Novel P62 ligand compound and composition for preventing, ameliorating or treating protein conformational diseases comprising the same
阅读说明：本技术 新颖的p62配体化合物及包含其的用于预防、改善或治疗蛋白质构象病的组合物 (Novel P62 ligand compound and composition for preventing, ameliorating or treating protein conformational diseases comprising the same ) 是由 权容兑 池昌勋 斯利尼瓦斯劳·甘尼皮瑟蒂 金希妍 文修兰 郑灿勋 郑宜静 成基云 于 2019-07-24 设计创作，主要内容包括：本发明涉及一种新颖的p62配体化合物或者其立体异构体、水合物、溶剂化物或前药；以及包含其作为活性成分的用于预防或治疗蛋白质构象病的药物或食品组合物。根据本发明的p62配体化合物可有效地用作通过活化细胞中的自噬且因此选择性地消除活体内蛋白质、细胞器合聚集体来预防、改善或治疗各种蛋白质构象病的药物组合物。(The present invention relates to a novel p62 ligand compound or a stereoisomer, hydrate, solvate or prodrug thereof; and a pharmaceutical or food composition for preventing or treating a protein conformation disease comprising the same as an active ingredient. The p62 ligand compound according to the present invention can be effectively used as a pharmaceutical composition for preventing, improving or treating various protein conformational diseases by activating autophagy in cells and thus selectively eliminating in vivo proteins, organelle aggregates.)
1. A p62 ligand compound represented by the following chemical formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof:
[ chemical formula 1]
In the chemical formula 1, the first and second,
w is a C6 to C10 aryl group;
l is-O- (CH)2)n1-CH (OH) -, with the proviso that the-O- (CH)2)n1O in-CH (OH) -wherein n1 is an integer of 1 to 4, is bonded to a benzene ring;
m is an integer of 0 to 2;
Rais R1OR-OR1，
Wherein R is1Is hydrogen or- (CH)2)n2-R'1，
R'1Is unsubstituted or substituted phenyl: hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino group(s),
n2 is an integer from 1 to 6;
Rbis-OR2，
Wherein R is2Is hydrogen or- (CH)2)n3-R'2，
R'2Is unsubstituted or substituted phenyl: hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino group(s),
n3 is an integer from 1 to 6;
Rcis- (CH)2)n4-OH、-(CH2)n4-NH-C(＝NH)NH2、-C(＝NH)NH2、C1-6Alkyl, -CH (R)3)-COO-R4or-CH (COO-R)4)-CH2CH2CH2-NH-C(＝NH)NH2、-(CH2)n4-O-(CH2)n4-OR4、-CONH(CH2)n4-OR4、-CO(CH2)n5-OR4、-(CH2)n5-CH(NH2)-COOR4、-(CH2)n5-CONHR4，
n4 is an integer from 2 to 4,
n5 is an integer from 1 to 4,
R3is hydrogen or C1-4Alkyl radical, and
R4is hydrogen or C1-4An alkyl group.
2. The compound of claim 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof, wherein W is phenyl and n1 is 1 or 2.
3. The compound, pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein RaIs hydrogen or-O- (CH)2)n2-R'1，R'1Is unsubstituted or substituted phenyl: hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino, and n2 is an integer from 1 to 4.
4. The compound, pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein RbIs hydroxy or-O- (CH)2)n3-R'2，R'2Is unsubstituted or substituted phenyl: hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino, and n3 is an integer from 1 to 4.
5. The compound, pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof according to claim 1, wherein RcIs- (CH)2)n4-OH、-(CH2)n4-NH-C(＝NH)NH2、-C(＝NH)NH2Methyl, ethyl or isopropyl, and n4 is an integer from 2 to 3.
6. The compound of claim 1, a pharmaceutically acceptable salt, a stereoisomer, a hydrate, a solvate, or a prodrug thereof, wherein the compound of chemical formula 1 is selected from the group consisting of the following compounds 1) to 19):
1) (R) -1- (3,4- (bis (benzyloxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YOK-1106);
2) (R) -1- (3,4- (bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-1);
3) (R) -1- (3- ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-2);
4) (R) -1- (3- ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-7);
5) (R) -1- (3- ((4-fluorobenzyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-8);
6) (R) -1- (2- ((3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-1109);
7) (R) -1- (2- ((3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-2209);
8) (R) -1- (2- ((3- (3, 4-bis (4-chlorophenylmethyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-1);
9) (R) -1- (2- ((3- (3, 4-bis (4-fluorobenzyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-2);
10) (R) -1- (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) guanidine (YOK-1107);
11) (R) -1- (3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) guanidine (YOK-2207);
12) (R) -1- (3, 4-bis (benzyloxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1104);
13) (R) -1- (3, 4-diphenylethoxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-2204);
14) (R) -1- (3, 4-bis (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-3304);
15) (R) -1- (3, 4-bis (4-phenylbutoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-4404);
16) (R) -1- (4- (benzyloxy) -3-phenethyloxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-1204);
17) (R) -1- (4- (benzyloxy) -3- (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1304);
18) (R) -1- (3, 4-bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-1); and
19) (R) -1- (3, 4-bis ((4-fluorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-2).
7. A pharmaceutical composition for preventing, improving or treating a protein conformational disease, comprising the compound of chemical formula 1, pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof, according to any one of claims 1 to 6.
8. The pharmaceutical composition of claim 7, wherein the protein conformation disease is a neurodegenerative disease, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, or cystic fibrosis.
9. The pharmaceutical composition of claim 8, wherein the neurodegenerative disease is at least one selected from the group consisting of: lyme borreliosis, 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), spinocerebellar ataxia, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type 2 diabetes, injection of local amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, and genetic systemic amyloidosis of the finland type.
10. A pharmaceutical composition for enhancing autophagy activity of a misfolded protein, comprising the compound of chemical formula 1 of any one of claims 1 to 6, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof.
11. The pharmaceutical composition of claim 10, wherein the protein is at least one selected from the group consisting of: cancer inducing proteins, prion proteins, Amyloid Precursor Protein (APP), alpha-synuclein, superoxide dismutase 1, tau protein, immunoglobulin, amyloid A, transthyretin, beta 2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, amylin, ANF, peptin, insulin, lysozyme, fibrinogen, Huntington protein and spinocerebellar ataxia protein and other proteins with poly-Q extensions.
12. A food composition for preventing, improving or treating a protein conformational disease, comprising the compound of chemical formula 1, pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof, according to any one of claims 1 to 6.
13. The food composition of claim 12, wherein the protein conformation disease is a neurodegenerative disease, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, or cystic fibrosis.
14. The food composition of claim 13, wherein the neurodegenerative disease is at least one selected from the group consisting of: lyme borreliosis, 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), spinocerebellar ataxia, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type 2 diabetes, injection of local amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, alexander disease, and finnish-type hereditary systemic amyloidosis.
15. A method for increasing degradation of misfolded protein aggregates, comprising treating a cell or a p62 protein with the p62 ligand compound of any one of claims 1 to 6.
16. A method for activating autophagy comprising treating a cell or a p62 protein with a p62 ligand compound of any one of claims 1 to 6.
17. A method for preventing, ameliorating or treating a protein conformation disease comprising treating a cell or a p62 protein with the p62 ligand compound of any one of claims 1 to 6, comprising administering the compound to a subject in need thereof.
18. The method of claim 17, wherein the protein conformation disease is cancer, neurodegenerative disease, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, or cystic fibrosis.
19. The method of claim 18, wherein the neurodegenerative disease is at least one selected from the group consisting of: lyme borreliosis, 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), spinocerebellar ataxia, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type 2 diabetes, injection of local amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, alexander disease, and finnish-type hereditary systemic amyloidosis.
Technical Field
The present invention relates to a novel p62 ligand compound and a pharmaceutical or food composition for preventing or treating protein conformational diseases, comprising the same.
Background
The N-terminal canonical pathway is a proteolytic system in which a specific N-terminal residue of the protein serves as a degradation signal (fig. 1). The N-terminal regular degradation signal is through the type I basic residues including N-terminal arginine (Nt-Arg), lysine (Nt-Lys) and histidine (Nt-His); and type II hydrophobic residues including phenylalanine (Nt-Phe), leucine (Nt-Leu), tryptophan (Nt-Trp), tyrosine (Nt-Tyr), and isoleucine (Nt-Ile). These N-terminal residues bind to a specific N-recognizer (hereinafter referred to as N-ligand).
The present inventors have for the first time discovered or cloned previously known N recognizers, namely UBR1, UBR2, UBR4 and UBR5, and found that they utilize the UBR cassette as a substrate recognition domain (Tasaki, T., et al, Mol Cell Biol 25,7120-36 (2005)). The inventors have also found that the UBR cassette binds to type I N-terminal canonical ligands (Nt-Arg, Nt-Lys, Nt-His) such as the N-terminal arginine residue to recognize the substrate and link the ubiquitin chain to this substrate. It was further found that UBR1 and UBR2 have N domains (Sriram, S.M., Kim, B.Y. & Kwon, Y.T., Nat Rev Mol Cell Biol 12,735-47(2011)) that play an important role in the binding of type 2N-terminal canonical ligands (Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, and Nt-Ile). The ubiquitinated substrate resulting from the binding between the N-recognizer and the N-terminal canonical ligand is delivered to the proteasome where it is 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) are the primary determinants of binding, as the N-recognizer provides the majority of the hydrogen bonds required to target the N-terminal regular substrate (Sriram, S.M. & Kwon, y.t., Nat Struct Mol Biol 17,1164-5 (2010)).
If a misfolded protein that is poorly folded in a cell is retained for a long time, it is aggregated and blocks proteasome function or reduces other cellular functions that cause cytotoxic substances, and thus the misfolded protein is ubiquitinated by ubiquitin conjugating enzymes and delivered to proteasomes for degradation (Ji, C.H. & Kwon, y.t., Mol Cells 40,441-449 (2017)). In normal cells, this process is smooth and minimizes damage caused by misfolded proteins, while in senescent neurons it is slowed down so that ubiquitinated misfolded proteins accumulate and these excess protein waste is converted back into aggregates (Ciechanover, a. & Kwon, y.t., Exp Mol Med 47, e147 (2015)). In addition, neuronal cells of patients suffering from degenerative brain diseases such as huntington's disease, parkinson's disease, human mad cow disease, greige's disease, and the like have a strong property of converting specific mutant proteins into aggregates even in protein conformation diseases, and thus are not degraded by the proteasome as described above. The reason for this is that, since the proteasome has a narrow inner diameter of about 13 angstroms, a misfolded protein must be unfolded, and when the protein is aggregated, it is not unfolded.
Meanwhile, autophagy (autophagy) is a major intracellular protein degradation system in addition to the ubiquitin-proteasome system. Autophagy is a protein degradation process that substantially maintains cell homeostasis and gene stability by degrading organelles of aging or damaged Cells or proteins that are damaged or abnormally folded (Ji, C.H. & Kwon, y.t., Mol Cells 40,441- & 449 (2017)). In particular, when misfolded protein aggregates accumulate in the cytoplasm, they may become cytotoxic substances and therefore should be received and degraded by autophagy. The mechanisms of autophagy are mainly divided into major autophagy (macroautophagy), minor autophagy (microaautophagy) and concomitant protein-mediated autophagy, and they are divided into major autophagy and selective autophagy depending on the purpose of degrading intracellular substrates (Dikic, I. & Elazar, z., Nat Rev Mol Cell Biol 19, 349-. Among these, selective autophagy and the accompanying protein-mediated autophagy cause the selective degradation of undesirable intracellular proteins and dysfunctional organelles. The development of new therapies based on diseases that accumulate pathologically misfolded proteins and dysfunctional organelles by inducing selective autophagy is currently establishing a new paradigm. The p62/SQSTM1/Sequestosome-1 protein is crucial for initiating the formation of autophagosomes as mediators in the mechanism of selective autophagy and for delivering the contents. At this time, p62/SQRSM1/Sequestosome-1 binds to misfolded proteins and aggregates thereof, and is thus delivered to autophagosomes. p62 undergoes self oligomerization as a key process in delivering misfolded proteins to autophagosomes (Ji & Kwon, Y.T., Mol Cells 40, 441-' 449 (2017)). At this point, the misfolded proteins are concentrated together to reduce volume, thereby promoting degradation using autophagy. The PB1 region regulates the self-oligomerization of p62, but the mechanism of regulation is not well known. Misfolded protein-p 62 conjugates delivered to autophagosomes can be degraded by lysosomal enzymes when the autophagosomes bind to lysosomes. Through the mechanisms described above, autophagy is critical for maintaining cell homeostasis by modulating intracellular changes in damaged proteins and cellular organelles. When autophagy function is diminished, it leads to accumulation and aggregation of misfolded proteins, resulting in protein conformation disease (2015) (Ciechanover, a. & Kwon, y.t., Exp Mol Med 47, e 147). The key technology of the present invention is to provide a method for effectively eliminating misfolded proteins or aggregates thereof that cause protein conformational diseases. For this purpose, it is necessary to activate only selective autophagy and not to activate host autophagy, which has a broad effect on various biological pathways.
Active studies have been conducted on activation of autophagy to treat protein conformational diseases. The regulator that normally inhibits autophagy in subjects is mTOR. The method of activating autophagy using mTOR inhibitors is the most widely used (Jung, c.h., Ro, s.h., Cao, j., Otto, N.M. & Kim, d.h., FEBS Lett 584,1287-95 (2010)). In particular, amyloid β (Ab) and tau were eliminated while cognitive ability was simultaneously improved in AD animal models overexpressing APP by treatment with rapamycin (rapamycin) (Caccamo, a., Majumder, s., Richardson, a., Strong, R. & oldo, s., J Biol Chem 285,13107-20 (2010)); elimination of tau protein in an animal model of AD overexpressing tau protein (Rodriguez-Navarro, j.a. et al Neurobiol Dis 39,423-38 (2010); and elimination of overexpressed mutant alpha-synuclein protein aggregates in a PD mouse model (Webb, j.l., Ravikumar, b., Atkins, J., Skepper, J.N. & Rubinsztein, d.c., J Biol Chem 278,25009-13 (2003)). It was confirmed that huntingtin aggregates were effectively eliminated by using CCI-779 (a rapamycin-like substance) in HD mice, thereby improving animal behavior and cognitive ability (Ravikumar, b., Duden, R. & Rubinsztein, d.c., Hum Mol gene 11,1107-17 (2002)). However, mTOR plays an extremely important role in a variety of intracellular pathways including NF-kB. Thus, although it exhibits excellent activity to eliminate misfolded protein aggregates of protein conformation diseases, there is a limit in that these host autophagy activators (mTOR known to regulate host autophagy is a drug target) are used as therapeutic agents.
As described above, no effective therapeutic agent currently exists for the treatment of most protein conformational diseases. In the case where ubiquitin ligase ligands are used to eliminate misfolded proteins as a major cause, it is difficult to eliminate misfolded proteins when they are aggregated. Furthermore, mTOR inhibitors (which are the most commonly used compounds as a major autophagy activator) play a broad role in regulating overall gene expression in cells in response to stimuli from various external environments in addition to regulation of autophagy, and thus have disadvantages in terms of being unsuitable as therapeutic agents. Therefore, there is a need to develop a method for eliminating misfolded protein aggregates without reducing the activity of mTOR (a regulator of host autophagy) by activating p62 (a key regulator of selective autophagy).
Disclosure of Invention
Under these circumstances, the inventors have conducted extensive studies to find a preventive and therapeutic agent for protein conformational diseases, which uses a material that activates autophagy independently of mTOR, and thus found that a ligand that binds to p62, more particularly, to the ZZ domain of p62 binds to LC3 and activates autophagy, resulting in effective elimination of pathogenic protein aggregates such as mutant huntingtin and α -synuclein, and thus it can be used for the prevention, amelioration or treatment of various protein conformational diseases. The present invention has been completed based on these findings.
The invention aims to provide a novel p62 ligand compound for inducing the activation and oligomerization of p62 protein.
It is another object of the present invention to provide a method for elimination by using the aforementioned novel compounds to deliver p62 and a misfolded protein that binds p62 to autophagosomes and finally to lysosomes.
It is another object of the present invention to provide a method for increasing macroautophagy activity via p62 protein by using the aforementioned novel compound.
It is a further object of the present invention to provide a pharmaceutical or food composition for eliminating misfolded protein aggregates comprising the aforementioned novel compound as an active ingredient.
It is another object of the present invention to provide a pharmaceutical or food composition for preventing, ameliorating or treating a protein conformational disease, which comprises the aforementioned novel compound as an active ingredient.
To achieve the above object, one embodiment of the present invention provides a novel compound that acts as a ligand of p62 protein. Preferably, the novel p62 ligand according to the invention binds to the ZZ domain of p 62.
Another embodiment of the present invention provides a pharmaceutical composition for preventing and treating a protein conformation disease such as a neurodegenerative disease; or a healthy functional food for preventing or improving a protein conformational disease, comprising as an active ingredient a ligand that binds to the ZZ domain of the p62 protein.
Another embodiment of the present invention provides: (1) methods of inducing p62 oligomerization and structural activation; (2) a method of enhancing p62-LC3 binding; (3) a method of increasing the delivery of p62 to the autophagosome; (4) a method of activating autophagy; and (5) a method of eliminating misfolded protein aggregates, the method comprising the step of treating a cell or p62 protein with a ligand that binds to the ZZ domain of p 62.
Yet another embodiment of the present invention provides a technique to eliminate misfolded protein aggregates, which are causative agents of degenerative brain diseases, by activating p62 that delivers the misfolded protein aggregates directly to autophagosomes.
The key technology of the present invention is to effectively eliminate misfolded protein aggregates (which cause degenerative brain diseases) by simultaneously activating p62 and autophagy.
The pharmacokinetics and key technology of the present invention are summarized in figure 1.
In particular, as shown in fig. 1, major pathogenic proteins of protein conformational diseases such as mutant huntingtin and α -synuclein are converted into water-insoluble misfolded proteins, and then aggregate with each other and grow into oligomeric aggregates. These misfolded proteins grow further while acting as cytotoxic substances in neurons and subsequently grow into large oligomeric or fibrous aggregates, eventually forming inclusion bodies. In the above process, endoplasmic reticulum associated protein (R) such as BiP is produced in large amounts of Nt-Arg by ATE 1R-transferase via N-terminal arginylation (R), and then arginylated BiP (R-BiP) is translocated into the cytoplasm and binds to misfolded huntingtin or α -synuclein (R). As a ligand, Nt-Arg of R-BiP binds to ZZ domain of p 62. Due to the binding, the abnormally activated closed form of p62 is changed to an open form, resulting in structural activation (iv) and thus exposing the region where PB1 and LC3 bind. This activation results in the formation of oligomers and high molecular weight aggregates due to the disulfide bond of p62 (c), and enhanced binding to the autophagosome marker LC3 that is ultimately delivered to the autolysosome (c). In addition, p62 bound to N-terminal arginine migrates to the endoplasmic reticulum membrane and activates PI 3P-mediated autophagosome biosynthesis ((c)), thereby enhancing intracellular autophagy ((b)).
p62 is the first type of target proposed by the present inventors for autophagy activation (fig. 1, viii). Furthermore, studies that suggest p62 as a drug target for autophagy activation or elimination of protein aggregates in degenerative brain diseases have not been completed before.
Autophagy is a mechanism used to degrade or recover unwanted or depleted cellular components in cells and can contribute to the production of energy and metabolites used in biosynthetic processes under conditions such as nutrient and energy deprivation. The mechanism of autophagy is mainly divided into major autophagy, minor autophagy and concomitant protein-mediated autophagy, and it is divided into bulk autophagy and selective autophagy depending on the purpose of degrading intracellular matrices. Among these, selective autophagy and the accompanying protein-mediated autophagy cause the selective degradation of undesirable intracellular proteins and dysfunctional organelles. The development of new therapies based on diseases that accumulate misfolded proteins and dysfunctional organelles by inducing selective autophagy is currently establishing a new paradigm.
The p62 protein is crucial for initiating the formation of autophagosomes (as a mediator in the mechanism of selective autophagy) and for delivering the contents. It was observed that significant p62 activation of the novel p62 ligand according to the invention induced p62 to oligomerize on its own. In addition, in view of the fact that autophagosome targeting of p62 was increased via this self oligomerization, this suggests that the novel p62 ligands according to the invention can induce targeting and degradation of the p62 protein by intracellular autophagy. These results suggest that the novel p62 ligand compounds according to the present invention can be used as more effective or complementary substitutes for existing anti-protein disease drugs.
Proteolytic Targeting chimeras (PROTAC) are compound chimeras that recognize the ligand for the target protein and the ligand for the E3 ubiquitinase. Since the paradigm for existing therapeutics for diseases is inhibition of proteinases, it is important to develop a new therapeutic for diseases where proteins misfolded that cannot be targeted with existing therapeutics. From these perspectives, PROTAC is an attractive new therapy development approach to achieve selective degradation under the ubiquitin-proteasome system relative to proteins that cannot be targeted using conventional enzyme inhibition approaches. However, current research on PROTAC is limited to the use of the ubiquitin-proteasome system by using only ligands that recognize the E3 ubiquitinase, and thus has folding problems associated with misfolded proteins in the aforementioned proteasome system. In contrast, since the novel p62 ligand according to the present invention is capable of inducing not only intracellular autophagy, but also autophagosome targeting of a cargo substrate protein interacting with p62, it can provide novel therapeutic agents capable of selective degradation under protein autophagy mechanisms that cannot be targeted using conventional enzyme inhibition methods.
The novel compounds according to the present invention act as ligands binding to the ZZ domain of the p62 protein, facilitate the delivery of p62 to autophagosomes, activate autophagy, and eliminate misfolded protein aggregates, and are therefore useful as drugs for the prevention, amelioration, and treatment of various protein conformational diseases.
Drawings
FIG. 1 is a schematic diagram illustrating the binding of an N-terminal regular arginylated protein to the p62ZZ domain and the degradation of intracellular substances such as proteins via intracellular autophagy activity.
FIG. 2 is the result of immunoblot assay showing the effect of increasing p62 protein oligomerization activity of p62 ligand compounds (YOK-1104, YOK-2204, YOK-3304, YOK-1204, YOK-4404, YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-6-2, YT-6-7, YT-6-8, YT-9-1 and YT-9-2) according to the present invention.
FIG. 3 is the result of immunoblot assay showing the effect of increasing autophagy activity of p62 ligand compounds (YOK-1107, YOK-4404, 3301, 3302, and YOK-1104) according to the present invention.
FIG. 4 is the result of immunoblot assay showing the effect of increasing the autophagy activity of p62 ligand compounds (YOK-1106, YOK-1107, YOK-1204, YOK-1304, YOK-2204, YOK-4404) according to the present invention.
Fig. 5 shows the results of immunoblot assays confirming that control compounds (1101, 1102, 1103, 1201, 1202, 1203, 1301, 1302, YTK-1105-1, 4402, 1401, 1402, 2201 and 2202) did not increase autophagy activity.
Fig. 6a and 6b are immunofluorescent staining assay results confirming that p62 ligand compounds (YOK-1106, YOK-1204, YOK-1504, YOK-2204, YOK-3304, and YOK-4404) according to the present invention activate and oligomerize p62 protein, and then show its efficacy in delivering it to autophagy bodies while increasing autophagy activity.
FIG. 7 is the results of immunofluorescence staining assays confirming that control compounds (YOK-A-1104, YOK-G-1104, YTK-1005 and YTK-1105-1) activate and oligomerize the p62 protein and subsequently show its efficacy in delivering it to autophagosomes while simultaneously increasing autophagy activity.
Figures 8a and 8b and figures 9a and 9b are immunofluorescent staining assays showing the efficacy of delivering a target protein for autophagy. This is a result showing that the markers of the target protein, FK2 (FIGS. 8a and 8b) or Huntington Q103-GFP (FIGS. 9a and 9b) were gradually increased in the co-presence of p62 protein and intracellular spots after treatment with the compound.
Detailed Description
Hereinafter, the present invention will be described in more detail.
Definitions of groups used herein are detailed. Unless otherwise indicated, each group has the following definitions.
As used herein, the term "halo" includes fluoro, chloro, bromo, and iodo.
As used herein, "alkyl" refers to a straight or branched chain aliphatic hydrocarbon group, and may preferably be an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 to 4 carbon atoms. Examples of such alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1-dimethylbutyl, 2-dimethylbutyl, 3-dimethylbutyl, and 2-ethylbutyl.
In one aspect of the present invention, there is provided a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof:
[ chemical formula 1]
In the chemical formula 1, the first and second,
w is a C6 to C10 aryl group;
l is-O- (CH)2)n1-CH (OH) -, with the proviso that-O- (CH)2)n1O in-CH (OH) -wherein n1 is an integer of 1 to 4, is bonded to a benzene ring;
m is an integer of 0 to 2;
Rais R1OR-OR1，
Wherein R is1Is hydrogen or- (CH)2)n2-R'1，
R'1Is unsubstituted or substituted phenyl: hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino group(s),
n2 is an integer from 1 to 6;
Rbis-OR2，
Wherein R is2Is hydrogen or- (CH)2)n3-R'2，
R'2Is unsubstituted or substituted benzeneBase: hydroxy, halogen, C1-4Alkyl radical, C1-4Alkoxy, nitro, amino, (C)1-4Alkyl) amino or di (C)1-4Alkyl) amino group(s),
n3 is an integer from 1 to 6;
Rcis- (CH)2)n4-OH、-(CH2)n4-NH-C(＝NH)NH2、-C(＝NH)NH2、C1-6Alkyl, -CH (R)3)-COO-R4or-CH (COO-R)4)-CH2CH2CH2-NH-C(＝NH)NH2、-(CH2)n4-O-(CH2)n4-OR4、-CONH(CH2)n4-OR4、-CO(CH2)n5-OR4、-(CH2)n5-CH(NH2)-COOR4、-(CH2)n5-CONHR4，
n4 is an integer from 2 to 4,
n5 is an integer from 1 to 4,
R3is hydrogen or C1-4Alkyl radicals, and
R4is hydrogen or C1-4An alkyl group.
Preferably, W may be phenyl.
Preferably, n1 can be 1 or 2.
Preferably, RaCan be hydrogen or-O- (CH)2)n2-R'1。
Preferably, R'1Phenyl which may be unsubstituted or substituted by: hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino.
Preferably, n2 may be an integer from 1 to 4.
Preferably, RbCan be hydroxyl or-O- (CH)2)n3-R'2。
Preferably, R'2Phenyl which may be unsubstituted or substituted by: hydroxy, fluoro, chloro, bromo, methyl, ethyl, methoxy, ethoxy, nitro, amino or dimethylamino.
Preferably, n3 may be an integer from 1 to 4.
Preferably, RcCan be- (CH)2)n4-OH、-(CH2)n4-NH-C(＝NH)NH2、-C(＝NH)NH2Methyl, ethyl or isopropyl.
Preferably, n4 may be an integer from 2 to 3.
Preferably, n5 may be an integer from 1 to 3. Preferably, R4May be hydrogen or methyl.
Specifically, representative examples of the compound represented by chemical formula 1 are as follows:
1) (R) -1- (3,4- (bis (benzyloxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YOK-1106);
2) (R) -1- (3,4- (bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-1);
3) (R) -1- (3- ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-2);
4) (R) -1- (3- ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-7);
5) (R) -1- (3- ((4-fluorobenzyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-8);
6) (R) -1- (2- ((3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-1109);
7) (R) -1- (2- ((3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-2209);
8) (R) -1- (2- ((3- (3, 4-bis (4-chlorophenylmethyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-1);
9) (R) -1- (2- ((3- (3, 4-bis (4-fluorobenzyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-2);
10) (R) -1- (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) guanidine (YOK-1107);
11) (R) -1- (3- (3, 4-diphenylethoxyphenoxy) -2-hydroxypropyl) guanidine (YOK-2207);
12) (R) -1- (3, 4-bis (benzyloxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1104);
13) (R) -1- (3, 4-diphenylethoxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-2204);
14) (R) -1- (3, 4-bis (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-3304);
15) (R) -1- (3, 4-bis (4-phenylbutoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-4404);
16) (R) -1- (4- (benzyloxy) -3-phenethyloxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-1204);
17) (R) -1- (4- (benzyloxy) -3- (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1304);
18) (R) -1- (3, 4-bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-1); and
19) (R) -1- (3, 4-bis ((4-fluorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-2).
Also, the compounds of the present invention may exist in the form of pharmaceutically acceptable salts. As salts, addition salts formed by pharmaceutically acceptable free acids can be used. The term "pharmaceutically acceptable salt" as used herein refers to any organic or inorganic addition salt of the compound represented by chemical formula 1, wherein the adverse effects caused by the salt do not impair the beneficial effects of the compound at concentrations that exhibit relatively non-toxic and non-harmful effective activity to patients.
Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the resulting salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, water or an alcohol containing equimolar amounts of the compound and the acid (e.g., ethylene glycol monomethyl ether) may be heated, and then the resulting mixture may be dried by evaporation or the precipitated salt may be filtered under suction.
In this case, the free acid may be an inorganic acid or an organic acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and stannic acid. Examples of organic acids 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, carbonic acid, vanillic acid, and hydroiodic acid.
In addition, bases can be used to prepare pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt and subsequently evaporating the filtrate until drying. At this time, as the metal salt, sodium, potassium or calcium salt is particularly suitable in the pharmaceutical field, but the present invention is not limited thereto. Furthermore, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).
Unless otherwise indicated herein, pharmaceutically acceptable salts of the compounds of the present invention include salts of acidic or basic groups, which may be present in the compounds of formula 1. For example, pharmaceutically acceptable salts include sodium, calcium, and potassium salts of hydroxyl groups; and other pharmaceutically acceptable salts of the amino group including hydrobromide, sulphate, bisulphate, phosphate, hydrogenphosphate, dihydrogenphosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulphonate (methanesulphonate/mesylate) and p-toluenesulphonate (p-tolenesulphonate/tosylate). Salts can be prepared using salt preparation methods known in the art.
The salt of the compound of chemical formula 1 of the present invention is a pharmaceutically acceptable salt, and may be used without particular limitation so long as it is a salt of the compound of chemical formula 1, which exhibits pharmacological activity equivalent to that of the compound of chemical formula 1, for example, neurodegenerative diseases can be prevented or treated by inducing autophagic degradation of intracellular neurodegenerative diseases and tumor-associated proteins through the ligand of p 62.
In addition, the compound represented by chemical formula 1 according to the present invention includes, but is not limited to, not only pharmaceutically acceptable salts thereof, but also all solvates or hydrates and all possible stereoisomers that may be prepared therefrom. All stereoisomers of the compounds of the present invention (e.g., those that may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of the invention. Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as pure or substantially pure optical isomers with the indicated activity) or may, for example, be as racemates or blended with all other stereoisomers or other selected stereoisomers. The chiral center of the compounds of the invention may have an S or R configuration as defined by the IUPAC 1974 recommendation. The racemic forms can be analyzed by physical methods such as fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers may be obtained from the racemates by any suitable method, including but not limited to salt formation with an optically active acid followed by crystallization.
Solvates and stereoisomers of the compound represented by chemical formula 1 may be prepared from the compound represented by chemical formula 1 using methods known in the art.
In addition, the compound represented by chemical formula 1 according to the present invention may be prepared in a crystalline form or an amorphous form, and when the compound is prepared in a crystalline form, it may be optionally hydrated or solvated. In the present invention, the compound of chemical formula 1 may include not only a stoichiometric hydrate but also a compound containing various amounts of water. The solvate of the compound of chemical formula 1 according to the present invention includes both stoichiometric and non-stoichiometric solvates.
The compound of chemical formula 1 according to the present invention may be prepared by the same method as in reaction formulas 1 to 5, which is an exemplary method below, and specific examples thereof are the same as those described in the following examples.
[ reaction formula 1]
[ reaction formula 2]
[ reaction formula 3]
[ reaction formula 4]
[ reaction formula 5]
In the preparation method of the present invention, the reactants used in the reaction formula may be commercially available compounds, or may be synthesized by performing one or more reactions thereof known in the art or by appropriately modifying the reactions. For example, the reactants may be synthesized by performing one or more reactions in a series of orders in consideration of the presence, type and/or position of the reactive functional group and/or hetero element contained in the skeleton structure, but is not limited thereto.
The compound of chemical formula 1 according to the present invention is characterized by functioning as a ligand binding to the ZZ domain of p62 and thus activating the function of p 62. The compound of chemical formula 1 according to the present invention can activate autophagy by activating the function of p 62.
Accordingly, in another aspect, the present invention provides a pharmaceutical composition for autophagy activation, comprising a compound of chemical formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof.
The compound of chemical formula 1 according to the present invention can eliminate aggregated proteins associated with diseases associated with aggregation of misfolded proteins due to activation of autophagy. In addition, the compound of chemical formula 1 is a p62 ligand, which binds to the p62ZZ domain and activates the PB1 region and the LIR region of the p62 protein, such that it induces p62 oligomerization and aggregate formation and increases autophagosome formation by inducing p62 aggregate formation. Through the above process, misfolded proteins can be effectively eliminated (see fig. 1). These proteins may be the major proteins of protein conformational diseases, more preferably at least one selected from the group consisting of: prion protein (prion protein), Amyloid Precursor Protein (APP), alpha-synuclein, superoxide dismutase 1, tau protein (tau), immunoglobulin, amyloid A, transthyretin, beta 2-microglobulin, cystatin C, apolipoprotein A1, TDP-43, islet amyloid polypeptide, ANF, peptin, insulin, lysozyme, fibrinogen, Huntington protein, alpha-1-antitrypsin Z, phacoelenteranin, C9 open reading frame 72(C9orf72), gliobulinic protein, cystic fibrosis transmembrane conductance regulator protein, rhodopsin (rhodosin) and spinocerebellar ataxia protein (ataxin), as well as other proteins with poly-Q extensions.
Accordingly, in a further aspect, the present invention provides a pharmaceutical composition for preventing, improving or treating a protein conformational disease, the pharmaceutical composition comprising a p62 ligand compound of the following chemical formula 1, a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate, or prodrug thereof.
The term "aggregation" according to the present invention refers to the formation of oligomeric or multimeric complexes of typically one or more types of proteins, which may be accompanied by the integration of additional biomolecules, such as carbohydrates, nucleic acids and lipids, into the complex. These aggregated proteins can form deposits in specific tissues, more preferably in neural tissues or brain tissues. The degree of aggregation depends on the particular disease.
The term "protein conformation disease" or "diseases associated with protein aggregation" as used herein refers to those diseases characterized by the presence of aggregated proteins, examples of which include, but are not limited to, neurodegenerative diseases, alpha-1 antitrypsin deficiency, keratopathy, retinitis pigmentosa, type 2 diabetes, cystic fibrosis, and the like.
The neurodegenerative disease herein is preferably selected from the group consisting of: lyme borreliosis (Lyme borreliosis), fatal familial insomnia, Curie's Disease (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), spinocerebellar ataxia, other poly-Q diseases, hereditary cerebral amyloid angiopathy, familial amyloid polyneuropathy, primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), injection of local amyloidosis, beta-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, Alexander Disease, and Finland-type hereditary systemic amyloidosis.
The dosage of the pharmaceutical composition of the present invention can vary widely depending on the body weight, age, sex or health condition of the patient, diet, administration period, administration method, excreta and severity of disease. However, effective doses are typically about 1ng to 10 mg/day, and especially about 1ng to 1 mg/day for an adult (60 kg). Since the dosage may vary depending on various conditions, it will be apparent to one skilled in the relevant art that the dosage may be increased or decreased. Thus, the scope of the invention is not limited in any way by the foregoing dosages. With respect to the number of administrations, the administration may be performed once per day or divided into several administrations within a desired range, and the administration period is not particularly limited or not.
As used herein, the term "treatment" refers to all measures for alleviating or advantageously altering the symptoms of various diseases associated with misfolded protein aggregation, such as protein conformation diseases, by administering the pharmaceutical composition of the invention.
As described above, the compounds of the present invention exhibit the following effects: (1) inducing p62 oligomerization and structural activation; (2) enhancing p62-LC3 binding, and (3) enhancing the delivery of p62 to the autophagosome; (4) activating autophagy; and finally (5) elimination of misfolded protein aggregates. Therefore, a pharmaceutical composition containing this compound as an active ingredient is useful for preventing, ameliorating or treating diseases associated with aggregation of various misfolded proteins.
For example, the compositions of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The composition may be used in various forms such as oral dosage forms of powder, granule, troche, capsule, suspension, emulsion, syrup, spray, and injection of sterile injectable solution, which are formulated by conventional methods according to the purpose of each of the intended uses. The compositions can be administered via a variety of routes, including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. Examples of suitable carriers, excipients, or diluents that may be included in such compositions may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In addition, the composition of the present invention may further include fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, preservatives and the like.
Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and these solid dosage forms are formulated by mixing the composition of the present invention with one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. In addition, lubricants other than simple excipients, such as magnesium stearate and talc, may be used.
Liquid preparations for oral administration may be illustrated as suspensions, solutions, emulsions, syrups and the like, and may include various excipients such as moisturizers, sweeteners, aromatics, preservatives and the like, and water and liquid paraffin as common diluents.
Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsion reagents, lyophilisates and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, Witepsol, polyethylene glycol, Tween 61, cacao butter, lauryl ester, glycerogelatin or the like can be used. In another aspect, the injectable formulation may include conventional additives such as solubilizers, isotonics agents, suspending agents, emulsifying agents, stabilizing agents, or preservatives.
The formulations may be prepared according to conventional mixing, granulating or coating methods and contain an amount of the active ingredient effective for medical treatment, particularly for the prevention, amelioration or treatment of diseases associated with misfolded protein aggregates.
In this case, the composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat a disease and insufficient to cause side effects at a reasonable benefit/risk ratio applicable to any medical treatment. The level of an effective amount may be determined depending on the patient's health, type of disease, severity of disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, rate of excretion, duration of treatment, combination, factors including concurrent use of other drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and it may be administered simultaneously or sequentially with conventional therapeutic agents, and administered one or more times. Taking all of the above factors into account, it is crucial that the minimum amount that can provide the maximum effect without side effects is administered, which can be readily determined by the person skilled in the art.
For example, the dose may be increased or decreased depending on the administration route, severity of disease, sex, body weight, age, and the like, and the scope of the present invention is not limited in any way by the aforementioned dose.
The preferred dosage of the compound according to the present invention may vary depending on the condition and body weight of the patient, the severity of the disease, the type of the drug, and the route and duration of administration, but it may be appropriately selected by those skilled in the art.
In yet another aspect, the present invention provides a method for preventing, ameliorating or treating a disease associated with misfolded protein aggregation comprising administering to a subject in need thereof a pharmaceutical composition according to the invention.
As used herein, the term "subject" refers to all animals including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, which have a disease associated with misfolded protein aggregation. Diseases associated with misfolded protein aggregation may be effectively prevented, ameliorated, or treated by administering to a subject a pharmaceutical composition of the invention. In addition, since the pharmaceutical composition of the present invention serves as a p62 ligand to activate autophagy, aggregates of misfolded proteins are eliminated due to autophagy activation, and thus, a prophylactic or therapeutic effect of diseases associated with these aggregated proteins is exhibited, which can exhibit a synergistic effect by being administered in combination with existing therapeutic agents.
As used herein, the term "administering" refers to introducing a prescribed amount of a substance into a patient in some appropriate way, and the composition of the invention can be administered via any of the general routes as long as it can reach the target tissue. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration may be performed, but the present invention is not limited to these exemplified modes of administration. Furthermore, the pharmaceutical compositions of the present invention may be administered using any device capable of delivering the active ingredient to the target cells. Preferred modes of administration and formulations are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous drip, and the like. Injectable formulations can be prepared using saline, aqueous solutions such as ringer's solution, and non-aqueous solutions such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Injectable formulations may include a pharmaceutical carrier, including stabilizers for preventing degenerative disorders (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and preservatives for inhibiting microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).
In another aspect, the present invention provides a food composition for preventing or improving a misfolded protein related disease, comprising a p62 ligand compound of chemical formula 1, a pharmaceutically acceptable salt, a stereoisomer, a hydrate, a solvate, or a prodrug thereof. The food composition is a health functional food and it can be used by itself via the preparation or included in other health functional foods as an additive to the health functional foods. The health functional food means a food having a body regulation function such as prevention or improvement of diseases, bio-defense, immunity, recovery of rehabilitation, aging inhibition, and the like, and it should not be harmful to the human body when taken for a long period of time. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (preventive, health care or therapeutic treatment).
The kind of food is not particularly limited. Examples of foods to which the above substances can be added are meat, sausage, bread, chocolate, candy, snack, cookie, pizza, stretched noodles, other noodles, jelly, dairy products including ice cream, soup, beverage, tea, drink, alcoholic drink, vitamin complex, etc., and they include all health functional foods in common knowledge.
The food composition of the present invention may comprise ingredients commonly used in the preparation of foods or food additives, in particular, a flavoring agent; natural sweeteners such as monosaccharides such as glucose, fructose and the like, disaccharides such as maltose, sucrose and the like, and dextrins, cyclodextrins as natural carbohydrates, or synthetic sweeteners such as saccharin, aspartame; nutrients; a vitamin; an electrolyte; a colorant; an organic acid; a protective colloid binder; a pH adjusting agent; a stabilizer; a preservative; glycerol; alcohol; carbonating agents used for carbonated drinks, and the like.
In a specific embodiment of the present invention, the compounds of examples 1 to 19, which are novel p62 ligands represented by chemical formula 1, were newly synthesized. Furthermore, to evaluate whether the novel p62 ligand compound according to the present invention can increase the autophagy phenomenon in cultured cells, a HeLa cell line (HeLa cell line), which is a cell line derived from cervical cancer patients, was treated with the novel p62 ligand compound according to the present invention and cultured, and then autophagy activity in the cultured cells was confirmed by immunoblotting. Therefore, it should be confirmed that the level of LC3, which is a marker of autophagy activity, gradually increases according to the time of treatment with the p62 ligand compound of the present invention, and the p62 ligand compound according to the present invention activates and oligomerizes the p62 protein and is delivered to autophagosome while enhancing autophagy activity, thereby effectively eliminating misfolded protein aggregates.
Examples
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are provided for illustrative purposes only, and the present invention is not intended to be limited to these examples.
The compounds shown in table 1 below were prepared by the methods according to examples 1 to 19 below.
[ Table 1]
As starting materials for the synthesis of the compounds of the invention, various synthetic methods are known and, if available on the market, the starting materials are commercially available from commercial suppliers. Examples of reagent suppliers include, but are not limited to, Sigma-Aldrich, TCI, Wako, Kanto, Fluorchem, Acros, Alfa, and Fluka, among others.
The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. With respect to typical or preferred process conditions (i.e., reaction temperature, time, mole ratios of reactants, solvents, pressures), etc., other process conditions may also be used unless otherwise specified. The optimum reaction conditions may vary depending on the particular reactants or solvents used. These conditions can be determined by one skilled in the art through routine optimization procedures.
Hereinafter, the production methods of examples 1 to 19 are described.
Preparation example 1) the compounds of examples 1, 2, 3, 12, 13, 14, 15, 18 and 19 were synthesized by the method shown in the following reaction formula 1.
[ reaction formula 1]
Example 1: preparation of (R) -1- (3,4- (bis (benzyloxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YOK-1106)
Step 1) preparation of 3, 4-bis (benzyloxy) phenol (1102)
After dichloromethane (15ml) was added to and dissolved in 3, 4-bis (benzyloxy) benzaldehyde (1101, 1.00g, 3.0mmol, 1 equiv.), mCPBA (0.78g, 4.5mmol, 1.5 equiv.) was added to the reaction and stirred at room temperature for 4 hours. The reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous solution of sodium carbonate, and the organic layer was separated. The organic layer was washed with an aqueous sodium chloride solution, then dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated and then re-dissolved in methanol (10ml), and 6N NaOH was added and stirred at room temperature for 30 minutes. 4N HCl solution was added to the reaction and stirred for further 30 minutes. The reaction mixture was diluted with ethyl acetate (50ml), washed with brine, and then dehydrated with 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 give 3, 4-bis (benzyloxy) phenol (1102, 0.87g, yield: 90%).
1H-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＝3.0and 9.0Hz),5.08(d,4H,J＝15Hz),4.55(s,1H)；ESIMS m/z:307.25[M+H]+。
Step 2) preparation of R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103)
3, 4-Diphenylmethoxyphenol (1102, 306mg, 1.0mmol) was diluted with ethanol (10ml), and then aqueous KOH (66 mg, 1.2mmol, 1ml) and (R) -2- (chloromethyl) oxirane (410. mu.l, 5.0mmol) were added in this order. The reaction mixture was stirred at room temperature for 5 hours, and then the organic solvent was removed under reduced pressure. Concentrated reaction mixtureThe compound was again diluted with ethyl acetate, washed with water and then brine. The extracted organic layer was dehydrated with anhydrous sodium sulfate and then 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, 297mg, yield: 82%). ESIMS M/z 363.5[ M + H ]]+。
Step 3) R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 9mg, 25nmol) was diluted with anhydrous ethanol (1ml), and 2-aminoethan-1-ol (7.6 μ L, 125nmol) was then added thereto and stirred at room temperature for 4 hours. After confirming the reaction by TLC, the reaction solvent was concentrated under reduced pressure, water was added to the concentrated reaction product and extracted with dichloromethane (3 × 5 mL). The extracted organic layer was dehydrated with 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- (3,4- (bis (benzyloxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YOK-1106, 9.0mg, yield: 85%).1H NMR(CDCl3,300MHz):δ7.42-7.40(m,2H),7.33-7.27(m,8H),6.82(d,1H,J＝9.0Hz),6.42(d,1H,J＝3.0Hz),6.28(dd,1H,J＝3.0and 9.0Hz),5.09(d,1H,J＝12.9Hz),5.06(s,2H),4.93(d,1H,J＝12.9Hz),4.35-4.25(m,1H),3.94(dd,1H,J＝3.6and 9.9Hz),3.89-3.72(m,3H),3.34(t,1H,J＝11.4Hz),2.93(brS,2H),2.80(d,1H,J＝10.8Hz)；ESIMS m/z:424.67[M+H]+。
Example 2: preparation of (R) -1- (3,4- (bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-1)
(R) -1- (3,4- (bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-1) was synthesized in the same manner as in the preparation method of example 19 by using 3, 4-bis ((4-chlorophenylmethyl) oxy) benzaldehyde (1101-1) instead of 3, 4-bis (benzyloxy) benzaldehyde (1101) as a starting material.1H NMR(400MHz,DMSO-d6)δ(ppm)2.59(m,4H),3.45(s,2H),3.81(m,3H),4.50(b,1H),4.85(b,1H),5.01(s,2H),5.11(s,2H),6.42(dd,J＝8Hz and 2.8Hz,1H),6.66(d,J＝2.8Hz,1H),6.92(d,J＝8Hz,1H),7.42(s,4H),7.45(s,4H)；C25H27Cl2NO5[M+2]+ESI-MS calculated value of m/z of 493.90, Experimental value of 492.39
Example 3: preparation of (R) -1- (3,4- (bis ((4-fluorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-2)
(R) -1- (3,4- (bis ((4-fluorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-2) was synthesized in the same manner as in the preparation method of example 19 by using 3, 4-bis ((4-fluorophenylmethyl) oxy) benzaldehyde (1101-2) instead of 3, 4-bis (benzyloxy) benzaldehyde (1101) as a starting material.1H NMR(400MHz,CDCl3)δ(ppm)2.82(m,4H),3.69(t,J＝5.2Hz,2H),3.91(m,2H),4.05(m,1H),5.00(s,2H),5.06(s,2H),6.41(m,1H),6.58(d,J＝2.8Hz,1H),6.84(d,J＝8Hz,1H),7.03(m,4H),7.37(m,4H)；C25H27F2NO5 M+2]+ESI-MS calculation of m/z [461.0, Experimental value 459.49 ]
Example 12: preparation of (R) -1- (3, 4-bis (benzyloxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1104)
(R) -1- (3, 4-bis (benzyloxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1104) was synthesized by using isopropylamine instead of 2-aminoethan-1-ol in preparation step 3 of example 1.1H NMR(300MHz,CDCl3)δ(ppm)1.12(d,J＝6Hz,6H),2.81(m,5H),3.88(d,J＝3Hz,2H),4.04(m,1H),5.09(d,J＝15Hz),6.38(dd,J＝9Hz and 3Hz,1H),6.59(s,1H),6.85(d,J＝9Hz,1H),7.35(m,10H；C26H31NO4[M+H]ESI-MS calculation of m/z of + 422.42, Experimental value 421.54
Example 13: preparation of (R) -1- (3, 4-Diphenylethoxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-2204)
(R) -1- (3, 4-Diphenylethoxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-2204) was synthesized in the same manner as in the preparation method of example 1, except that: in step 1 of the preparation method of example 1,3, 4-diphenylethoxybenzaldehyde (2201) was used as a starting material instead of 3, 4-bis (benzyloxy) benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol.1H NMR(300MHz,CDCl3)δ(ppm)1.17(d,J＝6Hz,6H),2.79(dd,J＝9Hz and 3Hz,1H),2.95(m,2H),3.08(m,4H),3.34(b,2H),3.89(m,2H),4.13(m,5H),6.37(dd,J＝6Hz and 3Hz,1H),6.52(d,J＝3Hz,1H),6.78(d,J＝9Hz,1H),7.25(m,10H)；C28H35NO4[M+H]+ESI-MS calculated value of m/z of 451.00, Experimental value of 449.59
Example 14: preparation of (R) -1- (3, 4-bis (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-3304)
(R) -1- (3, 4-bis (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-3304) was synthesized in the same manner as in the preparation of example 1, except that: in step 1 of the preparation method of example 1,3, 4-bis (3-phenylpropoxy) benzaldehyde (3301) was used as a starting material instead of 3, 4-bis (benzyloxy) benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol.1H NMR(300MHz,CDCl3)δ(ppm)1.21(d,J＝6Hz,6H),2.12(m,4H),2.83(t,J＝9Hz,5H),3.01(m,2H),3.30(b,3H),3.93(m,6H),4.18(m,1H),6.38(dd,J＝6Hz and 3Hz,1H),6.52(d,J＝3Hz,1H),6.80(d,J＝9Hz,1H),7.25(m,10H)；C30H39NO4[M+H]+ESI-MS calculated value of m/z of 479.09, Experimental value of 477.65
Example 15: preparation of (R) -1- (3, 4-bis (4-phenylbutoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-4404)
(R) -1- (3, 4-bis (4-phenylbutoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-4404) was synthesized in the same manner as in the preparation method of example 1, except that: in step 1 of the preparation method of example 1,3, 4-bis (4-phenylbutoxy) benzaldehyde (4401) was used as a starting material instead of 3, 4-bis (benzyloxy) benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol.1H NMR(300MHz,CDCl3)δ(ppm)1.26(dd,J＝6Hz and 3Hz,8H),1.81(m,7H),2.66(t,J＝6Hz,4H),2.85(m,1H),3.06(m,2H),3.46(b,2H),3.93(m,6H),4.24(m,1H),6.36(dd,J＝6Hz and 3Hz,1H),6.52(d,J＝3Hz,1H),6.78(d,J＝9Hz,1H),7.23(m,10H),C32H43NO4[M+H]+ESI-MS meter of m/z of (1)Calculated 505.70 and Experimental 503.45
Example 18: preparation of (R) -1- (3, 4-bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-1)
(R) -1- (3, 4-bis ((4-chlorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-1) was synthesized in the same manner as in the preparation of example 1, except that: in step 1 of the production method of example 1,3, 4-bis (4-chlorophenylmethoxy) benzaldehyde (1101-1) was used as a starting material instead of 3, 4-bis (benzyloxy) benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol.1H NMR(400MHz,CDCl3)δ(ppm)1.08(d,J＝6.4Hz,6H),2.68(dd,J＝12Hz and 8Hz,1H),2.83(m,2H),3.88(d,J＝5.2Hz,2H),3.95(m,1H),5.01(s,2H),5.06(s,2H),6.40(dd,J＝8Hz and 4Hz,1H),6.56(d,J＝2.8Hz,1H),6.82(d,J＝8Hz,1H),7.31(m,8H)；C26H29Cl2NO4[M+H]+ESI-MS of m/z of (1) 491.40 and experimental 490.42
Example 19: preparation of (R) -1- (3, 4-bis ((4-fluorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-2)
(R) -1- (3, 4-bis ((4-fluorophenylmethyl) oxy) phenoxy) -3- (isopropylamino) propan-2-ol (YT-4-2) was synthesized in the same manner as in the preparation of example 1, except that: in step 1 of the preparation method of example 1,3, 4-bis (4-fluorophenylmethoxy) benzaldehyde (1101-2) was used as a starting material instead of 3, 4-bis (benzyloxy) benzaldehyde (1101), and in step 3, isopropylamine was used instead of 2-aminoethan-1-ol.1H NMR(400MHz,CDCl3)δ(ppm)1.08(d,J＝6.4Hz,6H),2.68(dd,J＝12Hz and 8Hz,1H),2.83(m,2H),3.89(d,J＝4Hz,2H),3.95(m,1H),5.00(s,2H),5.05(s,2H),6.40(dd,J＝8Hz and 2.8Hz,1H),6.59(d,J＝2.8Hz,1H),6.83(d,J＝9.6Hz,1H),7.03(m,4H),7.37(m,4H)；C26H29F2NO4[M+H]+ESI-MS calculated value of m/z of 457.90, Experimental value of 457.52
Preparation example 2) the compounds of examples 6 to 11 were synthesized according to the method shown in the following reaction formula 2.
[ reaction formula 2]
Example 6: preparation of (R) -1- (2- ((3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-1109)
Step 1) after R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 500mg, 1.38mmol) was dissolved in methanol (10ml), tert-butyl 2-aminoethylcarbamate (442mg, 2.76mmol) was added thereto and stirred at 50 ℃ for 10 hours. When the reaction was complete, the reaction solvent was concentrated under reduced pressure.
Step 2) after dissolving the tert-butyl (R) - (2- ((3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethyl) carbamate (1105, 600mg, 1.15mmol) in methanol (3ml), methanol/hydrochloric acid (3N, 2ml) was added thereto and then stirred at room temperature for 4 hours. When the reaction was complete, the reaction mixture was concentrated under reduced pressure (white solid, 1106) and used as starting material in the next step without purification.
Step 3) 1- ((2-aminoethyl) amino) -3- (3, 4-bis (benzyloxy) phenoxy) propan-2-ol (1106, 400g, 0.95mmol) was dissolved in DMF (3ml), and then 1H-pyrazole-1-carboxamidine hydrochloride (553mg, 3.8mmol) and diisopropylethylamine (0.3g, 2.4mmol) were additionally added thereto. The reaction mixture was stirred at 30 ℃ for 12 hours, concentrated under reduced pressure and purified by high resolution liquid chromatography to synthesize (R) -1- (2- ((3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-1109, 22mg) as a white solid.1H NMR(400MHz,DMSO+D2O)δ(ppm)3.13(m,4H),3.47(s,2H),3.89(s,2H),5.04(s,2H),5.12(s,2H),6.46(d,J＝8Hz,1H),6.67(s,1H),6.97(d,J＝8Hz,1H),7.39(m,10H)；C26H32N4O4[M+H]+ESI-MS of m/z of (g) calculated 465.00, Experimental value 464.57.
Example 7: preparation of (R) -1- (2- ((3- (3, 4-Diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-2209)
(R)-1-(2-((3-(3,4-Diphenylethoxyphenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YOK-2209) was synthesized in the same manner as in the preparation method of example 6, except that: r-2- ((3, 4-diphenylethoxyphenoxy) methyl) oxirane (2203) was used instead of R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103) in step 1 of the preparation process of example 6.1H NMR(400MHz,CD3OD)δ(ppm)3.00(t,J＝6.8Hz,2H),3.06(t,J＝6.8Hz,2H),3.23(m,1H),3.28(m,3H),3.62(t,J＝6.4Hz,2H),6.46(dd,J＝8Hz and 2.8Hz,1H),6.60(d,J＝2.8Hz,1H),6.85(d,J＝8Hz,1H),7.25(m,10H)；C28H36N4O4[M+H]+ESI-MS calculated value of m/z of 493.00, Experimental value of 492.62
Example 8: preparation of (R) -1- (2- ((3- (3, 4-bis (4-chlorophenylmethyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-1)
(R) -1- (2- ((3- (3, 4-bis (4-chlorophenylmethyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-1) was synthesized in the same manner as in the preparation method of example 6, except that: r-2- ((3, 4-bis (4-chlorophenylmethyl) oxy) phenoxy) methyl) oxirane (1103-1) was used instead of 1103 in step 1 of the preparation method of example 6.1H NMR(400MHz,DMSO+D2O)δ(ppm)3.13(m,4H),3.47(t,J＝6Hz,2H),3.90(m,2H),4.13(m,1H),5.03(s,2H),5.10(s,2H),6.47(dd,J＝8Hz and 2Hz,1H),6.66(d,J＝2.4Hz,1H),6.97(d,J＝8.8Hz,1H),7.42(s,4H),7.45(s,4H)；C26H30Cl2N4O4[M+H]+ESI-MS calculated value of m/z of 532.90, Experimental value of 533.45
Example 9: preparation of (R) -1- (2- ((3- (3, 4-bis (4-fluorobenzyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-2)
(R) -1- (2- ((3- (3, 4-bis (4-fluorophenylmethyl) oxy) phenoxy) -2-hydroxypropyl) amino) ethyl) guanidine (YT-9-2) was synthesized in the same manner as in the preparation method of example 6, except that: r-2- ((3, 4-bis (4-fluorophenylmethyl) oxy) phenoxy) methyl) oxirane (1103-2) was used instead of 1103 in step 1 of the preparation method of example 6.1H NMR(400MHz,DMSO-d6)δ(ppm)3.13(m,4H),3.45(t,J＝6Hz,2H),3.89(m,2H),4.15(m,1H),5.01(s,2H),5.09(s,2H),6.46(dd,J＝8Hz and 2.8Hz,1H),6.68(d,J＝2.8Hz,1H),6.96(d,J＝9.2Hz,1H),7.20(m,4H),7.46(m,4H)；C26H30Cl2N4O4[M+H]+ESI-MS calculated value of m/z of 501.00, Experimental value of 500.55
Example 10: preparation of (R) -1- (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) guanidine (YOK-1107)
Step 1) after R-2- ((3, 4-bis (benzyloxy) phenoxy) methyl) oxirane (1103, 500mg, 1.38mmol) was dissolved in methanol (10ml), ammonia/methanol ((2N, 3ml) was added thereto and stirred at 50 ℃ for 10 hours. When the reaction was complete, the reaction solvent was concentrated under reduced pressure.
Step 2) (R) -1-amino-3- (3, 4-bis (benzyloxy) phenoxy) propan-2-ol (1107, 300mg, 0.8mmol) was dissolved in DMF (3ml) and then 1H-pyrazole-1-carboxamidine hydrochloride (570mg, 3.9mmol) and diisopropylethylamine (0.3g, 2.4mmol) were added in that order. The reaction mixture was stirred at 30 ℃ for 12 hours, concentrated under reduced pressure and purified by high resolution liquid chromatography to synthesize (R) -1- (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) guanidine (YOK-1107, 23 mg).1H NMR(400MHz,DMSO-d6)δ(ppm)3.20(m,1H),3.31(m,1H),5.03(s,2H),5.12(s,2H),6.44(dd,J＝8.8Hz and 2.8Hz,1H),6.68(d,J＝2.8Hz,1H),6.95(d,J＝8Hz,1H),7.38(m,10H)；C24H27N3O4[M+H]+ESI-MS of m/z of (g) calculated 422.00, Experimental value 421.50.
Example 11: preparation of (R) -1- (3- (3, 4-Diphenylethoxyphenoxy) -2-hydroxypropyl) guanidine (YOK-2207)
(R) -1- (3- (3, 4-Diphenylethoxyphenoxy) -2-hydroxypropyl) guanidine (YOK-2207, 25mg) was synthesized in the same manner as in the preparation method of example 10, except that: r-2- ((3, 4-diphenoxyphenoxy) methyl) oxirane (2203) was used instead of 1103 in step 1 of the preparation method of example 10.1H NMR(400MHz,CD3OD)δ(ppm)3.00(t,J＝8Hz,2H),3.06(t,J＝8Hz,2H),3.33(m,1H),3.42(dd,J＝24Hz and 4Hz,1H),3.91(m,2H),4.08(m,3H),4.16(t,J＝6.4Hz,2H),6.46(dd,J＝8Hz and 2Hz,1H),6.60(d,J＝2.8Hz,1H),6.84(d,J＝8Hz,1H),7.25(m,10H)；C26H31N3O4[M+H]+ESI-MS of m/z of (g) calculated 450.00, Experimental value 449.55.
Preparation example 3) the compounds of examples 4 and 5 were synthesized by the method shown in reaction formula 3 below.
[ reaction formula 3]
Example 4: preparation of (R) -1- (3- ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-7)
Step 1) 1, 3-dihydroxybenzene (5.0g, 45.4mmol) was dissolved in acetonitrile (100ml) and then K was added thereto2CO3. Subsequently, 1- (bromomethyl) -4-chlorobenzene (9.3g, 45.4mmol) was added to the reaction and subsequently stirred at 80 ℃ for 16 h. After cooling to room temperature, the reaction solvent was concentrated under reduced pressure. Water was added to the concentrated residue, followed by extraction with ethyl acetate. The organic layer was dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtrate was taken out, concentrated under reduced pressure, and then purified by column chromatography (ethyl acetate/petroleum ether 1:15 to 1:8) to synthesize 3- ((4-chlorophenylmethyl) oxy) phenol (2011, 2.3g) as a pale yellow oil. 1H NMR (400MHz, CD)3OD)δ(ppm)5.03(s,2H),6.35-6.43(m,3H),7.04(t,1H),7.45(s,4H),9.41(s,1H)。
Step 2) after 3- ((4-chlorophenylmethyl) oxy) phenol (2011, 1.0g, 4.2mmol) was dissolved in ethanol (10ml), (R) -2- (chloromethyl) oxirane (1.17g, 12.7mmol), purified water (1ml) and KOH (0.24g, 4.2mmol) were added, and then stirred at room temperature for 16 hours. Water (30ml) was added to the reaction and extracted with ethyl acetate (30 ml). The aqueous layer was extracted more than twice with ethyl acetate, and then the organic layer was dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated under reduced pressure and purified by column chromatography (ethyl acetate/petroleum ether 1:15 to 1:8) to synthesize (R) -2- ((3- ((4-chlorophenylmethyl) oxy) phenoxy) as a colorless oilMethyl) oxirane (3011, 0.85 g).1H NMR(400MHz,CD3OD)δ(ppm)2.74(m,1H),2.90(m,1H),3.34(m,1H),3.91-3.95(m,1H),4.18-4.22(m,1H),5.01(s,2H),6.52-6.59(m,3H),7.16-7.26(m,1H),7.35(s,4H)。
Step 3) (R) -2- ((3- ((4-chlorophenylmethyl) oxy) phenoxy) methyl) oxirane (3011, 300mg, 1.03mmol) was dissolved in methanol (5ml), and 2-aminoethanol (189mg, 3.09mmol) was subsequently added thereto. The reaction mixture was stirred at 65 ℃ for 5 hours. After cooling to room temperature, the reaction solvent was concentrated under reduced pressure and purified by high resolution liquid chromatography to synthesize (R) -1- (3- ((4-chlorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-7, 15mg) as a white solid.1H NMR(400MHz,CDCl3)δ(ppm)2.83(m,4H),3.69(m,2H),2.96(m,2H),4.08(m,1H),5.00(s,2H),6.55(m,3H),7.18(t,J＝8Hz,1H),7.35(s,4H)；C18H22ClNO4[M]+ESI-MS calculated value of m/z of 352.00, Experimental value of 351.83
Example 5: preparation of (R) -1- (3- ((4-fluorobenzyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-8)
(R) -1- (3- ((4-fluorophenylmethyl) oxy) phenoxy) -3- ((2-hydroxyethyl) amino) propan-2-ol (YT-6-8, 20mg) was synthesized in the same manner as in the preparation of example 4, except that: 1- (bromomethyl) -4-fluorobenzene was used instead of 1- (bromomethyl) -4-chlorobenzene in step 1 of the preparation method of example 4.1H NMR(400MHz,CDCl3)δ(ppm)2.03(b,3H),2.83(m,4H),3.69(t,J＝4.8Hz,2H),3.96(m,2H),4.08(m,1H),4.99(s,2H),6.56(m,3H),7.07(m,2H),7.18(t,J＝8Hz,1H),7.40(m,2H)；C18H22FNO4[M+H]+ESI-MS calculated value of m/z of 336.00, Experimental value of 335.38
Preparation example 4) the compounds of examples 16 and 17 were synthesized by the method shown in reaction formula 4 below.
[ reaction formula 4]
Example 16: preparation of (R) -1- (4- (benzyloxy) -3-phenethyloxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-1204)
Step 1) after 4- (benzyloxy) -3-phenethyloxybenzaldehyde (1201, 332mg, 1.0mmol) was dissolved in dichloromethane, m-chloroperbenzoic acid (mCPBA, 260mg, 1.5mmol) was added thereto and then stirred at room temperature for 4 hours. Excess ethyl acetate was added to the reaction and then washed with saturated aqueous sodium carbonate solution, and the organic layer was separated. The organic layer was washed with an aqueous sodium chloride solution, then dehydrated with anhydrous sodium sulfate and filtered under reduced pressure. The filtered solution was concentrated and re-dissolved in methanol (10ml), followed by addition of 6N NaOH and stirring at room temperature for 30 minutes. 4N HCl solution was added to the reaction and then stirred for further 30 minutes. The reaction mixture was diluted with ethyl acetate (50ml), followed by washing with brine, dehydration with anhydrous sodium sulfate, and filtration under reduced pressure. The filtered solution was concentrated and then purified by column chromatography (hexane/ethyl acetate ratio: 7/3) to give 4- (benzyloxy) -3-phenethyloxyphenol (1202, 310mg, yield: 90%). 1H NMR (cdcl3,300mhz) δ (ppm) 7.23-7.42(m,10H),6.77(d,1H, J ═ 9.0Hz),6.46(d,1H),6.28(dd,1H, J ═ 3.0and 9.0Hz),4.98(s,2H),4.58(s,1H),4.20(t,2H, J ═ 6Hz),3.14(t,2H, J ═ 6 Hz).
Step 2)4- (Phenylmethoxy) -3-phenethyloxyphenol (1202, 320mg, 1.0mmol) was diluted with ethanol (5ml), and then aqueous KOH (66 mg, 1.2mmol, 1ml) and (R) -2- (chloromethyl) oxirane (410. mu.l, 5.0mmol) were added in that order. The reaction mixture was stirred at room temperature for 5 hours, and then the organic solvent was removed under reduced pressure. The concentrated reaction mixture was again diluted with ethyl acetate, washed with water and then brine. The extracted organic layer was dehydrated with anhydrous sodium sulfate and then filtered under reduced pressure. The filtered organic layer was concentrated and purified by column chromatography to give pure R-2- ((4- (benzyloxy) -3-phenethyloxyphenoxy) methyl) oxirane (1203, 308mg, yield: 82%).1H NMR(300MHz,CDCl3)δ(ppm)7.44-7.47(m,2H),7.26-7.39(m,5H),7.19-7.22(m,3H),6.84(d,1H,J＝6.0Hz),6.54(d,1H,J＝3.0Hz),6.36(dd,1H),5.07(s,2H),4.15(dd,1H),4.00(t,2H,J＝6.0Hz),2.84(t,2H,J＝9.0Hz),2.73(dd,1H),2.10-2.19(m,2H)；ESIMS m/z:363.5[M+H]+。
Step 3) after R-2- ((4- (benzyloxy) -3-phenethyloxyphenoxy) methyl) oxirane (1203, 9.4mg, 25nmol) was diluted with absolute ethanol (1ml), isopropylamine (10 μ L, 125nmol) was added thereto and stirred at room temperature for 4 hours. After confirming the reaction by TLC, the reaction solvent was concentrated under reduced pressure, and water was added to the concentrated reaction product and extracted with dichloromethane (3 × 5 mL). The extracted organic layer was dehydrated with 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- (4- (phenylmethoxy) -3-phenylethoxyphenoxy) -3- (isopropylamino) propan-2-ol (YOK-1204, 9.3mg, yield: 86%).1H NMR(300MHz,CDCl3)δ(ppm)1.15(d,J＝6Hz,6H),2.71(m,4H),2.80(m,2H),3.14(t,J＝6Hz,2H),3.91(d,J＝3Hz,2H),4.09(m,1H),4.21(t,J＝6Hz,2H),4.99(s,2H),6.35(dd,J＝6Hz and 3Hz,1H),6.54(d,J＝3Hz,1H),6.82(d,J＝9Hz,1H),7.26(m,10H)；C27H33NO4[M+H]+ESI-MS of m/z of (g) calculated 437.00, Experimental value 435.56.
Example 17: preparation of (R) -1- (4- (benzyloxy) -3- (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1304)
(R) -1- (4- (benzyloxy) -3- (3-phenylpropoxy) phenoxy) -3- (isopropylamino) propan-2-ol (YOK-1304, 9.5mg, yield: 85%) as a white solid was synthesized in the same manner as in the preparation method of example 16 by using 4- (benzyloxy) -3- (3-phenylpropoxy) benzaldehyde (1301, 346mg, 1.0mmol) instead of 4- (benzyloxy) -3-phenylethoxybenzaldehyde (1201) in step 1 of the preparation method of example 16 as a starting material. 1H NMR (300MHz, CDCl)3)δ(ppm)1.13(d,J＝6Hz,6H),2.15(m,2H),2.33(b,3H),2.80(m,5H),3.91(d,J＝6Hz,2H),4.01(m,3H),5.07(s,2H),6.36(dd,J＝6Hz and 3Hz,1H),6.53(d,J＝3Hz,1H),6.84(d,J＝9Hz,1H),7.33(m,10H)；C28H35NO4[M+H]+ESI-MS of m/z of (g) calculated 451.00, Experimental value 449.59.
Preparation example 5) the compounds of comparative examples 1 and 2 were synthesized by the method shown in reaction formula 5 below.
[ reaction formula 5]
Comparative example 1: preparation of (R) - (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) glycine methyl ester (YOK-G-1104)
(R) - (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) glycine methyl ester (YOK-G-1104) was synthesized in the same manner as in the preparation method of example 19, except that: glycine methyl ester was used instead of 2-aminoethyl-1-ol in step 3 of the preparation method of example 19.1H NMR(300MHz,DMSO-d6)δ(ppm)2.55(m,1H),2.81(m,1H),3.51(s,2H),3.66(s,3H),4.05(m,1H),4.20(m,2H),5.16(s,2H),5.18(s,2H),5.37(br s,1H),5.52(br s,1H),6.57(s,1H),6.67(d,1H),6.96(d,1H),7.32-7.48(m,10H)。
Comparative example 2: preparation of (R) - (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) alanine ethyl ester (YOK-A-1104)
(R) - (3- (3, 4-bis (benzyloxy) phenoxy) -2-hydroxypropyl) alanine ethyl ester (YOK-A-1104) was synthesized in the same manner as in the preparation method of example 19, except that: alanine ethyl ester was used instead of 2-aminoethan-1-ol in step 3 of the preparation method of example 19.1H NMR(300MHz,DMSO-d6)δ(ppm)1.21(t,3H),1.27(m,3H),2.56-2.81(m,2H),3.56(m,1H),4.05(m,1H),3.95-4.20(m,4H),5.16(s,2H),5.18(s,2H),5.37(br s,1H),6.57(s,1H),6.67(d,1H),6.96(d,1H),7.32-7.48(m,10H)。
Experimental example 1 evaluation of oligomerization Activity of p62 protein in cultured cells Using immunoblotting
To evaluate the efficacy of p62 protein oligomerization activity using the compounds (examples 1 to 19), HEK293 cell line (which is human embryonic kidney-derived cells) was collected. As representative compounds of the current compounds, YOK-1104, YOK-2204, YOK-3304, YOK-1204, YOK-4404, YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-6-2, YT-6-7, YT-6-8, YT-9-1 and YT-9-2 are selected. To measure intracellular p62 protein activation and oligomerization upon treatment with these selected representative compounds, the corresponding cells were dispensed into 100pi dishes. The cells were harvested after further incubation for 24 hours, allowing the cells to fully attach to the surface of the disc. Mu.l of lysis buffer (20mM Tris, pH 7.4), 150mM NaCl, 1% Triton X-100, 2mM NaF, 2mM EDTA, 2mM beta-glycerophosphate, 5mM sodium orthovanadate, 1mM PMSF, leupeptin, aprotinin) was injected into each sample and the cells were lysed. Based on the total protein concentration measured, each sample was treated with the test compound at room temperature for 2 hours, followed by addition of sample buffer and allowing to react at 95 ℃ for 10 minutes. After the reaction 25 μ l was taken from the sample and dispensed into each well of the acrylamide gel, followed by immunoblotting. Immunoblots show representative results from three or more independent experiments. The results are shown in figure 2.
As seen in fig. 2, when treated with the p62 ligand compound according to the present invention, it was confirmed that treatment with the compound resulted in a decrease in monomers of p62 protein and an increase in oligomers and polymer aggregates at the same time.
EXAMPLE 2 evaluation of autophagy Activity in cultured cells Using immunoblotting
To evaluate the efficacy of autophagy activity using the compounds (examples 1 to 19), hela cell lines (which are cell lines derived from cervical cancer patients) were cultured in an incubator with 5% carbon dioxide using DMEM medium containing 10% FBS and 1% streptomycin/penicillin. As representative compounds among the current compounds, YOK-1107, YOK-4404 and YOK-1104 were selected. To measure autophagy activity based on treatment with these selected representative compounds, the corresponding cells were dispensed into 6-well plates. An additional 24 hours of incubation was performed to allow complete attachment of the cells to the surface of the disc. To find that the corresponding compounds can increase the concentration under autophagy phenomenon, test compounds were diluted to 1, 2, 5, 10 and 20 μ M and processed. After the treatment with the corresponding compound, the cells were again cultured in the cell incubator for 24 hours, followed by collection of the cells. To extract proteins from the collected cells, 100. mu.l of lysis buffer (20mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 2mM NaF, 2mM EDTA, 2mM beta-glycerophosphate, 5mM sodium orthovanadate, 1mM PMSF, leupeptin, aprotinin) was injected into each sample and the cells were lysed. Based on the total protein concentration measured, sample buffer was added to each sample and allowed to react for 5 minutes at 100 ℃. After the reaction 5 μ l was taken from the sample and dispensed into each well of the acrylamide gel, followed by immunoblotting. Immunoblots show representative results from three or more independent experiments. The results are shown in figure 3.
As seen in fig. 3, when treated with the p62 ligand compound according to the present invention, it was confirmed that the level of LC3 (a marker of large autophagy activity) gradually increased according to the concentration of the compound.
EXAMPLE 3 evaluation of autophagy Activity in cultured cells Using immunoblotting
To investigate the efficacy of autophagy activity using the compounds (examples 1 to 19), immunoblotting was performed in the same manner as in experimental example 2 using LC3 as a marker. Regarding the difference, in order to evaluate the treatment time required for activation and the activity retention time, 5. mu.M of YOK-1107, YOK-1204, YOK-1304, YOK-2204, YOK-3304, YOK-4404 was treated for 1,3, 6, 12, 24, 48 hours as a representative compound selected from the compounds of the present invention. On the other hand, as a representative compound selected from the compounds of the present invention, 10. mu.M of YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-6-2, YT-6-8, YT-6-7, YT-9-1, YT-9-2 was treated for 24 hours. Immunoblots show representative results from three or more independent experiments. The results are shown in fig. 4. As seen in fig. 4, when treated with the p62 ligand compound according to the present invention, it was confirmed that the level of LC3 (a marker of large autophagy activity) gradually increased according to the treatment with the compound.
EXAMPLE 4 evaluation of autophagy Activity in cultured cells Using immunoblotting
To investigate the efficacy of autophagy activity of the control compound, immunoblotting was performed by using LC3 as a marker in the same manner as in experimental example 2. As a control compound, a compound having the following structure was used.
[ Table 2]
The aforementioned compounds were confirmed by treatment at different concentrations and at different times, and immunoblots showed representative results from three or more independent experiments. The results are shown in fig. 5. As shown in fig. 5, it was confirmed that the control compound did not increase the level of LC3 (a marker of large autophagy activity) regardless of the treatment time or concentration.
EXAMPLE 5 assessment of autophagy Activity in cultured cells Using immunofluorescence staining and confocal microscopy
In order to confirm the activity of p62 protein and the level of autophagy activity using the compounds (examples 1 to 19), immunofluorescent staining was performed using p62 and LC3 as markers. In order to confirm the activity of p62 and the level of autophagy using a novel p62 ligand and its isomer in cultured cells, HeLa cell lines, which are cell lines derived from cervical cancer patients, were treated with novel p62 ligand compounds (YOK-1106, YOK-1204, YOK-1504, YOK-2204, YOK-3304, YOK-4404, YOK-1107, YOK-1109, YOK-2207, YOK-2209, YT-4-1, YT-4-2, YT-6-1, YT-9-1 and YT-9-2) and cultured. Thereafter, as a marker of autophagy phenomenon, the expression level and position of LC3 spot and local coexistence with p62 spot were observed.
For immunofluorescent staining, glass shields were placed on 24-well plates, cells were dispensed, cultured for 24 hours, and then treated with 5 μ M of the novel p62 ligand according to the invention. For the compound to work, an additional incubation for 24 hours was performed, followed by removal of the medium. Cells were fixed with formaldehyde at room temperature. To prevent non-specific staining, cells were reacted with blocking solution at room temperature for 1 hour, then treated with LC3 antibody diluted with blocking solution at a specific ratio, and allowed to react at room temperature for 1 hour. The antibody-treated cells were washed three times with PBS, and goat-derived secondary antibody was diluted with blocking solution at a specific ratio, and then allowed to react at room temperature for 30 minutes. The cells were washed again three times with PBS, and for intracellular nuclear staining, the expression level, intracellular spot formation and intracellular coexistence level of p62 and LC3 were observed via confocal microscope after DAPI staining. The results are shown in fig. 5. Immunofluorescent staining showed representative results from three or more independent experiments.
As seen in fig. 6, it was confirmed that intracellular spot formation of p62 protein, intracellular spot and local coexistence of LC3 as autophagosome markers, and intracellular spot formation of LC3 increased after treatment with the p62 ligand compound according to the present invention.
EXAMPLE 6 assessment of autophagy Activity in cultured cells Using immunofluorescence staining and confocal microscopy
In order to confirm the p62 protein activity and the level of autophagy activity of the control compounds (YOK-A-1104, YOK-G-1104, YTK-1005, YTK-1105-1), immunofluorescent staining was performed in the same manner as in Experimental example 4 by using p62 and LC3 as markers. YOK-A-1104 and YOK-G-1104 are compounds having the following structures.
[ Table 3]
Compounds were treated at different concentrations and intracellular spots (i.e., the extent to which autophagosomes formed and delivered p 62) of p62 or LC3 were observed using confocal microscopy. The results are shown in fig. 7. Immunofluorescent staining showed representative results from three or more independent experiments.
As shown in fig. 7, it was confirmed that intracellular spot formation of p62 protein, intracellular spot formation of LC3 as an autophagosome marker and local coexistence were increased, and intracellular spot formation of LC3 was not increased regardless of the concentration treated with the control compound.
EXAMPLE 7 Activity of P62-mediated delivery of self-ubiquitinated protein by autophagy in cultured cells evaluated by immunofluorescence staining and confocal microscopy
To confirm the level of activity of P62-mediated delivery of self-ubiquitinated protein in cultured cells as autophagy after treatment with compounds (examples 1 to 19), immunofluorescent staining was performed in the same manner as in experimental example 4 by using P62 and FK2 as markers. As the compounds, YT-4-1, YT-4-2, YT-6-1, YT-6-7, YT-6-8, YOK-4404, YOK-1106, YOK-1107, YOK-1109, YOK-1204 and YOK-1204 are used. Intracellular spotting of p62 or FK2 (i.e., the extent to which p62 and ubiquitinated protein are delivered to autophagosomes) was observed using confocal microscopy after treatment with compound. The results are shown in fig. 8a and 8 b. Immunofluorescent staining showed representative results from three or more independent experiments.
As shown in fig. 8a and 8b, it was confirmed that intracellular spots of FK2 (marker of p62 protein and ubiquitinated protein) were formed and local coexistence of spots was increased after treatment with the p62 ligand compound according to the present invention.
Experimental example 8 Activity of P62-mediated delivery of misfolded Huntington protein by autophagy in cultured cells was evaluated by immunofluorescence staining and confocal microscopy
To confirm the level of activity of P62-mediated misfolded huntingtin protein (Htt-Q103), the major protein of huntington's disease, after treatment of degenerative brain diseases with compounds (examples 1 to 19), immunofluorescent staining was performed in the same manner as in experimental example 4 by using P62 and Htt-Q103-GFP as markers. As the compounds, YOK-1106, YOK-1107, YOK-1109, YOK-1204, YOK-2204, YOK-4404, YT-4-1, YT-4-2, YT-6-1, YT-6-7 and YT-9-1 are used. After treatment with the compounds, intracellular spots of p62 or Htt-Q103-GFP (i.e., the extent to which p62 and misfolded huntingtin protein were delivered to the autophagosomes) were observed using confocal microscopy. The results are shown in fig. 9a and 9 b. Immunofluorescent staining showed representative results from three or more independent experiments.
As shown in fig. 9a and 9b, intracellular spot formation and local coexistence of spots of FK2 (marker of p62 protein and ubiquitinated protein) were confirmed to be increased after treatment with the p62 compound according to the present invention.