nutrients; vitamins; electrolytes; coloring agents; organic acids; protective colloid thickeners; pH adjusters; stabilizers; preservatives; glycerin; alcohol; and carbonation agents used in carbonated beverages.

[0087] In a specific example of the present invention, novel AutoTAK chimeric compounds 1 to 13 represented by Chemical Formula 1 were newly synthesized. To evaluate whether the novel AutoTAK chimeric compounds according to the present invention can increase autophagy in cultured cells, cell lines (MCF7, NTERA-2, ACHN, U87-MG, LNCaP, HEK293T) in which the target protein is naturally expressed and recombinant cell lines (SH-SY5Y-tau, HeLa-HttQ97, PC12-a-synA30P) were treated with the AutoTAK chimeric compounds according to the present invention and cultured, and the target protein degradation activity in the cultured cells was confirmed by immunoblotting. As a result, the AutoTAC compound of the present invention was confirmed to have a gradually increasing autophagy-mediated degradation effect on each target protein depending on the treatment concentration, and to have superior degradation efficacy to the p62 ligand and target protein ligands composed of chimeric compounds. It was confirmed that the AutoTAC compound of the present invention activates and oligomerizes p62 protein, delivering it to autophagosomes, while selectively and effectively removing proteins and their aggregates that are targeted for various diseases, such as cancer-related proteins or proteins associated with abnormal proteinase activity. [Example]

[0088] The present invention will be described in more detail with reference to the following examples, which are provided to illustrate the present invention in more detail and are not intended to limit the scope of the present invention.

[0089] The compounds of formulas 1 to 13 according to the present invention were prepared in Examples 1 to 13 below.

[0090] [Table 3] TIFF0007733394000012.tif74168

[0091] Various synthetic methods are known for starting materials for synthesizing the compounds of the present invention, and if the starting materials are commercially available, they can be purchased from suppliers such as, but not limited to, Aldrich, Sigma, TCI, Wako, Kanto, Fluorchem, Acros, Alfa, and Fluka.

[0092] The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. Typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given unless otherwise stated; other process conditions can also be used. Optimum reaction conditions may vary with the specific reactants or solvents used; such conditions can be determined by one skilled in the art by routine optimization procedures.

[0093] The manufacturing methods of Examples 1 to 13 will be described below.

[0094] Example 1. Preparation of (2E,4E,6E,8E)-N-(2-(2-(2-(((R)-3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide (RTEG-1104)

[0095] [ka]

[0096] (Step 1) Preparation of 3,4-bis(benzyloxy)benzaldehyde (1101) 3,4-Dihydroxybenzaldehyde (0.50 g, 3.62 mmol) was dissolved in anhydrous DMF (5 mL) and potassium carbonate (K2CO3, 1.50 g, 10.86 mmol) was added. Benzyl bromide (0.92 mL, 7.96 mmol) was then slowly added to the reaction mixture and stirred at 60°C for 4 hours. Upon completion of the reaction, the reaction mixture was cooled to room temperature, diluted with purified water, and extracted twice with diethyl ether (50 mL). The organic layer was washed twice with purified water (50 mL) and once more with saturated aqueous sodium chloride solution (50 mL). Anhydrous sodium sulfate was then added to the organic layer, stirred, and filtered under reduced pressure. The filtered solution was concentrated and purified by column chromatography to obtain 3,4-bis(benzyloxy)benzaldehyde (1101, 1.04 g, 90% yield). 1 H NMR (CDCl3, 300MHz) δ9.81(s, 1H), 7.49-7.31(m, 12H), 7.04(d, J=8.3Hz, 1H), 5.27(s, 2H), 5.22(s, 2H);ESIMS m / z:319.33[M+H] + .

[0097] (Step 2) Step 1) Preparation of 3,4-bis(benzyloxy)phenol (1102) Dichloromethane (15 ml) was added to 3,4-bis(benzyloxy)benzaldehyde (1101, 1.00 g, 3.0 mmol, 1 equiv.) and then mCPBA (0.78 g, 4.5 mmol, 1.5 equiv.) was added to the reaction mixture and stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous sodium carbonate, and the organic layer was separated. The organic layer was washed with aqueous sodium chloride, dried over anhydrous sodium sulfate, and filtered under reduced pressure. The filtered solution was concentrated and redissolved in methanol (10 ml). 6N NaOH was added and stirred at room temperature for 30 minutes. 4N HCl solution was added to the reaction mixture and stirred for an additional 30 minutes. The reaction mixture was diluted with ethyl acetate (50 ml) and washed with brine, then dried over anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated and then purified by column chromatography (hexane / ethyl acetate ratio = 7 / 3) to obtain 3,4-bis(benzyloxy)phenol (1102, 0.87 g, yield 90%).

[0098] 1 H-NMR (CDCl3, 300MHz): δ7.25-7.42(m, 10H), 6.80(d, 1H, J=9.0Hz), 6.48(d, 1H, J=3.0Hz), 6.29(dd, 1H, J=3.0 and 9.0Hz), 5.08(d, 4H, J=15Hz), 4.55(s, 1H);ESIMS m / z:307.25[M+H] + .

[0099] (Step 3) Preparation of R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103) 3,4-Dibenzyloxyphenol (1102, 306 mg, 1.0 mmol) was diluted in ethanol (10 mL), followed by the sequential addition of aqueous KOH solution (66 mg, 1.2 mmol, 1 mL) and (R)-2-(chloromethyl)oxirane (410 μL, 5.0 mmol). The reaction mixture was stirred at room temperature for 5 hours, after which the organic solvent was removed under reduced pressure. The concentrated mixture was again diluted with ethyl acetate and washed with water, followed by brine. The extracted organic layer was dried over anhydrous sodium sulfate and filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography to give pure R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 297 mg, 82% yield). ESIMS m / z: 363.5 [M+H] + .

[0100] (Step 4) Preparation of (R)-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino-3-(3,4-bis(benzyloxy)phenoxy)propan-2-ol (AL-1) R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 270 mg, 0.75 mmol) was dissolved in absolute ethanol (5 mL), and then 2,2'-(ethane-1,2-diylbis(oxy))bis(ethan-1-amine) (880 mg, 5.9 mmol) was added and stirred at room temperature for 8 hours. After confirming the reaction by TLC, the reaction solvent was concentrated under reduced pressure. Water was added to the concentrated reaction mixture and extracted with dichloromethane (3 x 5 mL). The extracted organic layer was dried over anhydrous sodium sulfate and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography (dichloromethane:methanol = 19:1) to give (R)-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino-3-(3,4-bis(benzyloxy)phenoxy)propan-2-ol (AL-1, 267 mg, 70% yield). ESIMS m / z: 511.5 [M+H] + .

[0101] (Step 5) (R)-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino-3-(3,4-bis(benzyloxy)phenoxy)propan-2-ol (AL-1, 100 mg, 0.19 mmol) was dissolved in DMF (4 ml), and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 44 mg, 0.285 mmol), hydroxybenzotriazole (HOBt, 38.5 mg, 0.285 mmol), and retinoic acid (60 mg, 0.2 mol) were added sequentially, followed by N-N-diisopropylethylamine (DIPEA, 0.6 ml) and stirring at room temperature for 12 hours. The reaction mixture was added with water and extracted twice with ethyl acetate. The organic layer was then washed once with brine. The organic layer was dried over anhydrous sodium sulfate and filtered under reduced pressure. The filtrate was concentrated under reduced pressure and purified by high-resolution liquid chromatography to produce a white solid, (2E,4E,6E,8E)-N-(2-(2-(2-(((R)-3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide (RTEG-1104, 60 mg, 41% yield).

[0102] 1 H NMR (400MHz, DMSO-d6) δ(ppm) 1.01(s, 6H), 1.53(m, 2H), 1.74-1.79(m, 5H), 1.96(m, 2H), 2.12(s, 3H), 2.42(s, 3H), 2.56-2.81(m, 4H), 3.04(m, 2H), 3.52-3.54(m, 6H), 3.67(m, 2H), 3.95-4.05(m, 3H), 5.16(s, 4H), 5.37(br s, 1H), 5.91(br ESI-MS Calcd m / z for C 49 H 64 N2O7[M+H] + 794.35 Found 793.06.

[0103] Example 2 Preparation of (2E,4E,6E,8E)-N-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide (RTEG-1105)

[0104] [ka]

[0105] (Step 1) Preparation of 2-(2-(2-aminoethoxy)ethoxy)-N-(3,4-bis(benzyloxy)benzyl)ethan-1-amine (AL-2) 3,4-Bis(benzyloxy)benzaldehyde (1101, 0.5 g, 1.57 mmol) was dissolved in acetonitrile (CAN, 10 mL), followed by the addition of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (468 mg, 1.88 mmol) and stirring at 60-70 °C for 5 hours. After cooling to room temperature, sodium borohydride (NaBH4, 106 mg, 2.82 mmol) was slowly added to the reaction mixture and stirred at room temperature for approximately 5 hours. Water was added to quench the reaction, and the compound was extracted with ethyl acetate (50 mL x 3). The extracted organic layer was washed with brine and dried over sodium sulfate. The filtered solvent was concentrated and then redissolved in dichloromethane (6 mL). Trifluoroacetic acid (TFA, 2 mL) was added and stirred at room temperature for 2 hours, after which the solvent was concentrated under reduced pressure. The residue was purified by column chromatography (methylene chloride / methanol = 15:1) to obtain a pale yellow liquid, 2-(2-(2-aminoethoxy)ethoxy)-N-(3,4-bis(benzyloxy)benzyl)ethan-1-amine (AL-2, 590 mg, 84% yield).

[0106] 1H NMR (400MHz, DMSO-d6) δ (ppm) 2.59 (t, 2H), 3.32-3.61 (m, 12H), 4.62 (br s, 1H), 5.10(s, 4H), 6.82(d, 1H), 6.97(d, 1H), 7.06(s, 1H), 7.30-7.46(m, 10H).

[0107] (Step 2) (2E,4E,6E,8E)-N-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide (RTEG-1105) was prepared by a method similar to that of Step 5 of Example 1.

[0108] 1 H NMR (400MHz, DMSO-d6) δ (ppm) 1.01 (s, 6H), 1.54 (m, 2H), 1.75-1.80 (m, 5H), 1.98 (m, 2H), 2.12 (s, 3H), 2.42(s, 3H), 2.72(t, 2H), 3.04(t, 2H), 3.51-3.54(m, 6H), 3.67(t, 2H), 3.76(s, 2H), 5.14(s, 4H), 6.22(m, 2H), 6.37(br s, 1H), 6.51(s, 4H), 6.80(d, 1H), 6.87(d, 1H), 6.99(s, 1H), 7.31-7.46(m, 10H), 8.41(br s, 1H); ESI-MS Calcd m / z for C 47 H 60 N2O5[M+H] + 734.3 Found 733.01.

[0109] Example 3. Preparation of (R)-N-(15-(3,4-bis(benzyloxy)phenoxy)-14-hydroxy-3,6,9-trioxa-12-azapentadecyl)-4-phenylbutanamide (PBA-1104)

[0110] [ka]

[0111] (Step 1) 2,2'-(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))diethanamine (21 g, 73.1 mmol) was dissolved in dichloromethane (400 ml), followed by the sequential addition of hydroxybenzotriazole (HOBt, 12.3 g, 91.5 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 17.6 g, 91.5 mmol). Triethylamine (EtN, 18.5 g, 180 mmol) was then added and the mixture was cooled to 0°C. 4-Phenylbutyric acid (15 g, 91.5 mmol) was dissolved in dichloromethane (200 ml) and added to the reaction mixture. The mixture was stirred at room temperature for 12 hours. The mixture was diluted with water and extracted with dichloromethane (50 ml x 3). The organic layer was then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography to produce a yellow liquid, N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-phenylbutanamide (TL-1, 12 g). ESIMS m / z: 339.1 [M+H] + .

[0112] (Step 2) N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-phenylbutanamide (TL-1, 100 mg, 0.295 mmol) was dissolved in methanol (6 mL), and R-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 128 mg, 0.354 mmol) was added and stirred at 50 °C for 10 hours. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography to produce a white solid, (R)-N-(15-(3,4-bis(benzyloxy)phenoxy)-14-hydroxy-3,6,9-trioxa-12-azapentadecyl)-4-phenylbutanamide (PBA-1104, 103 mg, 50% yield).

[0113] 1H NMR (400MHz, DMSO-d6) δ (ppm) 1.25 (s, 1H), 1.92 (m, 2H), 2.31 (t, 2H), 2.56-2.81 (m, 4H) , 3.28(m, 2H), 3.51-3.53(m, 10H), 3.67(t, 2H), 3.95-4.20(m, 3H), 5.14(s, 4H), 5.37(br E SI-MS Calcd m / z for C 41 H 52 N2O8[M+H] + 701.5 Found 700.87.

[0114] Example 4. Preparation of N-(1-(3,4-bis(benzyloxy)phenyl)-5,8,11-trioxa-2-azatridecan-13-yl)-4-phenylbutanamide (PBA-1105)

[0115] [ka]

[0116] (Step 1) Preparation of N-(1-(3,4-bis(benzyloxy)phenyl)-5,8,11-trioxa-2-azatridecan-13-yl)-4-phenylbutanamide (PBA-1105) N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-phenylbutanamide (TL-1) (10 g, 29.6 mmol) was dissolved in methanol (MeOH, 150 mL), and 3,4-dihydroxybenzaldehyde (10.3 g, 32.3 mmol) was added. The mixture was then stirred at 65 °C for 5 hours. After cooling to room temperature, sodium borohydride (NaBH4, 2.2 g, 57.9 mmol) was added and stirred at room temperature for 5 hours. Water was added to quench the reaction, and the compound was extracted with ethyl acetate (50 mL × 3). The extracted organic layer was washed with brine, and water was removed using sodium sulfate. The filtered solvent was concentrated and purified by silica gel column chromatography (methylene chloride / methanol = 15:1). Thereafter, 10.3 g of yellow liquid N-(1-(3,4-bis(benzyloxy)phenyl)-5,8,11-trioxa-2-azatridecan-13-yl)-4-phenylbutanamide (PBA-1105) was obtained.

[0117] 1 H NMR(CDCl3,400MHz)δ(ppm)7.45-7.42(m, 4H), 7.36-7.29(m, 7H), 7.27-7.26(m, 1H ), 7.18-7.15(m, 3H), 6.99(d, 1H), 6.83(d, 1H), 6.41(brs, 1H), 5.15(d, 4H), 3.71( s, 2H), 3.60-3.55(m, 10H), 3.55-3.49(m, 2H), 3.42-3.39(m, 2H), 2.75-2.73(m, 2H) ), 2.65-2.61(m, 2H), 2.18-2.15(m, 2H), 1.97-1.93(m, 2H), 1.25(brs, 1H); ESI-MS Calcd m / z for C 39 H 48 N2O6[M+H] + 641.00 Found 640.82.

[0118] Example 5. Preparation of 3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(2-(2-(2-((3-((4-fluorobenzyl)oxy)benzyl)amino)ethoxy)ethoxy)ethyl)aniline (Anle138b-F105)

[0119] [ka]

[0120] (Step 1) Preparation of 3-((4-fluorobenzyl)oxy)benzaldehyde (YT-102) Potassium carbonate (K2CO3, 112.5g, 0.81mol) and 1-(bromomethyl)-4-fluorobenzene (95g, 0.50mol) were added to 3-hydroxybenzaldehyde (50g, 0.41mol) dissolved in acetonitrile (ACN, 500mL). The mixture was stirred at 60°C for approximately 10 hours. Upon completion of the reaction, the solution was filtered and concentrated. Petroleum ether / ethyl acetate (PE / EA, 20:1, 20mL) was added to the mixture, and the mixture was stirred for an additional hour. The solution was then filtered and concentrated. 25g of 3-((4-fluorobenzyl)oxy)benzaldehyde (YT-102) was obtained as an off-white solid.

[0121] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 9.98 (s, 1H), 7.54-7.50 (m, 5H), 7.36 (m, 1H), 7.23 (m, 2H), 5.18 (s, 2H).

[0122] (Step 2) Preparation of tert-butyl 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole-1-carboxylate (T-1) 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole (200 mg, 0.58 mmol) was dissolved in methylene chloride (DCM, 4 mL), followed by the addition of triethylamine (TEA, 88 mg, 0.87 mmol) and di-tert-butyl dicarbonate (BocO, 153 mg, 0.70 mmol). The mixture was stirred at room temperature for approximately 4 hours. After the reaction was complete, the filtered solution was concentrated and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:5). tert-Butyl 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole-1-carboxylate (T-1, 200 mg) was obtained as a white solid.

[0123] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 8.09-7.43 (m, 5H), 7.11-6.94 (m, 3H), 6.08 (s, 2H), 1.34 (d, J=20Hz, 9H).

[0124] (Step 3) Preparation of tert-butyl 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-(2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)amino)phenyl)-1H-pyrazole-1-carboxylate (TL-2) tert-Butyl 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole-1-carboxylate (T-1, 200 mg, 0.45 mmol) was dissolved in dimethyl sulfoxide (DMSO, 4 mL), followed by the addition of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (L-1, 168 mg, 0.68 mmol) and potassium tert-butoxide (t-BuOK, 101 mg, 0.90 mmol). The mixture was stirred at 120 °C for approximately 16 hours. After the reaction was completed, the product was filtered, concentrated, and purified by preparative high-performance liquid chromatography (Prep-HPLC). Thereafter, a pale yellow solid, tert-butyl 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)amino)phenyl)-1H-pyrazole-1-carboxylate (TL-2, 180 mg), was obtained.

[0125] (Step 4) Preparation of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)aniline (TL-3) tert-Butyl 3-(benzo[d][1,3]dioxol-5-yl)-5-(3-((2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl)amino)phenyl)-1H-pyrazole-1-carboxylate (TL-2, 180 mg, 0.29 mmol) was dissolved in ethyl acetate (EA, 4 mL), and then a solution of hydrochloric acid / ethyl acetate (1 mL, 3N) was added. The mixture was stirred at room temperature for approximately 2 hours. After the reaction was completed, the compound was filtered and concentrated. Subsequently, a white solid, N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)aniline (TL-3, 100 mg), was obtained.

[0126] (Step 5) Preparation of 3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(2-(2-(2-((3-((4-fluorobenzyl)oxy)benzyl)amino)ethoxy)ethoxy)ethyl)aniline (Anle138b-F105) N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)aniline (TL-3, 90 mg, 0.22 mmol) was dissolved in methanol (MeOH, 2 mL). 3-((4-fluorobenzyl)oxy)benzaldehyde (YT-102, 50 mg, 0.22 mmol) was added and the mixture was stirred at 65 °C. Sodium borohydride (NaBH4, 16 mg, 0.44 mmol) was then added at 5 °C and the mixture was stirred for an additional hour. After the reaction was completed, the compound was concentrated and purified by preparative high-performance liquid chromatography (Prep-HPLC). 3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(2-(2-(2-((3-((4-fluorobenzyl)oxy)benzyl)amino)ethoxy)ethoxy)ethyl)aniline (Anle138b-F105, 15 mg) was obtained as a colorless liquid.

[0127] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 13.08 (brs, 1H), 7.47 (q, J=6Hz, 2H), 7.37-7.31 (m, 2H), 7.21-7.17(m, 3H), 7.11(t, J=8Hz, 1H), 7.00-6.96(m, 5H), 6.88-6.82(m, 2H), 6.56(d, J=8Hz, 1H), 6.04(s, 2H), 5.59(brs, 1H), 5.04(s, 2H), 3.66(s, 2H), 3.60- 3.52(m, 6H), 3.47(t, J=6Hz, 2H), 3.26-3.22(m, 2H), 2.61(t, J=5.6Hz, 2H);ESI-MS Calcd m / z for C 36 H 37 FN4O5[M+H] + 625.10 Found 624.71.

[0128] Example 6. Preparation of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-1-yl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl(13E,15E,17E,19E)-1-(3-(benzyloxy)phenyl)-12-oxo-5,8-dioxa-2,11-diazahenicosa-13,15,17,19-tetraen-21-oate (Fumagillin-105)

[0129] [ka]

[0130] (Step 1) Preparation of 3-(benzyloxy)benzaldehyde (201) Potassium carbonate (K2CO3, 112.5g, 0.81mol) and bromomethylbenzene (85g, 0.50mol) were added to 3-hydroxybenzaldehyde (50g, 0.41mol) dissolved in acetonitrile (ACN, 500mL). The mixture was stirred at 60°C for approximately 10 hours. Upon completion of the reaction, the solution was filtered and concentrated. Petroleum ether / ethyl acetate (PE / EA, 20:1, 20mL) was then added and the mixture was stirred for an additional hour, after which it was filtered and concentrated. 3-(benzyloxy)benzaldehyde (201, 25g) was obtained as an off-white solid.

[0131] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 9.97 (s, 1H), 7.54-7.51 (m, 3H), 7.47 (d, J=7.2Hz, 2H), 7.42-7.34 (m, 4H), 5.19 (s, 2H).

[0132] (Step 2) Preparation of 2-(2-(2-aminoethoxy)ethoxy)-N-(3-(benzyloxy)benzyl)ethan-1-amine (AL-3) 3-(Benzyloxy)benzaldehyde (201, 50 g, 235.8 mmol) was dissolved in methanol (MeOH, 500 mL) and 2,2'-(ethane-1,2-diylbis(oxy))diethanamine (L-2, 34.9 g, 235.8 mmol) was added. The mixture was stirred at 65 °C for 6 hours. After cooling to room temperature, sodium borohydride (NaBH4, 8.95 g, 235.8 mmol) was added and the mixture was stirred at 50 °C overnight. Water was added to quench the reaction, and the compound was extracted with ethyl acetate (EtOAc, 50 mL x 3). The extracted organic layer was washed with brine, and sodium sulfate (Na2SO4) was added to remove water. The solution was concentrated and purified by silica gel column chromatography (methylene chloride / methanol = 12:1). Thereafter, a yellow liquid, 2-(2-(2-aminoethoxy)ethoxy)-N-(3-(benzyloxy)benzyl)ethan-1-amine (AL-3, 20 g), was obtained.

[0133] 1 H NMR (DMSO+D2O, 400MHz) δ(ppm)7.41-7.29(m, 5H), 7.18(t, J=8Hz, 1H), 6.94(s, 1H), 6.86-6.81(m, ESI-MS Calcd m / z for C 20 H 28 N2O3[M+H] + 345.10 Found 344.46.

[0134] (Step 3) Preparation of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-1-yl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl(13E,15E,17E,19E)-1-(3-(benzyloxy)phenyl)-12-oxo-5,8-dioxa-2,11-diazahenicosa-13,15,17,19-tetraen-21-oate (Fumagillin-105) 2-(2-(2-aminoethoxy)ethoxy)-N-(3(benzyloxy)benzyl)ethan-1-amine (AL-3, 70 mg, 0.22 mmol) was dissolved in methylene chloride (DCM, 2 mL) and fumagillin (100 mg, 0.22 mmol), hydroxybenzotriazole (HOBT, 34 mg, 0.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 48 mg, 0.25 mmol), and triethylamine (EtN, 44 mg, 0.44 mmol) were added. The mixture was stirred at 30 °C for approximately 10 hours. Upon completion of the reaction, the compound was extracted with methylene chloride (DCM, 50 mL x 3). The extracted organic layer was washed with brine and then dehydrated using sodium sulfate. The filtered solvent was concentrated and purified by preparative high-performance liquid chromatography (Prep-HPLC). This resulted in a yellow solid (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-1-yl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl(13E,15E,17E,19E)-1-(3-(benzyloxy)phenyl)-12-oxo-5,8-dioxa-2,11-diazahenicosa-13,15,17,19-tetraen-21-oate (Fumagillin-105, 3 mg). ESI-MS Calcd m / z for C 46 H 60 N2O9[M] + 784.90 Found 784.99.

[0135] Example 7. 3-(3,5-dichlorophenyl)-5-((R)-15-(3,4-diphenyl)- Preparation of (netoxyphenoxy)-14-hydroxy-6,9-dioxa-3,12-diazapentadecyl)-5-methyloxazolidine-2,4-dione (Vinclozolin-2204)

[0136] [ka]

[0137] (Step 1) Preparation of 3-hydroxy-4-phenethoxybenzaldehyde (A-1) 3,4-Dihydroxybenzaldehyde (200 g, 1.4 mol) was dissolved in acetonitrile (ACN, 2 L), followed by the addition of potassium carbonate (K2CO3, 260 g, 1.9 mol) and 1-(2-bromoethyl)benzene (267 g, 1.4 mol). The mixture was stirred at 80 °C for 16 hours. The reaction was quenched with hydrochloric acid (1.5 L, 1 N), and the compound was extracted with ethyl acetate (EA, 1 L x 3). The extracted organic layer was washed with brine, removed with sodium sulfate, and filtered. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 20:1 to 10:1). 3-Hydroxy-4-phenethoxybenzaldehyde (A-1, 320 g) was obtained as a white solid. ESI-MS Calcd m / z for C 15 H 14 O3 [M+H] + 243.0 Found 242.27.

[0138] (Step 2) Preparation of 3,4-diphenethoxybenzaldehyde (2201) 3-Hydroxy-4-phenethoxybenzaldehyde (A-1, 320 g, 1.32 mol) was dissolved in tetrahydrofuran (THF, 5 L), followed by the addition of 2-phenylethanol (193.5 g, 1.59 mol), triphenylphosphine (PPh3, 520 g, 1.98 mol), and diisopropyl azodicarboxylate (DIAD, 400 g, 1.98 mol). The mixture was stirred at 65 °C for 16 hours. The reaction was terminated by adding water (100 mL) and extracted with ethyl acetate (100 mL x 2). The extracted organic layer was added with sodium sulfate to remove water, filtered, and concentrated. The concentrated compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:10). 3,4-diphenethoxybenzaldehyde (2201, 160 g) was obtained as a white solid.

[0139] 1H NMR (DMSO-d6, 400MHz) δ (ppm) 9.81 (s, 1H), 7.51 (dd, J=8Hz and 1.6Hz, 1H), 7.40-7.17(m, 12H), 4.27(t, J=6.8Hz, 2H), 4.22(t, J=6.8Hz, 2H), 3.06(q, J=6.4Hz, 4H);ESI-MS Calcd m / z for C 23 H 22 O3 [M+H] + 346.90 Found 346.43.

[0140] (Step 3) Preparation of 3,4-diphenethoxyphenol (2202) 3,4-Diphenethoxybenzaldehyde (2201, 160 g, 0.46 mmol) was dissolved in methylene chloride (DCM, 2 L), and meta-chloroperoxybenzoic acid (m-CPBA, 119 g, 0.69 mmol) was added. The mixture was stirred at room temperature for approximately 16 hours. Upon completion of the reaction, the solution was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:5). 3,4-Diphenethoxyphenol (2202, 100 g) was obtained as an off-white solid. ESI-MS Calcd m / z for C 22 H 22 O3 [M+H] + 335.00 Found 334.42.

[0141] (Step 4) Preparation of (R)-2-((3,4-diphenethoxyphenoxy)methyl)oxirane (A-2) 3,4-Diphenethoxyphenol (2202) (5 g, 15 mmol) was dissolved in ethanol (EtOH, 50 mL), followed by the addition of water (2.5 mL) and potassium hydroxide (KOH, 1.93 g, 34.5 mmol). (R)-2-(chloromethyl)oxirane (8 g, 87 mmol) was added and stirred at room temperature for approximately 16 hours. Water (50 mL) was added to terminate the reaction, and the compound was extracted with ethyl acetate (EA, 20 mL x 3). The extracted organic solvent was washed with brine, removed with sodium sulfate, filtered, and concentrated. The concentrated compound was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:10). A white solid, (R)-2-((3,4-diphenethoxyphenoxy)methyl)oxirane (A-2, 2.5 g), was obtained. ESI-MS Calcd m / z for C 25 H 26 O4 [M+H] + 391.00 Found 390.48.

[0142] (Step 5) Preparation of (R)-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-3-(3,4-diphenethoxyphenoxy)propan-2-ol (AL-4) (R)-2-((3,4-diphenethoxyphenoxy)methyl)oxirane (A-2, 20 g, 51.2 mmol) was dissolved in acetonitrile (ACN, 200 mL), and 2-(2-(2-aminoethoxy)ethoxy)ethanamine (L-2, 15.2 g, 102.5 mmol) was added. The mixture was stirred at 70 °C for approximately 40 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (methylene chloride / methanol = 100:1 to 30:1). A yellow liquid, (R)-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-3-(3,4-diphenethoxyphenoxy)propan-2-ol (AL-4, 12 g), was obtained.

[0143] 1H NMR (CD3OD, 400MHz) δ(ppm)8.52(s, 3H), 7.30-7.20(m, 10H), 6.85(d, J=8Hz, 1H), 6.60(d, J=2.8Hz, 1H), 6.47(dd, J=8Hz and 2Hz, 1H), 4.16(t, J=6.8Hz, 3H), 4.08(t, J=6.8Hz, 2H), 3.96(q, J=4Hz, 2H), 3. 79(t, J=5.2Hz, 2H), 3.70(m, 7H), 3.32-3.25(m, 3H), 3.17-2.98(m, 8H);ESI-MS Calcd m / z for C 31 H 42 N2O6[M+H] + 539.20 Found 538.69.

[0144] (Step 6) Preparation of Liquid 3-(3,5-Dichlorophenyl)-5-((R)-15-(3,4-diphenethoxyphenoxy)-14-hydroxy-6,9-dioxa-3,12-diazapentadecyl)-5-methyloxazolidine-2,4-dione (Vinclozolin-2204) (R)-1-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-3-(3,4-diphenethoxyphenoxy)propan-2-ol (AL-4, 120 mg, 0.22 mmol) was dissolved in methanol (MeOH, 2 mL), and then vinclozolin (117 mg, 0.33 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. Upon completion of the reaction, the solution was filtered and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). Thereafter, 15 mg of colorless liquid 3-(3,5-dichlorophenyl)-5-((R)-15-(3,4-diphenethoxyphenoxy)-14-hydroxy-6,9-dioxa-3,12-diazapentadecyl)-5-methyloxazolidine-2,4-dione (Vinclozolin-2204) was obtained.

[0145] 1H NMR (DMSO-d6, 400MHz) δ (ppm) 1.65 (m, 3H), 2.65 (m, 4H), 2.97 (m, 4H), 3.17 (m, 2H), 3.42(m, 8H), 3.57(d, J=8Hz, 1H), 3.80(m, 3H), 4.02(t, J=8Hz, 2H), 4.13(t, J=8Hz, 2H), 4.90(b, 1H), 5.33(m, 2H), 6.20(m, 1H), 6.38(d, J=8Hz, 1H), 6.54(m, 1H), 6.82 (d, J=8Hz, 1H), 7.25(m, 11H), 7.49(d, J=1Hz, 1H), 7.76(s, 1H), 9.70(s, 1H);ESI-MS Calcd m / z for C 43 H 51 Cl2N3O9[M+H] + 825.00 Found 824.79.

[0146] Example 8. Preparation of (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-((2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)amino)propan-2-ol (PHTPP-1304)

[0147] [ka]

[0148] (Step 1) Preparation of 4-(benzyloxy)-3-hydroxybenzaldehyde (A-3) 3,4-Dihydroxybenzaldehyde (500 g, 3.62 mol) was dissolved in acetonitrile (ACN, 7 L), followed by the addition of sodium bicarbonate (NaHCO3, 395 g, 4.71 mol) and benzyl bromide (BnBr, 619 g, 3.62 mol). The mixture was stirred at 80 °C for 16 hours. The reaction was quenched with hydrochloric acid (3 L, 1 N), and the compound was extracted with ethyl acetate (3 L × 3). The extracted organic layer was washed with brine, and the remaining water was removed with sodium sulfate. The mixture was filtered to remove impurities and concentrated. The concentrated solution was purified by silica gel column chromatography (EA / PE = 20:1 to 10:1). 250 g of 4-(benzyloxy)-3-hydroxybenzaldehyde (A-3) was obtained as a white solid.

[0149] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)9.76(s, 1H), 9.65(s, 1H), 7.49(d, J=6.8Hz, 2H), 7.42-7.34(m, 4H), 7.29(d, J=2Hz, 1H), 7.19(d, J=8Hz, 1H), 5.23(s, 2H).

[0150] (Step 2) 4-(benzyloxy)-3-(3-phenylpropoxy)benzaldehyde (1301) Production 4-(benzyloxy)-3-hydroxybenzaldehyde (A-3, 120 g, 526 mmol) was dissolved in tetrahydrofuran (THF, 2 L), followed by the addition of 3-phenylpropan-1-ol (85.9 g, 631 mmol), triphenylphosphine (PPh3, 206.9 g, 789 mmol), and diisopropyl azodicarboxylate (DIAD, 159.5 g, 789 mmol). The mixture was stirred at 65°C for approximately 16 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:10). 4-(benzyloxy)-3-(3-phenylpropoxy)benzaldehyde (1301, 100 g) was obtained as a white solid.

[0151] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 9.82 (s, 1H), 7.55-7.50 (m, 3H), 7.38 (t, J=7.2Hz, 3H), 7.36-7.34 (m, 1H), 7.29- 7.25(m, 3H), 7.21-7.16(m, 3H), 5.26(s, 2H), 4.04(t, J=6.4Hz, 2H), 2.76(t, J=8Hz, 2H), 2.04(t, J=7.2Hz, 2H).

[0152] (Step 3) Preparation of 4-(benzyloxy)-3-(3-phenylpropoxy)phenol (1302) 4-(benzyloxy)-3-(3-phenylpropoxy)benzaldehyde (1301, 100 g, 289 mmol) was dissolved in methylene chloride (DCM, 900 mL), and meta-chloroperoxybenzoic acid (m-CPBA, 74.7 g, 433 mmol) was added. The mixture was stirred at room temperature for approximately 16 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:5). 4-(benzyloxy)-3-(3-phenylpropoxy)phenol (1302, 66 g) was obtained as an off-white solid.

[0153] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)9.00(s, 1H), 7.44-7.18(m, 10H), 6.81(d, J=8Hz, 1H), 6.39(d, J=2.8Hz, 1H), 6.22(dd, J=8.8Hz and 2.8Hz, 1H), 4.96(s, 2H), 3.90(t, J=6Hz, 2H), 2.74(t, J=8Hz, 2H), 2.01(q, J=5.6Hz, 2H).

[0154] (Step 4) Preparation of (R)-2-((4-(benzyloxy)-3-(3-phenylproxy)phenoxy)methyl)oxirane (A-4) 4-(Benzyloxy)-3-(3-phenylpropoxy)phenol (1302, 40 g, 60 mmol) was dissolved in ethanol (EtOH, 800 mL), followed by the addition of water (40 mL) and potassium hydroxide (KOH, 8.0 g, 143 mmol). (R)-2-(Chloromethyl)oxirane (33.2 g, 359 mmol) was then added and stirred at room temperature for approximately 16 hours. Upon completion of the reaction, water (1600 mL) was added to terminate the reaction, and the compound was extracted with ethyl acetate (1600 mL x 3). The extracted organic layer was washed with brine, and the remaining water was removed with sodium sulfate. Impurities were removed by filtration and then concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:10). Thereafter, a white solid (R)-2-((4-(benzyloxy)-3-(3-phenylproxy)phenoxy)methyl)oxirane (A-4, 30 g) was obtained.

[0155] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)7.46-7.17(m, 10H), 6.93(d, J=8.8Hz, 1H), 6.61(d, J=3.2Hz, 1H), 6.43(dd, J=8.8Hz and 2.8Hz, 1H), 5.02(s, 2H), 4.23(dd, J=7.6Hz and 2.8Hz, 1H), 3.97(t, J=6.4Hz, 2H), 3.75(dd, J=7.2Hz and 1.6Hz, 1H), 3.28(m, 1H), 2.82(dd, J=5.2Hz and 4Hz, 1H), 2.75(t, J=7.6Hz, 2H), 2.73-2.67(m, 1H), 2.01(m, 2H).

[0156] (Step 5) Preparation of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (L-4) 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol (L-3, 5 g, 33 mmol) was dissolved in methylene chloride (DCM, 100 mL), and triethylamine (TEA, 4.1 g, 40 mmol) and di-tert-butyl dicarbonate (BocO, 8.1 g, 37 mmol) were added. The mixture was stirred at room temperature for approximately 16 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (EA / PE = 1:3 to 1:1). The resulting product was tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (L-4, 4.2 g) as a colorless liquid.

[0157] 1 H NMR (DMSO-d6, 400MHz) δ(ppm) 6.74(m, 1H), 4.56(t, J=5.6Hz, 1H), 3.51-3.46(m, 6H), 3.42-3.37(m, 4H), 3.07-3.03(m, 2H), 1.37(s, 9H).

[0158] Step 6) Preparation of tert-butyl (2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (TL-4) tert-Butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (L-4, 300 mg, 1.2 mmol) was dissolved in tetrahydrofuran (THF, 5 mL), followed by the addition of 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP, 509 mg, 1.2 mmol), triphenylphosphine (PPh3, 377 mg, 1.44 mmol), and diisopropyl azodicarboxylate (DIAD, 291 mg, 1.44 mmol). The mixture was stirred at 65 °C for 16 hours. After completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (DCM / MeOH = 100:1 to 50:1). Thereafter, a yellow solid of tert-butyl (2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (TL-4, 200 mg) was obtained.

[0159] (Step 7) Preparation of 2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethan-1-amine (TL-5) tert-Butyl (2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (TL-4, 200 mg, 0.31 mmol) was dissolved in ethyl acetate (EA, 4 mL), and then hydrochloric acid (g) / ethyl acetate (1 mL) was added. The mixture was stirred at room temperature for approximately 2 hours. After the reaction was completed, the reaction was terminated, and the solution was filtered and concentrated. The concentrated solution was purified to obtain a yellow solid, 2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethan-1-amine (TL-5, 120 mg).

[0160] (Step 8) Preparation of (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-((2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)amino)propan-2-ol (PHTPP-1304) 2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethan-1-amine (TL-5, 120 mg, 0.22 mmol) was dissolved in methanol (MeOH, 2 mL), and then (R)-2-((4-(benzyloxy)-3-(3-phenylproxy)phenoxy)methyl)oxirane (A-4, 117 mg, 0.33 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. After the reaction was completed, the mixture was filtered and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). Thereafter, a yellow solid (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-((2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)amino)propan-2-ol (PHTPP-1304, 15 mg) was obtained.

[0161] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 1.99 (m, 2H), 2.73 (m, 6H), 3.55 (m, 8H), 3.80 (m, 5H), 3.94 (t, J=8Hz, 2H), 4.13 (t, J=4Hz, 2H), 4.9 9(s, 2H), 6.40(d, J=8Hz, 1H), 6.55(d, J=4Hz, 1H), 6.89(d, J=8Hz, 1H), 7.04(d, J=8Hz, 2H), 7.18(t, J=4Hz, 3H), 7.27(dd, J=4Hz) and 4Hz, 3H), 7.35(m, 4H), 7.44(m, 5H)7.61(m, 2H), 8.07(s, 1H), 8.42(s, 1H);ESI-MS Calcd m / z for C 51 H 50F6N4O7[M+H] + 945.10 Found 944.97.

[0162] Example 9. Preparation of (R,Z)-4-((2-(2-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol (Baicalein-2204)

[0163] [ka]

[0164] (Step 1) Preparation of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (L-1) 2,2'-(ethane-1,2-diylbis(oxy))diethanamine (L-2, 5 g, 33.7 mmol) was dissolved in methylene chloride (DCM, 100 mL), and di-tert-butyl dicarbonate (BocO, 7.36 g, 33.7 mmol) was added. The mixture was stirred at room temperature for approximately 16 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:3 to 1:1). The resulting product was tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (L-2, 2.2 g) as a colorless liquid.

[0165] (Step 2) Preparation of tert-butyl (Z)-(2-(2-(2-(5,6,7-trihydroxy-2-phenyl-4H-chromen-4-ylidene)amino)ethoxy)ethoxy)ethyl)carbamate (TL-6) tert-Butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (TL-6, 300 mg, 1.21 mmol) was dissolved in methanol (MeOH, 5 mL), and then 5,6,7-trihydroxyflavone (baicalein, 326 mg, 1.21 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). A yellow solid, tert-butyl (Z)-(2-(2-(2-((5,6,7-trihydroxy-2-phenyl-4H-chromen-4-ylidene)amino)ethoxy)ethoxy)ethyl)carbamate (TL-6, 120 mg), was obtained.

[0166] (Step 3) Preparation of (Z)-4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol (TL-7) tert-Butyl (Z)-(2-(2-(2-((5,6,7-trihydroxy-2-phenyl-4H-chromen-4-ylidene)amino)ethoxy)ethoxy)ethyl)carbamate (TL-6, 120 mg, 0.24 mmol) was dissolved in ethyl acetate (EA, 2 mL), and then hydrochloric acid (g) / ethyl acetate (1 mL) was added. The mixture was stirred at room temperature for approximately 2 hours. Upon completion of the reaction, the mixture was extracted with ethyl acetate, filtered, and concentrated. A yellow solid, (Z)-4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol (TL-7, 90 mg), was obtained.

[0167] (Step 4) Preparation of (R,Z)-4-(2-(2-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol (Baicalein-2204) (Z)-4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol (TL-7, 90 mg, 0.22 mmol) was dissolved in methanol (MeOH, 2 mL), and then (R)-2-((3,4-diphenethoxyphenoxy)methyl)oxirane (A-2, 87 mg, 0.22 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. After the reaction was completed, the mixture was filtered and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). Then, a yellow solid (R,Z)-4-((2-(2-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol (Baicalein-2204, 12 mg) was obtained.

[0168] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 2.91 (m, 7H), 3.35(m, 11H), 3.80(m, 3H), 3.96(t, J=8Hz, 2H), 4.08(t, J=8Hz, 2H), 6.32(dd, J=4Hz and 4Hz, 1H), 6.37(s, 1H), 6.47(s, 1H), 6.76(d, J=12Hz, 2H), 7.25(m, 10H), 7.58(t, J=4Hz, 3H), 7.99(d, J=8Hz, 2H), 8.42(s, 1H);ESI-MS Calcd m / z for C 46 H 50 N2O 10 [M+H] + 791.20 Found 790.91

[0169] Example 10. Preparation of (E)-5-(4-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethoxy)styryl)benzene-1,3-diol (Resveratrol-1105)

[0170] [ka]

[0171] (Step 1) Preparation of 2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethan-1-ol (AL-5) 3,4-Bis(benzyloxy)benzaldehyde (1101, 25 g, 78.6 mmol) was dissolved in methanol (MeOH, 250 mL), followed by the addition of 2-(2-(2-aminoethoxy)ethoxy)ethanol (L-3, 11.7 g, 78.6 mmol). The mixture was stirred at 65 °C for 6 hours. After cooling to room temperature, sodium borohydride (NaBH4, 3 g, 78.6 mmol) was added and the mixture was stirred at 50 °C overnight. Water was added to terminate the reaction, and the compound was extracted with ethyl acetate (EtOAc, 50 mL x 3). The extracted compound was washed with brine and the remaining water was removed with sodium sulfate. After filtration, the solution was concentrated, and the concentrated solution was purified by silica gel column chromatography (methylene chloride / methanol = 20:1). Thereafter, a yellow liquid, 2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethan-1-ol (AL-5, 13 g) was obtained.

[0172] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)7.46-7.30(m, 10H), 7.06(d, J=1.6Hz, 1H), 6.97(d, J=8Hz, 1H), 6.82 (d, J=1.2Hz, 1H), 5.10(d, J=4Hz, 4H), 3.61(s, 2H), 3.50-3.39(m, 11H), 2.58(t, J=6Hz, 2H)ESI-MS Calcd m / z for C 27 H 33 N5O5[M+H] + 452.10 Found 451.56

[0173] (Step 2) Preparation of tert-butyl (3,4-bis(benzyloxy)benzyl)(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (AL-6) 2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethan-1-ol (AL-5, 500 mg, 1.11 mmol) was dissolved in methylene chloride (DCM, 6 mL), and triethylamine (TEA, 168 mg, 1.66 mmol) and di-tert-butyl dicarbonate (BocO, 290 mg, 1.33 mmol) were added. The mixture was stirred at room temperature for approximately 4 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:5 to 1:1). Colorless tert-butyl (3,4-bis(benzyloxy)benzyl)(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (AL-6, 520 mg) was obtained.

[0174] (Step 3) Preparation of tert-butyl (E)-(3,4-bis(benzyloxy)benzyl)(2-(2-(2-(4-(3,5-dihydroxystyryl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (ATL-1) tert-Butyl (3,4-bis(benzyloxy)benzyl) (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (AL-6, 300 mg, 0.54 mmol) was dissolved in tetrahydrofuran (THF, 5 mL), followed by the addition of resveratrol (149 mg, 0.65 mmol), triphenylphosphine (PPh3, 214 mg, 0.82 mmol), and diisopropyl azodicarboxylate (DIAD) (165 mg, 0.82 mmol). The mixture was stirred at 65°C for 16 hours. After the reaction was complete, the mixture was filtered and concentrated. The concentrated solution was purified by silica gel column chromatography (methylene chloride / methanol = 100:1 to 50:1). Thereafter, a yellow solid of tert-butyl (E)-(3,4-bis(benzyloxy)benzyl)(2-(2-(2-(4-(3,5-dihydroxystyryl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (ATL-1, 200 mg) was obtained.

[0175] (Step 4) Preparation of (E)-5-(4-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethoxy)styryl)benzene-1,3-diol (Resveratrol-1105) tert-Butyl (E)-(3,4-bis(benzyloxy)benzyl)(2-(2-(2-(4-(3,5-dihydroxystyryl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (ATL-1, 200 mg) was dissolved in ethyl acetate (EA, 4 mL), and then hydrochloric acid (g) / ethyl acetate (1 mL) was added. The mixture was stirred at room temperature for approximately 2 hours. Upon completion of the reaction, the mixture was filtered and concentrated. The concentrated solution was purified by high-resolution liquid chromatography (Prep-HPLC). This afforded a white solid, (E)-5-(4-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethoxy)styryl)benzene-1,3-diol (Resveratrol-1105, 15 mg).

[0176] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 2.58 (t, J=4Hz, 2H), 3.45 (t, J=4Hz, 2H), 3.51 (m, 2H), 3.57 (m, 4H), 3.73 (m, ESI-MS Calcd m / z for C 41 H 43 NO7[M+H] + 662.10 Found 661.80.

[0177] Example 11. Preparation of (R)-2-(4-(benzo[d]thiazol-2-yl)phenyl)-14-(3,4-bis(benzyloxy)phenoxy)-5,8-dioxa-2,11-diazatetradecan-13-ol (BTA-1-1104)

[0178] [ka]

[0179] (Step 1) Preparation of 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl methanesulfonate (L-5) tert-Butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (L-4, 1 g, 4.0 mmol) was dissolved in methylene chloride (DCM, 15 mL), and triethylamine (TEA, 0.486 g, 4.8 mmol) and methanesulfonyl chloride (MSCl, 0.504 g, 4.4 mmol) were added. The mixture was stirred at room temperature for approximately 4 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:5 to 1:1). 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl methanesulfonate (L-5, 1 g) was obtained as a colorless liquid.

[0180] (Step 2) Preparation of tert-butyl (2-(2-(2-((4-(benzo[d]thiazol-2-yl)phenyl)(methyl)amino)ethoxy)ethoxy)ethyl)carbamate (TL-8) 2,2-Dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl methanesulfonate (L-5, 300 mg, 0.92 mmol) was dissolved in dimethyl sulfoxide (DMSO, 5 mL) and then 2-(4'-methylaminophenyl)benzyl Benzothiazole (BTA-1, 220 mg, 0.92 mmol) and potassium tert-butoxide (t-BuOK, 154 mg, 1.37 mmol) were added to the mixture, which was then stirred at 120°C for approximately 16 hours. After the reaction was completed, the mixture was filtered and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). A yellow liquid, tert-butyl (2-(2-(2-((4-(benzo[d]thiazol-2-yl)phenyl)(methyl)amino)ethoxy)ethoxy)ethyl)carbamate (TL-8, 180 mg), was obtained.

[0181] (Step 3) Preparation of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(benzo[d]thiazol-2-yl)-N-methylaniline (TL-9) tert-Butyl (2-(2-(2-((4-(benzo[d]thiazol-2-yl)phenyl)(methyl)amino)ethoxy)ethoxy)ethyl)carbamate (TL-8, 180 mg, 0.38 mmol) was dissolved in ethyl acetate (EA, 4 mL), and then hydrochloric acid (g) / ethyl acetate (1 mL) was added. The mixture was stirred at room temperature for approximately 2 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. A yellow solid, N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(benzo[d]thiazol-2-yl)-N-methylaniline (TL-9, 120 mg), was obtained.

[0182] (Step 4) Preparation of (R)-2-(4-(benzo[d]thiazol-2-yl)phenyl)-14-(3,4-bis(benzyloxy)phenoxy)-5,8-dioxa-2,11-diazatetradecan-13-ol (BTA-1-1104) N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(benzo[d]thiazol-2-yl)-N-methylaniline (TL-9, 120 mg, 0.33 mmol) was dissolved in methanol (MeOH, 2 mL), and then (R)-2-((3,4-bis(benzyloxy)phenoxy)methyl)oxirane (1103, 117 mg, 0.33 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). Thereafter, a yellow solid, (R)-2-(4-(benzo[d]thiazol-2-yl)phenyl)-14-(3,4-bis(benzyloxy)phenoxy)-5,8-dioxa-2,11-diazatetradecan-13-ol (BTA-1-1104, 8 mg), was obtained.

[0183] 1H NMR (DMSO-d6, 400MHz) δ (ppm) 1.15 (d, J=8Hz, 3H), 1.23 (s, 1H), 2.67 (m, 4H) , 3.50(m, 11H), 3.81(m, 4H), 5.01(s, 2H), 5.10(s, 2H), 6.27(d, J=8Hz, 1H), 6.41(d, J=8Hz, 1H), 6.69(m, 3H), 6.91(d, J=12Hz, 1H), 7.38(m, 12H), 7.79( d, J=8Hz, 2H), 7.89(d, J=8Hz, 1H), 7.90(d, J=8Hz, 1H), 8.40(s, 1H);ESI-MS Calcd m / z for C 43 H 47 N3O6S[M+H] + 734.10 Found 733.92.

[0184] Example 12. Preparation of (1E,6E)-1-(4-(2-(2-(2-(((R)-3-(3-(benzyloxy)-4-phenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (Curcumin-1204)

[0185] [ka]

[0186] (Step 1) Preparation of 4-(benzyloxy)-3-phenethoxybenzaldehyde (1201) 4-(Benzyloxy)-3-hydroxybenzaldehyde (A-3, 50 g, 219 mmol) was dissolved in tetrahydrofuran (THF, 1 L), followed by the addition of 2-phenylethanol (32.1 g, 263 mmol), triphenylphosphine (PPh3, 86.2 g, 329 mmol), and diisopropyl azodicarboxylate (DIAD, 66.4 g, 329 mmol). The mixture was stirred at 65°C for approximately 16 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:10). 4-(benzyloxy)-3-phenethoxybenzaldehyde (1201, 25 g) was obtained as a white solid.

[0187] 1 H NMR (DMSO-d6, 400MHz) δ (ppm) 9.82 (s, 1H), 7.52 (dd, J=8Hz and 2Hz, 1H), 7.43-7.40(m, 5H), 7.37-7.32(m, 3H), 7.28-7.21(m, 4H), 5.21(s, 2H), 4.26(t, J=6.4Hz, 2H), 3.05(t, J=6.4Hz, 2H)

[0188] (Step 2) Preparation of 4-(benzyloxy)-3-phenethoxyphenol (1202) 4-(benzyloxy)-3-phenethoxybenzaldehyde (1201, 50 g, 150 mmol) was dissolved in methylene chloride (DCM, 500 mL), followed by the addition of meta-chloroperoxybenzoic acid (m-CPBA, 39 g, 225 mmol). The mixture was stirred at room temperature for approximately 16 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:5). 4-(benzyloxy)-3-phenethoxyphenol (1202, 32 g) was obtained as an off-white solid.

[0189] 1H NMR (DMSO-d6, 400MHz) δ(ppm)9.00(s, 1H), 7.36-7.20(m, 10H), 6.78(d, J=8Hz, 1H), 6.43(d, J=2.8Hz, 1H), 6.22(dd, J=8Hz and 2.8Hz, 1H), 4.86(s, 2H), 4.13(t, J=6.8Hz, 2H), 3.02(t, J=6.4Hz, 2H)

[0190] (Step 3) Preparation of (R)-2-((4-(benzyloxy)-3-phenethoxyphenoxy)methyl)oxirane (1203) 4-(Benzyloxy)-3-phenethoxyphenol (1203, 40 g, 64 mmol) was dissolved in ethanol (EtOH, 800 mL), followed by the addition of water (40 mL) and potassium hydroxide (KOH, 8.2 g, 146 mmol). (R)-2-(Chloromethyl)oxirane (34.6 g, 374 mmol) was then added and the mixture was stirred at room temperature for an additional 16 hours. The reaction was terminated by the addition of water (1600 mL) and extracted with ethyl acetate (1600 mL x 3). The extracted organic layer was washed with brine, and the remaining water was removed with sodium sulfate. The mixture was filtered to remove impurities and concentrated. The concentrated solution was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:15 to 1:10). Thereafter, a white solid (R)-2-((4-(benzyloxy)-3-phenethoxyphenoxy)methyl)oxirane (1203, 34 g) was obtained.

[0191] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)7.38-7.20(m, 10H), 6.90(d, J=8.8Hz, 1H), 6.64(d, J=2Hz, 1H), 6.41(dd, J=8.8Hz and 2.8Hz, 1H), 4.93(s, 2H), 4.25-4.17(m, 3H), 3.75(dd, J=11.2Hz and 6.4Hz, 1H), 3.28(m, 1H), 3.03(t, J=6.8Hz, 2H), 2.81(t, J=4.4Hz, 1H), 2.67(dd, J=5.2Hz and 2.8Hz, 1H)

[0192] (Step 4) Preparation of tert-butyl (2-(2-(2-(4-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxohepta-1,6-dien-1-yl)-2-methoxyphenoxy)ethoxy)ethoxy)ethyl)carbamate (TL-10) tert-Butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (L-4, 300 mg, 1.2 mmol) was dissolved in tetrahydrofuran (THF, 5 mL), followed by the addition of curcumin (442 mg, 1.2 mmol), triphenylphosphine (PPh3, 377 mg, 1.44 mmol), and diisopropyl azodicarboxylate (DIAD, 291 mg, 1.44 mmol). The mixture was stirred at 65°C for approximately 16 hours. After the reaction was complete, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by silica gel column chromatography (methylene chloride / methanol = 100:1 to 50:1). Thereafter, a yellow solid, tert-butyl (2-(2-(2-(4-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxohepta-1,6-dien-1-yl)-2-methoxyphenoxy)ethoxy)ethoxy)ethyl)carbamate (TL-10, 200 mg) was obtained. .

[0193] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)9.87(brs, 1H), 7.57(dd, J=16Hz and 4Hz, 2H), 3.34(dd, J=12Hz and 1.6Hz, 2H), 7.24(dd, J=8Hz and 1.2Hz, 1H), 7.16(dd, J=8Hz and 1.2Hz, 1H), 7.02(d, J=8Hz, 1H), 6.85-6.74(m, 4H), 6.08(s, 1H), 4.13(t, J=4Hz, 2H), 3.83(m, 6H), 3.75( t, J=4Hz, 2H), 3.59(q, J=4Hz, 2H), 3.52(q, J=4Hz, 2H), 3.40-3.38(m, 4H), 3.06(qJ=6Hz, 2H), 1.36(s, 9H)

[0194] (Step 5) Preparation of (1E,6E)-1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (TL-11) tert-Butyl (2-(2-(2-(4-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxohepta-1,6-dien-1-yl)-2-methoxyphenoxy)ethoxy)ethoxy)ethyl)carbamate (TL-10, 200 mg, 0.33 mmol) was dissolved in ethyl acetate (EA, 4 mL), and then hydrochloric acid (g) / ethyl acetate (1 mL) was added. The mixture was stirred at room temperature for approximately 2 hours. After the reaction was complete, the compound was extracted, filtered, and concentrated. Thereafter, a yellow solid, (1E,6E)-1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (TL-11, 120 mg), was obtained.

[0195] (Step 6) Preparation of (1E,6E)-1-(4-(2-(2-(2-(((R)-3-(3-(benzyloxy)-4-phenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (Curcumin-1204) (1E,6E)-1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (TL-11, 120 mg, 0.24 mmol) was dissolved in methanol (MeOH, 2 mL), and then (R)-2-((4-(benzyloxy)-3-phenethoxyphenoxy)methyl)oxirane (1203, 90 mg, 0.24 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). Thereafter, a yellow solid, (1E,6E)-1-(4-(2-(2-(2-(((R)-3-(3-(benzyloxy)-4-phenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (Curcumin-1204, 12 mg), was obtained.

[0196] 1 H NMR (DMSO-d6+D2O, 400MHz) δ (ppm) 2.81 (m, 1H), 2.92 (m, 3H), 3.00 (t, J=6.4Hz, 2H), 3.56 (m, 9H) ), 3.80(m, 9H), 3.97(b, 2H), 4.09(m, 2H), 4.14(t, J=6.4Hz, 2H), 4.88(s, 2H), 6.36(dd, J=8.8Hz and ESI-MS Calcd m / z for C 51 H 57 NO 12 [M] + 876.10 Found 876.0 1

[0197] Example 13. Preparation of (R)-1-(3-phenethoxyphenoxy)-3-((2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)amino)propan-2-ol (Riluzole-204)

[0198] [ka]

[0199] (Step 1) Preparation of 3-phenethoxyphenol (A-5) Resorcinol (50 g, 0.45 mol) was dissolved in acetonitrile (ACN, 500 mL), and potassium carbonate (K2CO3, 112.5 g, 0.81 mol) and (2-bromoethyl)benzene (83.2 g, 0.45 mol) were added. The mixture was stirred at 60°C for approximately 10 hours. After the reaction was complete, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1). 25 g of 3-phenethoxyphenol (A-5) was obtained as a yellow liquid.

[0200] 1 H NMR(CDCl3,400MHz)δ(ppm)7.31-7.23(m, 5H), 7.11(t, J=8Hz, 1H), 6.50-6.39(m, 3H), 4.74(s, 1H), 4.14(m, 2H), 3.08(t, J=7.2Hz, 2H)

[0201] (Step 2) Preparation of (R)-2-((3-phenethoxyphenoxy)methyl)oxirane (A-6) 3-Phenethoxyphenol (A-5, 25 g, 116.8 mmol) was dissolved in ethanol (EtOH, 500 mL), and then water (25 mL) and potassium hydroxide (KOH, 11.1 g, 278.3 mmol) were added. (R)-2-(chloromethyl)oxirane (64.6 g, 698.8 mmol) was then added and the mixture was stirred at room temperature for approximately 16 hours. The reaction was terminated by adding water (1000 mL), and ethyl acetate (500 mL × The compound was extracted using 3). The extracted organic solvent layer was washed with brine, and the remaining water was removed with sodium sulfate. The solution was filtered to remove impurities and concentrated. The concentrated solution was purified by column chromatography using silica gel (ethyl acetate / petroleum ether = 1:15 to 1:10). After that, a yellow liquid, (R)-2-((3-phenethoxyphenoxy)methyl)oxirane (A-6, 18 g), was obtained.

[0202] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)7.32-7.28(m, 4H), 7.24-7.14(m, 2H), 6.52(m, 3H), 4.29(dd, J=11.2Hz and 2Hz, 1H), 4.16(t, J=6.8Hz, 2H), 3.79(m, 1H), 3.34(s, 1H), 3.01(t, J=6Hz, 2H), 2.82(t, J=4Hz, 1H), 2.69-2.67(m, 1H)

[0203] (Step 3) Preparation of tert-butyl (2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate (TL-12) 2,2-Dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl methanesulfonate (L-5, 300 mg, 0.92 mmol) was dissolved in dimethyl sulfoxide (DMSO, 5 mL), followed by the addition of riluzole (214 mg, 0.92 mmol) and potassium tert-butoxide (t-BuOK, 154 mg, 1.37 mmol). The mixture was stirred at 120 °C for approximately 16 hours. After the reaction was complete, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). A pale yellow liquid, tert-butyl (2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate (TL-12, 180 mg), was obtained.

[0204] 1 H NMR (DMSO-d6, 400MHz) δ(ppm)8.27-8.25(m, 1H), 7.78(d, J=1.6Hz, 1H), 7.41(d, J=8Hz, 1H), 7.18(dd, J=8Hz and 1.6Hz, 1H), 6.74(m, 1H), 3.63-3.51(m, 12H), 3.05(q, J=6Hz, 2H), 1.36(s, 11H).

[0205] (Step 4) Preparation of N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-6-(trifluoromethoxy)benzo[d]thiazol-2-amine (TL-13) tert-Butyl (2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate (TL-12, 180 mg, 0.39 mmol) was dissolved in ethyl acetate (EA, 4 mL), and then hydrochloric acid (g) / ethyl acetate (1 mL) was added. The mixture was stirred at room temperature for approximately 2 hours. Upon completion of the reaction, the compound was extracted, filtered, and concentrated. A yellow solid, N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-6-(trifluoromethoxy)benzo[d]thiazol-2-amine (TL-13, 120 mg), was obtained.

[0206] (Step 5) Preparation of (R)-1-(3-phenethoxyphenoxy)-3-((2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)amino)propan-2-ol (Riluzole-204) N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-6-(trifluoromethoxy)benzo[d]thiazol-2-amine (TL-13, 120 mg, 0.33 mmol) was dissolved in methanol (MeOH, 2 mL), and then (R)-2-((3-phenethoxyphenoxy)methyl)oxirane (A-6, 89 mg, 0.33 mmol) was added. The mixture was stirred at 65°C for approximately 16 hours. After the reaction was complete, the compound was extracted, filtered, and concentrated. The concentrated solution was purified by preparative high-performance liquid chromatography (Prep-HPLC). A colorless liquid, (R)-1-(3-phenethoxyphenoxy)-3-((2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)amino)propan-2-ol (Riluzole-204, 15 mg), was obtained.

[0207] 1 H NMR(400MHz, DMSO-d6)δ(ppm)2.60(m, 4H), 3.00(t, J=8Hz, 2H), 3.52(m, 10H), 3.85(m, 3H), 4.15(t, J=8Hz, 2H), 6.48(dd, J=12Hz and 8Hz, 3H), 7.18(m, 3H), 7.31(d, J=4Hz, 4H), 7.43(d, J=12Hz, 1H), 7.75(s, 1H);ESI-MS Calcd m / z for C 31 H 36 F3N3O6S[M+H] + 636.10 Found 635.70

[0208] Experimental Example 1: Evaluation of p62 protein oligomers and activity in cultured cells by immunoblotting To evaluate the efficacy of Compounds 1-13 (Examples 1-13) in the p62 protein oligomerization activity, a human embryonic kidney-derived cell line, HEK293, was prepared. The compounds of the Examples and the p62 ligand compound, YTK-1105, were treated, and DMSO was used as a control.

[0209] To measure the intracellular p62 protein activation and oligomerization upon treatment with these compounds, cells were plated into 100-well dishes. After an additional 24 hours of incubation to allow the cells to fully attach to the plate surface, the cells were collected and lysed by adding 100 μL of lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 2 mM NaF, 2 mM EDTA, 2 mM beta-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aproteinin) to each sample. Based on the measured total protein concentration, each sample was treated with the test compound for 2 hours at room temperature, and then sample buffer was added and incubated at 95°C for 10 minutes. After the incubation, 25 μL of each sample was dispensed into each well of an acrylamide gel and subjected to immunoblotting. Immunoblotting is shown as a representative graph from at least three independent experiments. Results are shown in Figures 2a-2c.

[0210] As can be seen from Figures 2a to 2c, when treated with the AutoTack chimeric compound according to the present invention, as with the p62 ligand compound YTK-1105, unlike the control group treated with DMSO, a decrease in p62 protein monomers was observed, along with an increase in oligomers and high-molecular aggregates.

[0211] Experimental Example 2: Evaluation of target protein degradation in cultured cells by immunoblotting To evaluate the target protein degradation efficacy of the compounds (Examples 1-13), cell lines (MCF7, NTERA-2, ACHN, U87-MG, LNCaP, HEK293T) or recombinant cell lines (SH-SY5Y-tau, HeLa-HttQ97, PC12-a-synA30P) expressing the target proteins were cultured in 12-well plates and treated with the corresponding AutoTak chimeric compounds at various concentrations. Immunoblotting was performed as described in Experimental Example 1. Immunoblotting results were shown as representative graphs from at least three independent experiments. The results are shown in Figures 3a, 3b, and 3c.

[0212] As can be seen from Figures 3a, 3b and 3c, the AutoTack chimeric compound of the present invention When the compound was treated, it was confirmed that the amount of the target protein gradually decreased depending on the concentration of the compound.

[0213] Experimental Example 3: Evaluation of target protein degradation mechanisms in cultured cells by immunoblotting To assess whether the target protein degradation mechanism of the compounds (Examples 1-13) is mediated by autophagy, cell lines or recombinant cell lines expressing the target proteins were cultured in 12-well plates and treated with 2.5 μM of the corresponding AutoTak chimeric compound alone or with 10 μM of hydroxychloroquine (HCQ), an inhibitor of the autophagy-lysosome pathway. Immunoblotting was performed as in Experimental Example 1. Immunoblotting was performed using representative data from at least three independent experiments. The results are shown in Figures 4a and 4b.

[0214] As can be seen from Figures 4a and 4b, when the AutoTack chimeric compound according to the present invention was treated alone, the amount of the target protein decreased, and when it was treated simultaneously with HCQ, an inhibitor of the autophagy-lysosome pathway, the amount of the decreased target protein increased again.

[0215] Experimental Example 4: Comparative evaluation of target protein degradation efficacy in cultured cells by immunoblotting To evaluate whether the target protein degradation efficacy of the compounds (Examples 1-13) was superior to that of the chimeric components, p62 ligand and target protein ligand, cell lines or recombinant cell lines expressing the target protein were cultured in 12-well plates and treated with the corresponding AutoTack chimeric compound, p62 ligand, or target protein ligand, followed by immunoblotting as in Experimental Example 1. Immunoblotting was performed using representative data from at least three independent experiments, and the results are shown in Figures 5a-c.

[0216] As can be seen from Figures 5a to 5c, after treatment with the AutoTack chimeric compound according to the present invention, the amount of target protein was significantly reduced compared to treatment with the p62 ligand or target protein ligand.

[0217] Experimental Example 5: Evaluation of p62-mediated autophagy transduction of target proteins in cultured cells by immunofluorescence staining and confocal microscopy To evaluate the efficacy of the compounds (Examples 1-13) in transferring target proteins to p62-mediated autophagy, immunofluorescence staining was performed using p62 and each target protein as a marker. For immunofluorescence staining, cell lines expressing the target proteins natively or recombinant cell lines were cultured on cover glasses in 24-well plates. The cells were then aliquoted and cultured for 24 hours, followed by treatment with 2.5 μM of the novel AutoTAK chimeric ligand according to the present invention. To confirm the activity of the compounds, the medium was removed after an additional 24-hour culture, and the cells were fixed with formaldehyde at room temperature. To prevent nonspecific staining, the cells were incubated with blocking solution for 1 hour at room temperature, followed by treatment with p62 antibody and target protein antibody diluted at a fixed ratio in blocking solution, followed by incubation at room temperature for 1 hour. After antibody treatment, the cells were washed three times with PBS and incubated with a chlorine-derived secondary antibody diluted at a fixed ratio in blocking solution for 30 minutes at room temperature. After washing again with PBS three times, cells were stained with DAPI for intracellular nuclei and then examined by confocal microscopy for the expression, intracellular puncta, and intracellular colocalization of p62 and LC3. The results are shown in Figures 6a-c. Immunofluorescence staining is shown as a representative graph from at least three independent experiments.

[0218] As can be seen from Figures 6a-c, after treatment with the AutoTAC chimeric compound according to the present invention, the intracellular speck formation of p62 protein, the intracellular speck formation of target proteins, and the colocalization of these were confirmed to increase.

Claims

[Claim 1] A compound represented by a compound selected from the group consisting of compounds 1 to 13 below. 1) (2E,4E,6E,8E)-N-(2-(2-(2-(((R)-3-(3,4-bis(benzyloxy)phenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide; 2) (2E,4E,6E,8E)-N-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethyl)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide; 3) (R)—N-(15-(3,4-bis(benzyloxy)phenoxy)-14-hydroxy-3,6,9-trioxa-12-azapentadecyl)-4-phenylbutanamide; 4) N-(1-(3,4-bis(benzyloxy)phenyl)-5,8,11-trioxa-2-azatridecan-13-yl)-4-phenylbutanamide; 5) 3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(2-(2-(2-((3-((4-fluorobenzyl)oxy)benzyl)amino)ethoxy)ethoxy)ethyl)aniline; 6) (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-1-yl)oxiran-2-yl)-1-oxaspiro[2.5]octan-6-yl(13E,15E,17E,19E)-1-(3-(benzyloxy)phenyl)-12-oxo-5,8-dioxa-2,11-diazahenicosa-13,15,17,19-tetraene-21-oate; 7) 3-(3,5-dichlorophenyl)-5-((R)-15-(3,4-diphenethoxyphenoxy)-14-hydroxy-6,9-dioxa-3,12-diazapentadecyl)-5-methyloxazolidine-2,4-dione; 8) (R)-1-(4-(benzyloxy)-3-(3-phenylpropoxy)phenoxy)-3-((2-(2-(2-(4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)ethoxy)ethoxy)ethyl)amino)propan-2-ol; 9) (R,Z)-4-((2-(2-(2-((3-(3,4-diphenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethyl)imino)-2-phenyl-4H-chromene-5,6,7-triol; 10) (E)-5-(4-(2-(2-(2-((3,4-bis(benzyloxy)benzyl)amino)ethoxy)ethoxy)ethoxy)styryl)benzene-1,3-diol; 11) (R)-2-(4-(benzo[d]thiazol-2-yl)phenyl)-14-(3,4-bis(benzyloxy)phenoxy)-5,8-dioxa-2,11-diazatetradecan-13-ol; 12) (1E,6E)-1-(4-(2-(2-(2-(((R)-3-(3-(benzyloxy)-4-phenethoxyphenoxy)-2-hydroxypropyl)amino)ethoxy)ethoxy)ethoxy)-3-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione; and, 13) (R)-1-(3-phenethoxyphenoxy)-3-((2-(2-(2-((6-(trifluoromethoxy)benzo[d]thiazol-2-yl)amino)ethoxy)ethoxy)ethyl)amino)propan-2-ol.

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