Tricyclic ketolactone compound, method for preparing the same, and pharmaceutical use thereof

Abstract

The present invention relates to a tricyclic ketolactone compound, a method for preparing the same, and a pharmaceutical use thereof, and more specifically, to a 3-dehydroxycollinoketone compound or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a composition containing the same for the prevention, improvement, or treatment of neurodegenerative diseases.

Description

Technology Field

[0001] The present invention relates to a tricyclic ketolactone compound, a method for preparing the same, and a pharmaceutical use thereof, and more specifically, to a 3-dehydroxycollinoketone compound or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a composition containing the same for the prevention, improvement, or treatment of neurodegenerative diseases. Background Technology

[0002] Alzheimer's disease (AD) is a representative neurodegenerative disease, and its main pathological features are known to be the formation of amyloid-beta (Aβ) plaques and hyperphosphorylated tau aggregates. These protein aggregates impair neuronal function, causing cognitive impairment and neuronal death, which eventually lead to severe memory loss and symptoms of dementia.

[0003] Currently, treatments for Alzheimer's disease are broadly divided into symptom relievers and disease-modifying therapies (DMTs). Existing treatments have focused on symptom relievers such as cholinesterase inhibitors (Donepezil, Rivastigmine, Galantamine) and NMDA receptor antagonists (Memantine). However, these drugs have limitations in that they are difficult to prevent the fundamental progression of the disease and have limited therapeutic effects.

[0004] Accordingly, monoclonal antibody-based therapies targeting Aβ or tau proteins (such as Aducanumab, Lecanemab, and Donanemab) have recently been developed, but issues such as high cost, difficulty in penetrating the blood-brain barrier (BBB), and side effects (ARIA: Amyloid-Related Imaging Abnormalities) have been raised. Therefore, research on small molecule therapies that efficiently remove Aβ and tau aggregates while simultaneously providing neuroprotective and cognitive function improvement effects is actively underway.

[0005] Meanwhile, collinolactone has been reported as a unique lactone compound isolated from strains of the genus Streptomyces, which has a 7 / 10 / 6 tricyclic (bicyclo[7.3.1]tridecane) structure. Collinolactone has the effect of dissociating Aβ and tau aggregates, and it has been confirmed to reduce Aβ plaques in APP / PS1 Alzheimer's disease model mice.

[0006] However, cholinolactone has limitations in that it does not effectively reach the cortical region due to its low blood-brain barrier (BBB) ​​permeability. Additionally, as a natural product-based compound, it is difficult to mass-produce, and the isolation and purification process through microbial culture yields only a very low amount (approximately 210 mg / 700 L culture medium), making it difficult to utilize as a therapeutic agent. Furthermore, although cholinolactone exhibits dissociative effects against Aβ and tau aggregates, it has the disadvantage of having a minimal aggregation inhibitory effect and limited neuroprotective and cognitive function improvement effects.

[0007] Therefore, to utilize colinolactone as a therapeutic agent, a strategy is required to improve BBB permeability through structural modification of the compound and to enhance the inhibition and dissociation effects of Aβ and tau aggregation. Prior art literature

[0008] (Patent Document 0001) US 2022-0396580 A1

[0009] Angew. Chem. Int. Ed. 2021, 60, 23212-23216 The problem to be solved

[0010] Accordingly, the inventors of the present invention conducted a thorough study to overcome the low blood-brain barrier (BBB) ​​permeability and limited pharmacological effects of colinolactone and to develop a compound having a more effective action against Aβ and tau aggregates. As a result, they established a method to synthesize 3-dehydroxycollinoketone, which can improve BBB permeability through structural modification of colinolactone and simultaneously exhibit inhibitory and dissociative effects on Aβ and tau aggregates. By confirming that this can increase the potential for application as a treatment for neurodegenerative diseases, they completed the present invention.

[0011] One aspect of the present invention provides a novel 3-dehydroxycollinoketone compound of a tricyclic ketolactone backbone or a pharmaceutically acceptable salt thereof that modulates both amyloid-beta (Aβ) and tau aggregates, which are major new drug targets for Alzheimer's disease.

[0012] Another aspect of the present invention provides a method for preparing the 3-dehydroxycholinoketone compound.

[0013] Another aspect of the present invention provides a pharmaceutical composition for the treatment and prevention of neurodegenerative diseases containing the 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another aspect of the present invention provides a method for preventing or treating a neurodegenerative disease, comprising the step of administering the 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt, or the pharmaceutical composition, to an individual who has developed or is at risk of developing a neurodegenerative disease.

[0015] Another aspect of the present invention provides a health functional food composition for the prevention or improvement of neurodegenerative diseases containing the 3-dehydroxycholinoketone compound or a food-grade acceptable salt thereof as an active ingredient. means of solving the problem

[0016] One embodiment of the present invention provides a 3-dehydroxycollinoketone compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0017] [Chemical Formula 1]

[0018]

[0019] Another aspect of the present invention provides a method for preparing a 3-dehydroxycholinoketone compound represented by Formula 1 using Module 1 represented by Formula 11, Module 2 represented by Formula 12, and Module 3 represented by Formula 13.

[0020] [Chemical Formula 11]

[0021]

[0022] [Chemical Formula 12]

[0023]

[0024] [Chemical Formula 13]

[0025]

[0026] In the above formula,

[0027] R1 to R6 are each independently C1-C6 alkyl;

[0028] TBS tert -butyldimethylsilyl( tert -butyldimethylsilyl)

[0029] Another aspect of the present invention provides a pharmaceutical composition for the treatment and prevention of neurodegenerative diseases containing the 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0030] Another aspect of the present invention provides a method for preventing or treating a neurodegenerative disease, comprising the step of administering the 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt, or the pharmaceutical composition, to an individual who has developed or is at risk of developing a neurodegenerative disease.

[0031] Another aspect of the present invention provides a health functional food composition for the prevention or improvement of neurodegenerative diseases containing the 3-dehydroxycholinoketone compound or a food-grade acceptable salt thereof as an active ingredient. Effects of the invention

[0032] A 3-dehydroxycholinoketone compound according to one embodiment exhibits a dual-action therapeutic effect that inhibits the formation of Aβ and tau aggregates and dissociates already formed aggregates. This may increase the potential for treating neurodegenerative diseases such as Alzheimer's disease.

[0033] A 3-dehydroxycolinoketone compound according to one embodiment has excellent blood-brain barrier (BBB) ​​permeability and can effectively reach the cerebral cortex through high lipophilicity compared to existing collinolactone. These characteristics enable a more effective action in the treatment of neurodegenerative diseases.

[0034] Furthermore, a 3-dehydroxycholinoketone compound according to one embodiment was found to provide neuroprotective effects, improve cognitive function in an AD mouse model, and alleviate synapse loss. Therefore, a 3-dehydroxycholinoketone compound according to one embodiment is expected to be a promising candidate substance applicable to the treatment of various neurodegenerative diseases as well as Alzheimer's disease.

[0035] The manufacturing method according to one embodiment provides an efficient chemical synthesis pathway for 3-dehydroxycolinoketone compounds, thereby solving the problem of low yield of existing colinolactones and enabling large-scale synthesis. In addition, the [6+4] cycloaddition reaction used in one embodiment is a method rarely utilized in existing synthesis methods, providing a new methodology for the synthesis of tricyclic compounds, thereby enabling more stable and reproducible synthesis. Brief explanation of the drawing

[0036] Fig. 1 - 3-dehydroxy-5- epi XRD analysis and NOE-based stereochemical results of -collinoketone (2) Fig. 2 - 3-dehydroxy-5- epi Evaluation of the inhibition and dissociation ability of -collinoketone (2) on Aβ aggregation [Aβ 42 Thioflavin-T (ThT) fluorescence assay-based efficacy evaluation for: a) 3-dehydroxy-5- epi -collinoketone (2)'s Aβ 42 ThT fluorescence analysis results demonstrating the inhibitory effect on aggregation, b) 3-dehydroxy-5- epi -Pre-formed Aβ of -collinoketone (2) 42 ThT fluorescence analysis results on dissociation effect on fibrils; Aβ 42 Efficacy evaluation based on amyloid aggregation analysis (A3) for oligomers: c) Aβ 42 Schematic diagram of oligomer aggregation analysis, d) Aβ 42 3-dehydroxy-5- for oligomers epi Evaluation of the dissociation rate of -collinoketone (8a)] Fig. 3 - 3-dehydroxy-5- for internal Aβ accumulation in 5XFAD mice epiPharmacological effects of -collinoketone (2) [a) PAMPA-BBB assay results to evaluate blood-brain barrier (BBB) ​​permeability, b) schematic diagram of the analysis of the effects of (2) on Aβ using a 5XFAD mouse model, c) half-brain images stained with Thioflavin-S (ThS) of the vehicle and 3-dehydroxy-5-epi-collinoketone (2) treatment groups, d) quantification of Aβ plaques in the cortical region of the ThS-stained brain, e) quantification of total Aβ (6E10) and oligomeric Aβ (A11) in RIPA-soluble lysates of the cortex, f) quantification of Aβ plaques in the hippocampal region of the ThS-stained brain, and g) total Aβ in RIPA-soluble lysates of the hippocampus Quantification of (6E10) and oligomer Aβ (A11).] Fig. 4 - 3-dehydroxy-5- epi Evaluation of the inhibitory and dissociative ability of -collinoketone (2) on Tau aggregation [Thioflavin-T (ThT) fluorescence assay-based efficacy evaluation on Tau K18 and Tau R3: a) Tau protein consists of 4 domains: N-terminus, proline-rich region, repeat domains (R1-R4), C-terminal domain, b) 3-dehydroxy-5- epi ThT fluorescence analysis results demonstrating the inhibitory effect of -collinoketone (2) on tau K18 aggregation, c) 3-dehydroxy-5- epi ThT fluorescence analysis results of the dissociation effect of -collinoketone (2) on pre-formed tau K18 aggregates, d) 3-dehydroxy-5- epi ThT fluorescence analysis results demonstrating the inhibitory effect of -collinoketone (2) on R3 aggregation, e) 3-dehydroxy-5- epi-Results of ThT fluorescence analysis of the dissociation effect of -collinoketone (2) on pre-formed R3 aggregates.] Fig. 5 - 3-dehydroxy-5- for hippocampus-dependent cognitive decline in tau-injected acute Alzheimer's disease mice and PS19 mice epi Therapeutic efficacy of -collinoketone (2) [3-dehydroxy-5- using an R3-injection acute AD mouse model epi Evaluation of the in vivo therapeutic efficacy of -collinoketone (2): a)-c) overall experimental overview of behavioral experiments and comparison of voluntary turnover rates and arm entry counts, d) analysis of the degree of aggregation of R3 injected into the brain, e) quantification of oligomer R3 (T22) injected into the brain; Evaluation of the in vivo therapeutic efficacy of 3-dehydroxy-5-epi-collinoketone (8a) using a PS19 mouse model: f) schematic diagram of the analysis of the effect of (2) on Tau-induced cognitive decline using a PS19 mouse model, g) expression levels of postsynaptic density protein 95 (PSD95) and synaptophysin in the hippocampus region of the PS19 mouse brain.] Specific details for implementing the invention

[0037] The present invention will be described in detail below. Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains, and descriptions of known functions and configurations that could unnecessarily obscure the essence of the invention are omitted in the following description.

[0038] The terms used in the description herein are merely for the purpose of effectively describing specific embodiments and are not intended to limit the invention.

[0039] The singular form used in this specification is intended to include the plural form unless specifically indicated otherwise in the context.

[0040] Throughout this specification, any description of a component as “comprising,” “having,” “containing,” or “having” is an open description meaning that, unless specifically stated otherwise, it does not exclude other components but rather implies that additional components may be included, and does not exclude elements, materials, or processes not additionally listed.

[0041] The numerical ranges used herein include lower and upper limits and all values ​​within the range, increments logically derived from the form and width of the defined range, all of which are limited, and all possible combinations of upper and lower limits of the numerical range defined in different forms. Unless otherwise specifically defined in this specification, values ​​outside the numerical range that may occur due to experimental error or rounding are also included in the defined numerical range.

[0042] Unless otherwise specifically defined in this specification, “about” may be considered to be a value within 30%, 25%, 20%, 15%, 10%, or 5% of the specified value.

[0043] The term “compound of the present invention” means including not only the compound of Formula 1, but also its solvates, hydrates, or prodrugs. Additionally, the term “compound of the present invention” means including the pharmaceutically acceptable salt of the compound of the present invention unless the pharmaceutically acceptable salt thereof is mentioned. In one embodiment, the compound of the present invention may exist as stereoisomerically pure compounds (e.g., substantially free of other stereoisomers (e.g., 85% ee or more, 90% ee or more, 95% ee or more, 97% ee or more, or 99% ee or more)). That is, if the compound according to the present invention or its salt is a tautomeric isomer and / or stereoisomer (e.g., geometrical isomer and conformational isomer)), their separated isomers and mixtures, respectively, may also be included in the category of the compound of the present invention. Where the compounds of the present invention or their salts have an asymmetric carbon within their structure, their optically active compounds and racemic mixtures may also be included within the scope of the compounds of the present invention. The compounds of the present invention include the R and S forms of such isomers, and mixtures of the R and S forms may also be included within the scope of the compounds of the present invention. Furthermore, the compounds of the present invention may exist in either a keto form or an enol form, and both of these forms may be included within the scope of the compounds of the present invention.

[0044] The term "solvent" means a compound of the present invention or a pharmaceutically acceptable salt thereof comprising a stoichiometric or non-stoichiometric amount of solvent bonded by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and can be administered to humans in minute amounts.

[0045] The term "hydrate" means a compound of the present invention or a pharmaceutically acceptable salt thereof comprising stoichiometric or non-stoichiometric amounts of water bonded by non-covalent intermolecular forces.

[0046] The term "prodrug" means a compound of the present invention that can be hydrolyzed, oxidized, and otherwise reacted under biological conditions (in vitro or in vivo) to supply an active compound, particularly a compound of the present invention. A prodrug can be used to modify or improve the physical and / or pharmacokinetic profile of a parent compound and may be formed if it contains a suitable group or substituent that can induce the parent compound to form a prodrug. Prodrugs can be readily prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers Gmfh).

[0047] The term "purified" means that when separated, the separated material is 90% or more pure, and in one embodiment, 95% or more pure, in another embodiment, 99% or more pure, and in yet another embodiment, 99.9% or more pure.

[0048] The term "pharmaceuticalally acceptable" means suitable for use as a pharmaceutical preparation, generally considered safe for such use, and means officially approved by a national regulatory agency for such use or listed in the Korean Pharmacopoeia or the United States Pharmacopoeia.

[0049] The term "pharmaceuticalally acceptable salt" refers to any organic or inorganic addition salt of the compound of the present invention, at a concentration having a relatively non-toxic and harmless active effect on the individual, such that side effects caused by this salt do not impair the beneficial efficacy of the compound of the present invention itself.

[0050] The terms "pharmaceuticalally acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that aid in the administration of an active agent and its absorption by a target.

[0051] The terms “ingredient,” “composition,” “composition of a compound,” “compound,” “drug,” “pharmaceutical active agent,” “active agent,” “healing,” “treatment,” “therapy,” or “drug” are used interchangeably to refer to a compound or a composition of compound(s) or substances that induces a desired pharmaceutical and / or physiological effect by local and / or systemic action when administered to a subject (human or animal).

[0052] The term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of neurodegenerative diseases.

[0053] The term "treatment" refers to any action that improves or beneficially alters the symptoms of a neurodegenerative disease.

[0054] The term "improvement" refers to any action that at least reduces parameters related to the condition being treated, such as the severity of symptoms.

[0055] The term "individual" means any animal, including humans, that has developed or is likely to develop a neurodegenerative disease. Such animals may include, but are not limited to, humans as well as mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms.

[0056] The term "administration" means introducing the pharmaceutical composition of the present invention into an individual by any appropriate method, and the route of administration of the composition of the present invention may be administered via various oral or parenteral routes as long as it can reach the target tissue.

[0057] The term "pharmaceutically / therapeutically effective dose (effective dose)" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level can be readily determined by a person skilled in the art based on factors including the patient's gender, age, weight, health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0058] The term "food" includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods and health foods, and includes all food in the conventional sense.

[0059] The term "health functional food" refers to food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body pursuant to Article 6727 of the Health Functional Foods Act, and "functionality" means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0060] The term "food-acceptable salt" refers to a formulation of a compound that does not cause severe irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound.

[0061] The present invention will be described in detail below. However, this is merely illustrative and the present disclosure is not limited to the specific embodiments described illustratively.

[0062] One aspect of the present invention provides a 3-dehydroxycollinoketone compound represented by the following formula 1 having a tricyclic ketolactone backbone, or a pharmaceutically acceptable salt thereof.

[0063] [Chemical Formula 1]

[0064]

[0065] A 3-dehydroxycolinoketone compound according to one embodiment is a novel compound that can overcome the limitations of collinolactone and has a tricyclic ketolactone backbone.

[0066] According to one embodiment, the 3-dehydroxycholinoketone compound may exhibit improved pharmacokinetics (PK) and promising therapeutic potential compared to collinolactone. The 3-dehydroxycholinoketone compound exhibited superior biological activity compared to collinolactone; specifically, it significantly inhibited and dissociated amyloid-beta (Aβ) and tau aggregates in vitro and was confirmed to show improvement in cognitive function in an AD mouse model. Therefore, since the 3-dehydroxycholinoketone compound has the potential to act as a dual-action therapeutic agent targeting both Aβ and tau aggregates, it can be usefully employed as an active ingredient for the prevention, improvement, or treatment of neurodegenerative diseases, including Alzheimer's disease.

[0067] A 3-dehydroxycholinoketone compound according to one embodiment has the ability to penetrate the blood-brain barrier (BBB) ​​with excellent efficiency, so that a rapid, fast, and more efficient therapeutic effect can be obtained by administering a low amount.

[0068] In one embodiment, the 3-dehydroxycollinoketone compound may be 3-dehydroxy-5-epi-collinoketone represented by the following chemical formula 2.

[0069] [Chemical Formula 2]

[0070]

[0071] Another aspect of the present invention provides a method for preparing the 3-dehydroxycholinoketone compound, specifically.

[0072] A method is provided for preparing a 3-dehydroxycholinoketone compound represented by Chemical Formula 1 using Module 1 represented by Chemical Formula 11, Module 2 represented by Chemical Formula 12, and Module 3 represented by Chemical Formula 13.

[0073] [Chemical Formula 1]

[0074]

[0075] [Chemical Formula 11]

[0076]

[0077] [Chemical Formula 12]

[0078]

[0079] [Chemical Formula 13]

[0080]

[0081] In the above formula,

[0082] R1 to R6 are each independently C1-C6 alkyl;

[0083] TBS tert -butyldimethylsilyl( tert-butyldimethylsilyl)

[0084] A method for preparing the 3-dehydroxycholinoketone compound according to one embodiment utilizes an approach based on a modular synthesis strategy to assemble multiple molecules to form a larger molecule. Through modular chemical synthesis, efficient large-scale synthesis is possible, and reaction steps can be minimized to secure a high yield. In addition, structural modification is easy, and in particular, the synthesis of cyclic lactones can be performed effectively, thereby lowering the difficulty of synthesis compared to existing methods.

[0085] In one embodiment, the method for preparing the 3-dehydroxycholinoketone compound is,

[0086] 1) A step of preparing a compound of the following formula 21 by reacting module 1 of formula 11 and module 2 of formula 12;

[0087] 2) A step of preparing a compound of the following chemical formula 22 by reacting the compound of the following chemical formula 21 with module 3 of the above chemical formula 13;

[0088] 3) A step of deprotecting the compound of Chemical Formula 22 below to produce the compound of Chemical Formula 23 below;

[0089] 4) A step of hydrolyzing the compound of Chemical Formula 23 below to produce the compound of Chemical Formula 24 below;

[0090] 5) a step of preparing a compound of the following formula 25 from a compound of the following formula 24; and

[0091] 6) A step of preparing a 3-dehydroxycolinoketone compound of the following formula 2 by performing a transannular [6+4] cycloaddition reaction on a compound of the following formula 25; may be included.

[0092] [Chemical Formula 2]

[0093]

[0094] [Chemical Formula 21]

[0095]

[0096] [Chemical Formula 22]

[0097]

[0098] [Chemical Formula 23]

[0099]

[0100] [Chemical Formula 24]

[0101]

[0102] [Chemical Formula 25]

[0103]

[0104] In one embodiment, step 1) may be a step of preparing a compound of Formula 21 by performing a Horner-Wadsworth-Emmons (HWE) olefinization reaction of Module 1 of Formula 11 and Module 2 of Formula 12 in the presence of a base. Module 1 (Formula 11) and Module 2 (Formula 12) may be used in equal equivalent amounts.

[0105] In one embodiment, the base may be one or more mixtures selected from the group consisting of barium hydroxide (Ba(OH)2), barium hydroxide octahydrate (Ba(OH)2·8H2O), sodium hydride (NaH), and potassium t-butoxide (KOtBu). The amount of the base used is not particularly limited, but may be used in the range of 1 to 3 equivalents, or 1.1 to 2 equivalents, or 1.1 to 1.5 equivalents for 1 equivalent of Module 1 (Chemical Formula 11).

[0106] In step 1) above, acetonitrile, methylene chloride, tetrahydrofuran, water, etc., may be used as the reaction solvent, and specifically, tetrahydrofuran or a mixed solvent of tetrahydrofuran and water may be used. In addition, the reaction temperature may be in the range of 10 to 30°C, specifically 20 to 25°C, and may be carried out under dark conditions.

[0107] In one embodiment, step 2) is a step of preparing a compound of formula 22 by Suzuki coupling reaction of the compound of formula 21 and module 3 of formula 13. Step 2) may be performed in the presence of a palladium catalyst and a base.

[0108] The amount of the above module 3 (chemical formula 13) used is not particularly limited, but may be in the range of 1 to 3 equivalents, specifically 1.1 to 2 equivalents, for every 1 equivalent of the compound of chemical formula 21.

[0109] In one embodiment, the palladium catalyst may be used without limitation as long as it is a catalyst commonly used in Suzuki coupling, but specifically, Pd(PPh3)4(Tetrakis(triphenylphosphine)palladium(0)), Pd(OAc)2(Palladium(II) Acetate), PdCl2(dppf)CH2Cl2([1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane), Pd(PPh3)2Cl2(Bis(triphenylphosphine)palladium(II) dichloride), Pd2(dba)3(Tris(dibenzylideneacetone)dipalladium(0)), PdCl2(CH3CN)2(Bis(acetonitrile)dichloropalladium(II)), Pd[P(t-Bu)3]2(Bis(tri-tert-butylphosphine)palladium(0)), It may be one or more mixtures selected from the group consisting of PdCl2((P(o-tolyl)3)2(Dichlorobis(tri-o-tolylphosphine)palladium(II)), PdCl2(dppf) ([1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)), PdCl2(dppe) ([1,2-Bis(diphenylphosphino)ethane]dichloropalladium(II)), PdCl2(dppp) (Dichloro(1,3-bis(diphenylphosphino)propane)palladium(II)) and PdCl2(TPP) (Bis(triphenylphosphine)palladium(II) dichloride). The amount of the palladium catalyst used is not particularly limited, but may be in the range of 0.01 to 1 equivalent, specifically 0.1 to 0.5 equivalents, per 1 equivalent of the compound of Formula 21.

[0110] In one embodiment, the base may be used without limitation as long as it is a base commonly used in Suzuki coupling, but specifically, it may be one or more mixtures selected from the group consisting of silver oxide (Ag2O), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), potassium carbonate (K2CO3), cesium carbonate (Cs2CO3), sodium carbonate (Na2CO3), potassium fluoride (KF), cesium fluoride (CsF), sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium methoxide (CH3ONa), sodium t-butoxide (tBuONa), cesium acetate (CsOAc), potassium acetate (KOAc), triethylamine, and diisopropylethylamine. The amount of the base used is not particularly limited, but may be in the range of 1 to 10 equivalents, specifically 3 to 7 equivalents, per 1 equivalent of the compound of Formula 21.

[0111] In one embodiment, step 2) may further include a palladium catalyst and a ligand other than a base. The above ligand may specifically be one or more mixtures selected from the group consisting of triphenyl arsine, triphenylphosphine (PPh3), tri(o-tolyl)phosphine, tri(t-butyl)phosphine, tri(cyclohexyl)phosphine, 1,1'-bis(diphenylphosphino)ferrocene (DPPF), 1,2-bis(diphenylphosphino)ethane (DPPE), 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (rac-BINAP), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (XantPhos), 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos), and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos). The amount of the above ligand used is not particularly limited, but may be in the range of 0.01 to 1 equivalent, specifically 0.1 to 0.5 equivalents, per 1 equivalent of the compound of Formula 21.

[0112] In one embodiment, step 2) above can be carried out in the presence of a palladium catalyst, a base and a ligand, specifically in the presence of Pd(PPh3)4, silver oxide (Ag2O) and triphenylarsenine.

[0113] In step 2) above, any catalyst commonly used for Suzuki coupling may be used as the reaction solvent without limitation, but methanol, N-methylpyrrolidine-2-one, dimethyl ether, dimethylformamide, tetrahydrofuran, 1,4-dioxane, or water may be used, and specifically, tetrahydrofuran or a mixed solvent of tetrahydrofuran and water may be used. In addition, the reaction temperature may be in the range of 10 to 50°C, specifically 20 to 30°C, and may be carried out under dark conditions.

[0114] In one embodiment, in step 3), the deprotection of the TBS group is performed using camphorsulfonic acid (CSA) or tetra-n-butylammonium fluoride (tetra- n- The process may be carried out in the presence of butylammonium fluoride (TBAF). The amount of camphosulfonic acid (CSA) or tetra-n-butylammonium fluoride (TBAF) used is not particularly limited, but may be in the range of 0.1 to 2 equivalents, specifically 0.3 to 1 equivalent, per 1 equivalent of the compound of Formula 22. In this case, methanol, acetonitrile, methylene chloride, diethyl ether, tetrahydrofuran, etc., may be used as the reaction solvent, and methanol is particularly preferred; the process may be carried out under dark conditions.

[0115] In one embodiment, in step 4), the hydrolysis of the compound of Formula 23 may be performed under acidic conditions. Suitable acids include, but are not limited to, trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, etc.

[0116] According to one embodiment, step 4) can be used immediately in the next reaction without separating and purifying the product.

[0117] In one embodiment, in step 5), the compound of Formula 24 can be subjected to a Yamaguchi esterification reaction in the presence of a base, 2,4,6-trichlorobenzoyl chloride, and 4-(dimethylamino)pyridine (4-(dimethylamino)pyridine, DMAP) to produce the compound of Formula 25.

[0118] In one embodiment, the base may be one or more mixtures selected from the group consisting of triethylamine, pyridine, N-methylimidazole, potassium carbonate (K2CO3), cesium carbonate (Cs2CO3), sodium carbonate (Na2CO3), DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) and sodium hydride (NaH), specifically triethylamine. The amount of the base used is not particularly limited, but may be in the range of 1 to 5 equivalents, specifically 2 to 4 equivalents, per 1 equivalent of the compound of Formula 23.

[0119] In one embodiment, in step 6), the cross-ring [6+4] ring addition reaction of the compound of Formula 25 can be carried out in a microwave reactor.

[0120] In one embodiment, module 1 of the formula 11 is,

[0121] a) a step of preparing a compound of the following formula 11-b by reacting the compound of the following formula 11-a with hydrozirconation and iodination; and

[0122] b) a step of preparing Module 1 of Formula 11 by reacting a compound of Formula 11-b with a C1-C10 alkyllithium compound of Formula 11-b below with a phosphonate compound of Formula 11-b below; may be prepared by including the above steps.

[0123] [Chemical Formula 11]

[0124]

[0125] [Chemical Formula 11-a]

[0126]

[0127] [Chemical Formula 11-b]

[0128]

[0129] [Chemical Formula 11-c]

[0130]

[0131] In the above equation, R1, R2, and R a Each is independently a C1-C6 alkyl.

[0132] In one embodiment, module 2 of the formula 12 is,

[0133] c) React the compound of the following chemical formula 12-a with isobutenylmagnesium bromide, and then, tert -Butyldimethylsilyl chloride ( tert A step of preparing a compound of the following chemical formula 12-b by reacting with -butyldimethylsilyl chloride (TBSCl); and

[0134] d) a step of selectively oxidizing the compound of Formula 12-b in the presence of SeO2 to produce Module 2 of Formula 12; may be prepared by including this step.

[0135] [Chemical Formula 12]

[0136]

[0137] [Chemical Formula 12-a]

[0138]

[0139] [Chemical Formula 12-b]

[0140]

[0141] In the above equation, TBS is tert -butyldimethylsilyl( tert -butyldimethylsilyl)

[0142] In one embodiment, module 3 of the formula 13 is,

[0143] e) a step of preparing a compound of the following formula 13-b by reacting the compound of formula 13-a below with zirconium-catalyzed carboalumination and iodination;

[0144] f) a step of preparing a compound of the following formula 13-c by oxidizing the compound of formula 13-b in the presence of manganese oxide;

[0145] g) a step of preparing a compound of the following formula 13-e by reacting the compound of the following formula 13-c with the compound of the following formula 13-d using the Wittig reaction; and

[0146] h) a step of reacting the compound of Formula 13-e below with a triC1-C10 alkyl borate in the presence of C1-C10 alkyl lithium, and then reacting it with a diol compound of Formula 13-f to prepare Module 3 of Formula 13; may be prepared by including this step.

[0147] [Chemical Formula 13]

[0148]

[0149] [Chemical Formula 13-a]

[0150]

[0151] [Chemical Formula 13-b]

[0152]

[0153] [Chemical Formula 13-c]

[0154]

[0155] [Chemical Formula 13-d]

[0156]

[0157] [Chemical Formula 13-e]

[0158]

[0159] [Chemical Formula 13-f]

[0160]

[0161] In the above formula, R3 to R6 are each independently C1-C6 alkyl.

[0162] The process for preparing a 3-dehydroxycholinoketone compound according to one embodiment is shown in Reaction Scheme 1 below.

[0163] [Reaction Equation 1]

[0164]

[0165] According to one embodiment, a 3-dehydroxycholinoketone compound, a promising therapeutic candidate targeting Aβ and tau aggregates, which are two major pathological features of Alzheimer's disease (AD), can be efficiently synthesized using a stereoselective trans-circular [6+4] cycloaddition reaction as a key step. In addition, efficient large-scale synthesis is possible by assembling multiple molecules using a modular synthesis strategy.

[0166] Another aspect of the present invention provides a pharmaceutical composition for the treatment and prevention of neurodegenerative diseases containing the 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0167] In one embodiment, the neurodegenerative disease refers to a disease or pathological condition in which the function of the subject's nervous system is impaired, meaning abnormalities in motor control ability, cognitive function, perceptual function, sensory function, and autonomic nervous system function, and is synonymous with 'degenerative brain disease'. Specifically, the aforementioned neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, Amyotrophic Lateral Sclerosis (ALS), mild cognitive impairment, amyloidosis, multiple system atrophy, multiple sclerosis, tauopathies, Pick's disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, and Lewy body dementia. It can be selected from a group consisting of dystonia, progressive supranuclear palsy, and frontotemporal dementia.

[0168] The above 3-dehydroxycholinoketone compound has been confirmed to have the effect of simultaneously reducing amyloid-beta (Aβ) and tau aggregates in vitro and in vivo, and can be usefully utilized as an active ingredient for the prevention, improvement, or treatment of neurodegenerative diseases, including Alzheimer's disease. In addition, the above 3-dehydroxycholinoketone compound has very high permeability to the blood-brain barrier (BBB), so it can act directly on the brain and exhibit excellent effects in the treatment of neurodegenerative brain diseases. Therefore, the above 3-dehydroxycholinoketone compound can be usefully utilized for the prevention or treatment of neurodegenerative diseases more effectively by targeting both Aβ and tau aggregates.

[0169] In one embodiment, the pharmaceutical composition may be formulated into a formulation conventional in the pharmaceutical field, namely an oral formulation or a parenteral formulation, by additionally including a conventional non-toxic pharmaceutically acceptable carrier and / or excipient in addition to the active ingredient. Additionally, it may further include a diluent such as a filler, a volume extender, a binder, a wetting agent, a disintegrant, or a surfactant.

[0170] The above-mentioned pharmaceutically acceptable carriers, excipients, or diluents may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0171] The above pharmaceutical composition can be formulated into various forms according to the usual method to suit the intended use, such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injectable formulations like sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and local administration.

[0172] The above pharmaceutical composition may additionally include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0173] Oral dosage forms include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc., and in addition to the above active ingredient, these dosage forms may use one or more diluents or excipients such as commonly used fillers, fillers, humectants, disintegrants, lubricants, binders, and surfactants. Agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate anhydrous, etc. may be used as disintegrants; silica, talc, stearic acid or its magnesium or calcium salt, polyethylene glycol, etc. may be used as lubricants; and magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, etc. may be used as binders. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. may be used as diluents, and in some cases, generally known boiling mixtures, absorbents, coloring agents, flavoring agents, sweeteners, etc. may be used together.

[0174] Examples of formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerol, gelatin, etc. may be used. Meanwhile, injectable formulations may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. To formulate an injectable formulation, the above-mentioned 3-dehydroxycolinoketone compound or a pharmaceutically acceptable salt thereof may be mixed in water with a stabilizer or buffer to prepare a solution or suspension, and this may be prepared in a unit dosage form in an ampoule or vial.

[0175] The above pharmaceutical composition may be sterilized or further include auxiliary agents such as preservatives, stabilizers, thickeners, wettable agents or emulsification promoters, salts and / or buffers for osmotic pressure regulation, and further include other therapeutically useful substances, and may be formulated according to conventional methods such as dissolution, dispersion, mixing, granulation, gelation, or coating.

[0176] The pharmaceutically effective amount of the above 3-dehydroxycholinoketone compound may be determined based on factors including the patient's health condition, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. Specifically, the effective amount of the 3-dehydroxycholinoketone compound in the above pharmaceutical composition may vary depending on the patient's age, gender, and weight, and is generally about 0.01 to 500 mg / kg / day, preferably 0.1 to 100 mg / kg / day, and may be administered daily or every other day, or divided into one or several doses per day. However, since the dosage may increase or decrease depending on the route of administration, severity of the disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0177] The above pharmaceutical composition may be administered orally or parenterally, and parenteral administration, such as subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection, is preferred.

[0178] The above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking into account all of the above factors, and this can be easily determined by a person skilled in the art.

[0179] Another aspect of the present invention provides a method for preventing or treating a neurodegenerative disease, comprising the step of administering the 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt, or the pharmaceutical composition, to an individual who has developed or is at risk of developing a neurodegenerative disease.

[0180] A 3-dehydroxycholinoketone compound or pharmaceutical composition according to one embodiment may be administered to an individual in a therapeutically effective or pharmaceutically effective amount.

[0181] The route and method of administration for administering the above-mentioned 3-dehydroxycholinoketone compound or pharmaceutical composition are not particularly limited and may follow any route and method of administration as long as the above-mentioned 3-dehydroxycholinoketone compound or pharmaceutical composition can reach the intended site. Specifically, the above-mentioned 3-dehydroxycholinoketone compound or pharmaceutical composition may be administered via various routes of oral or parenteral administration, and non-limiting examples of such routes of administration include administration via the mouth, rectum, topical, intravenous, intraperitoneal, intramuscular, intra-arterial, transdermal, nasal, or inhalation.

[0182] The above 3-dehydroxycholinoketone compound or pharmaceutical composition may be administered systemically or topically, and may be administered alone or in combination with other therapeutic agents.

[0183] Another aspect of the present invention provides a health functional food composition for the prevention or improvement of neurodegenerative diseases containing the 3-dehydroxycholinoketone compound or a food-grade acceptable salt thereof as an active ingredient.

[0184] The above food-grade acceptable salt may be an acid addition salt formed by a food-grade acceptable free acid or a salt formed by a base. As an example, inorganic acids and organic acids may be used as free acids. As inorganic acids, hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. may be used, and as organic acids, sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc. may be used. In addition, salts formed by a base may include alkali metal salts such as ammonium salts, sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, salts of organic bases such as dicyclohexylamine, N-methyl-D-glucarmine, and tris(hydroxymethyl)methylamine, and amino acid salts such as arginine and lysine. However, they are not limited thereto.

[0185] The above-mentioned health functional food composition may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage, and the health functional food may be used in combination with other foods or food additives in addition to the active ingredient, and may be used appropriately according to conventional methods. The amount of the mixture of the active ingredient may be appropriately determined according to its purpose of use, for example, prevention, health, or therapeutic treatment.

[0186] The above-mentioned health functional food composition may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0187] The above-mentioned health functional food may additionally contain food additives, and unless otherwise stipulated, suitability as a 'food additive' shall be determined in accordance with the specifications and standards for the relevant item, based on the general provisions and general test methods of the Food Additives Codex approved by the Korea Food and Drug Administration.

[0188] Examples of items listed in the above 'Food Additives Codex' include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0189] The above health functional food composition can be formulated into various forms such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0190] The content of the active ingredient in the above health functional food composition may be used in accordance with the effective dose of the above pharmaceutical composition, but in the case of long-term consumption for the purpose of health and hygiene or health control, it may be less than the above range, and since the active ingredient has no problem in terms of safety, it is obvious that it may be used in an amount greater than the above range.

[0191] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0192] All reactions were performed in oven-dried or flame-dried round-bottom flasks and vials. Unless otherwise specified, flasks were fitted with rubber septa, and reactions were performed under positive argon pressure; vials were tightly sealed with plastic septa, Teflon tape, and Parafilm. Stainless steel syringes or cannulas were used to transfer air and moisture-sensitive liquids. Flash column chromatography was performed using silica gel (60 Å pore size, 40–63 μm particle size, 4–6% H2O content, Merck) [Reference J. Org. Chem. 1978 , 43 It was performed according to [ , 2923-2925]. Thin-layer chromatography (TLC) for analysis was performed using glass plates pre-coated with 0.25 mm silica gel containing a fluorescent indicator (254 nm). The thin-layer chromatography plates were visualized by exposure to ultraviolet light and / or an aqueous CAM (ceric ammonium molybdate) solution.

[0193] Unless otherwise specified, all commercially available reagents and solvents were used without further purification, except as specified below. Dichloromethane and tetrahydrofuran were purchased from Merck and Daejung Inc., respectively, and reference [ Organometallics 1996 , 15, It was purified under positive argon pressure according to the method of [1518-1520].

[0194] 1 H and 1313C NMR spectra were recorded using a Bruker Avance NEO (500 MHz) or Agilent DD-2 (600 MHz) and corrected using residual deuterium chloroform (residual CHCl3in CDCl3, δH = 7.24 ppm) and CDCl3 (δC = 77.16 ppm) as internal standards. Data were expressed as follows: chemical shift (units ppm) [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m = multiplet, app = apparent, br = broad), coupling constant (units Hz), integral value]. To remove residual DCl (deuterium chloride), NMR solvent CDCl3 was taken from a stock containing anhydrous K2CO3. High-resolution mass spectra (HRMS) of the synthetic compound were obtained at the KAIST Analysis Center for Research Advancement (KARA) (Daejeon) using electrospray ionization (ESI) or atmospheric solids analysis probe (ASAP) ionization methods. Specific rotation [α] D T The spectra were obtained using a JASCO P-2000 polarimeter. The ECD (electronic circular dichroism) spectra were measured using a JASCO J-1500 CD spectrometer (JASCO, Easton, MD, USA).

[0195] [Manufacturing Example 1] Manufacturing of Module 1

[0196]

[0197] Preparation of Methyl (E)-6-iodohex-5-enoate (Compound 11-b)

[0198] A solution of Schwartz's reagent (20.1 g, 78 mmol, 1.3 equiv.) in tetrahydrofuran (THF) (180 mL) was mixed with methyl 5-hexynoate (compound) while stirring at room temperature 11-a ) (7.9 mL, 60 mmol, 1.0 equiv.) was added dropwise. After 35 minutes, iodine (19.80 g, 78 mmol, 1.3 equiv.) in THF (30 mL) was added dropwise to the reaction mixture at 0 °C over 10 minutes. The reaction mixture was heated to room temperature (rt) and stirred for 1 hour and 10 minutes. Afterward, 500 mL of saturated NaHCO3 solution was added to quench the reaction. The aqueous layer was extracted with ethyl acetate (EA) (3 × 300 mL). The combined organic layer was washed with saturated Na2S2O3 solution, dried with anhydrous MgSO4, filtered, and the filtrate concentrated under vacuum. The residue was purified by silica gel column chromatography (silica gel: diam. 10 cm, ht. 15 cm; eluent: ethyl acetate:hexane = 1:20), and the compound 11-b It was obtained as yellow oil (11.52 g, yield 75%).

[0199] 1 1H NMR (600 MHz, Chloroform- d ): δ 6.46 (dt, J = 14.4, 7.2 Hz, 1H), 6.02 (dt, J = 14.4, 1.5 Hz, 1H), 3.65 (s, 3H), 2.29 (t, J = 7.4 Hz, 2H), 2.08 (qd, J = 7.2, 1.4 Hz, 2H), 1.71 (p, J = 7.4 Hz, 2H).

[0200] 13C NMR (151 MHz, CDCl3): δ 173.75, 145.43, 77.37, 77.16, 76.95, 75.68, 51.73, 35.41, 33.18, 23.62.

[0201] HRMS (ESI): Calculated for C7H 11 IO2[M+H] + : 254.9877, found: 254.9884.

[0202] TLC (ethyl acetate:hexane = 1:20) R f : 0.5 (UV, CAM).

[0203] Preparation of Dimethyl (E)-(7-iodo-2-oxohept-6-en-1-yl)phosphonate (Compound 11, hereinafter referred to as 'Module 1')

[0204] dimethyl methylphosphonate (compound) in THF (25 mL) at -78 ℃ 11-c n-BuLi (2.116 M in hexane, 21.3 mL, 45 mmol, 1.8 equiv.) was added dropwise to a solution of ) (5.4 mL, 50 mmol, 2.0 equiv.). After stirring the reaction mixture for 40 minutes, the compound in THF (10 mL) 11-b A solution of 6.35 g (25 mmol) was added dropwise. The dark yellow solution was stirred at -78 °C for 4 hours, after which the reaction was quenched by adding 200 mL of saturated NH₄Cl solution. The mixture was heated to room temperature and diluted by adding 200 mL of H₂O. After separating the layers, the aqueous layer was extracted with EA (3 × 200 mL). The combined organic layer was dried with anhydrous MgSO₄, filtered, and concentrated under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 10 cm, height 15 cm; eluent: dichloromethane : acetone = 25 : 1) to obtain the compound 11 (Module 1) was obtained as yellow oil (6.31 g, yield 72%).

[0205] 1 H NMR (500 MHz, Chloroform- d ) δ 6.42 (dt, J = 14.4, 7.2 Hz, 1H), 5.99 (dt, J = 14.6, 1.5 Hz, 1H), 3.75 (s, 3H), 3.73 (s, 3H), 3.06 (s, 1H), 3.01 (s, 1H), 2.58 (t, J = 7.1 Hz, 2H), 2.03 (qd, J = 7.3, 1.4 Hz, 2H), 1.65 (p, J = 7.2 Hz, 2H).

[0206] 13 C NMR (126 MHz, CDCl3) δ 201.36, 201.31, 145.53, 77.41, 77.16, 76.91, 75.61, 53.20, 53.15, 42.92, 42.03, 41.02, 35.03, 21.93.

[0207] HRMS (ESI): Calculated for C9H 16 IO4P [M+H] + : 346.9904, found: 346.9909.

[0208] TLC (acetone : dichloromethane = 1 : 25) R f : 0.5 (UV, CAM).

[0209] [Manufacturing Example 2] Manufacture of Module 2

[0210]

[0211] (R)- tert Preparation of -Butyldimethyl((6-methylhept-5-en-3-yl)oxy)silane (Compound 12-b)

[0212] 2-Methyl-1-propenylmagnesium Grignard reagent (100 mL, 1.1 equiv., 0.5 M in THF) was added to a flame-dried Schlenk flask and cooled to -35 °C. Copper(I) iodide (432 mg, 2.27 mmol, 5 mol%) was added, and the mixture was stirred at -35 °C for 30 minutes. Then, (R)-(+)-1,2-Epoxybutane (compound 12-a ) (3.95 mL, 45.45 mmol, 1.0 equiv.) was added dropwise over 20 minutes, and the reaction mixture was stirred for 1 hour. The mixture was heated to room temperature (rt) and stirred for an additional 2.5 hours. The reaction mixture was poured into a separatory funnel containing 12 M HCl (50 mL) and brine (200 mL). After separating the layers, the aqueous layer was washed with EA (3 × 300 mL). The combined organic layer was dried with anhydrous MgSO4, filtered, and concentrated under vacuum. The resulting crude residue was used directly in the next reaction without further purification by column chromatography.

[0213] The crude residue was dissolved in dichloromethane (450 mL, 0.1 M), and tert-butyldimethylsilyl chloride (10.24 g, 68.18 mmol, 1.5 equiv.) and imidazole (9.283 g, 136.4 mmol, 3.0 equiv.) were added at 0 °C. After removing the ice bath, the reaction mixture was stirred overnight at room temperature. Subsequently, 500 mL of saturated aqueous NH4Cl solution was added to quench the reaction. The aqueous layer was extracted with dichloromethane (3 × 300 mL). The combined organic layer was dried with anhydrous MgSO4, filtered, and concentrated under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 10 cm, height 15 cm; eluent: ethyl acetate : hexane = 1 : 50) to obtain the compound 12-b It was obtained in the form of a transparent oil (10.1 g, yield 92%).

[0214] 1 1H NMR (500 MHz, Chloroform- d ) δ 5.11 (tp, J = 7.3, 1.4 Hz, 1H), 3.54 (qd, J = 6.2, 4.9 Hz, 1H), 2.13 - 2.08 (m, 2H), 1.68 (q, J = 1.3 Hz, 3H), 1.58 (d, J = 1.3 Hz, 3H), 1.49 - 1.32 (m, 2H), 0.87 (s, 9H), 0.84 (d, J = 7.5 Hz, 3H), 0.02 (s, 6H).

[0215] 13 C NMR (126 MHz, CDCl3) δ 132.80, 121.40, 77.41, 77.16, 76.90, 73.98, 35.87, 29.75, 26.06, 26.01, 18.33, 18.05, 9.95, 9.71, -4.31, -4.44.

[0216] HRMS (ESI): Calculated for C 22 H 30 N2O4[M+H] + : 243.2139, found: 243.2143.

[0217] TLC (ethyl acetate:hexane = 1:50) R f : 0.42 (UV, CAM).

[0218] Specific Rotation [α] D 25 : -2.5 (c 0.1, MeOH).

[0219] (R,E)-5-(( tert Preparation of -butyldimethylsilyl)oxy)-2-Methylhept-2-enal (Compound 12, hereinafter referred to as 'Module 2')

[0220] Compound in 95% EtOH (250 mL) 12-b SeO2 (10.09 g, 90.9 mmol, 2.0 equiv.) was added to a solution of (11.0 g, 45.45 mmol, 1.0 equiv.). The reaction mixture was heated under reflux for 18 hours. After cooling the reaction mixture to 23 °C, it was treated with a saturated aqueous NaHCO3 solution (100 mL). The aqueous layer was extracted with EA (3 × 300 mL), the combined organic layer was washed with brine, dried with anhydrous MgSO4, and filtered through a Celite plug. The filtrate was washed with EA and concentrated under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 10 cm, height 15 cm; eluent: ethyl acetate : hexane = 1 : 50) to determine the compound 12 (Module 2) was obtained in the form of a clear oil (10.1 g, yield 62%).

[0221] 1 1H NMR (500 MHz, Chloroform- d ) δ 9.40 (s, 1H), 6.57 (td,J = 7.3, 1.4 Hz, 1H), 3.78 (p, J = 5.8 Hz, 1H), 2.53 - 2.45 (m, 2H), 1.73 (d, J = 1.3 Hz, 3H), 1.47 (tdd, J = 7.3, 5.9, 1.2 Hz, 2H), 0.89 (t, J = 7.5 Hz, 3H), 0.87 (s, 9H), 0.04 (s, 3H), 0.02 (s, 3H).

[0222] 13 C NMR (126 MHz, CDCl3) δ 195.41, 151.62, 140.53, 77.41, 77.16, 76.91, 72.50, 36.39, 30.25, 25.95, 18.22, 9.82, 9.58, -4.36, -4.39.

[0223] HRMS (ESI): Calculated for C 14 H 28 O2Si [M+H] + : 257.1932, found: 257.1936.

[0224] TLC (ethyl acetate : hexane = 1 : 50) R f : 0.3 (UV, CAM).

[0225] Specific Rotation [α] D 25 : 16.7 (c 0.3, MeOH).

[0226] [Manufacturing Example 3] Manufacture of Module 3

[0227]

[0228] Preparation of allylic alcohol (compound 13-b)

[0229] A trimethylaluminum (Me3Al) solution (25 mL, 2 M in toluene, 40 mmol) was slowly added dropwise to a mixture of Cp2ZrCl2 (1.17 g, 4 mmol) in dry dichloromethane (CH2Cl2) (30 mL). Then, after cooling to 0 °C, propargylic alcohol (compound) in dry dichloromethane (20 mL) 13-a A solution of ) (1.12 g, 22 mmol) was slowly added dropwise over 30 minutes. After stirring the reaction mixture overnight, it was cooled to -30 °C, and a solution of iodine (7.60 g, 30 mmol) in anhydrous THF (25 mL) was slowly added. After stirring for an additional 20 minutes at -30 °C, a saturated NaHCO3 solution (5 mL) was slowly added (Caution: gas generation and heat release). The cooling bath was then removed. After gas generation ceased, an additional saturated NaHCO3 solution (40 mL) was added, and the layers were separated. The aqueous layer was extracted with ether (3 × 50 mL), the combined organic layer was washed with a saturated Na2S2O3 solution (3 × 50 mL), dried with anhydrous sodium sulfate (Na2SO4), and concentrated under vacuum to obtain the crude product (compound 13-b ) was obtained. The resulting crude product was used directly in the next reaction without further purification by column chromatography.

[0230] tert Preparation of -Butyl (2E,4E)-5-iodo-4-methylpenta-2,4-dienoate (Compound 13-e)

[0231] Allylic alcohol 13-b(9.23 g, 46.6 mmol, 1.0 equiv.) was dissolved in dry dichloromethane (0.1 M, 470 mL). Manganese dioxide (MnO2, 81.00 g, 932 mmol, 20.0 equiv.) was added, and the mixture was stirred at room temperature (rt) for 4 hours. The black solid was then removed by filtering through Celite, and the mixture was thoroughly washed with dichloromethane. The filtrate was transferred to a reaction flask, and (tert-butoxycarbonylmethylene)triphenylphosphorane (compound 13-d ) (19.3 g, 51.26 mmol, 1.1 equiv.) was added. The reaction mixture was stirred overnight at room temperature under light-blocking aluminum foil. After removing dichloromethane using a rotary evaporator, diethyl ether was added to the residue. The resulting solid was filtered and thoroughly washed with diethyl ether. The filtrate was concentrated using a rotary evaporator, and the resulting crude residue was purified by flash column chromatography (silica gel, diameter 10 cm, height 15 cm; eluent: ethyl acetate : hexane = 1 : 40) to obtain the compound 13-e It was obtained in the form of a yellow oil (11.4 g, yield 83%).

[0232] 1 1H NMR (500 MHz, Chloroform- d ) δ 7.21 (d, J = 15.6 Hz, 1H), 6.82 (d, J = 1.4 Hz, 1H), 5.85 (d, J = 15.6 Hz, 1H), 1.95 (d, J = 1.2 Hz, 3H), 1.46 (s, 9H).

[0233] 13C NMR (126 MHz, CDCl3) δ 166.34, 144.22, 143.45, 120.47, 92.56, 80.73, 77.41, 77.16, 76.91, 28.25, 19.84.

[0234] HRMS (ESI): Calculated for C 10 H 15 IO2[M+H] + : 295.0190, found: 236.1286.

[0235] TLC (ethyl acetate:hexane = 1:40) R f : 0.2 (UV, CAM).

[0236] tert Preparation of -Butyl (2E,4E)-4-methyl-5-(4,4,5,5-tetraethyl-1,3,2-dioxaborolan-2-yl)penta-2,4-dienoate (Compound 13, hereinafter referred to as 'Module 3')

[0237] compound 13-e (5.62 g, 18.85 mmol, 1.0 equiv.) and triisopropylborate (4.8 mL, 20.74 mmol, 1.1 equiv.) were dissolved in a mixed solvent of toluene (120 mL) and THF (20 mL), after which n-BuLi (1.75 M in hexane, 11.9 mL, 20.74 mmol, 1.1 equiv.) was added dropwise at -78 °C. After stirring the reaction mixture at -78 °C for 60 minutes, 3,4-diethylhexane-3,4-diol (compound 13-f) (4.93 g, 28.28 mmol, 1.5 equiv.) was added at -78 °C. The mixture was protected from light by aluminum foil, heated to 25 °C, and stirred for 34 hours. The reaction mixture was diluted with EA and washed with saturated aqueous NH4Cl solution, water, and brine. The organic layer was dried with anhydrous MgSO4, filtered, and concentrated under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diam. 10 cm, ht. 15 cm; eluent: ethyl acetate : hexane = 1 : 20) to extract the compound 13 (Module 3) was obtained in the form of a yellow oil (5.62 g, yield 85%).

[0238] 1 1H NMR (500 MHz, Chloroform- d ) δ 7.21 (d, J = 15.7 Hz, 1H), 5.88 (d, J = 15.7 Hz, 1H), 5.66 (s, 1H), 2.09 (s, 3H), 1.67 (dp, J = 21.7, 7.2 Hz, 8H), 1.47 (s, 9H), 0.90 (t, J = 7.5 Hz, 12H).

[0239] 13 C NMR (126 MHz, CDCl3) δ 166.50, 152.78, 148.96, 121.74, 88.37, 80.43, 77.28, 77.02, 76.77, 28.16, 26.34, 16.21, 8.85.

[0240] HRMS (ESI): Calculated for C 20 H 35 BO4[M+Na] + : 373.25, found: 373.2527.

[0241] TLC (ethyl acetate:hexane = 1:20) Rf : 0.3 (UV, CAM).

[0242] [Example 1] 3-Dehydroxy-5- epi Preparation of -collinoketone (compound 2)

[0243]

[0244] (R,1E,7E,9E)-12-(( tert Preparation of -butyldimethylsilyl)oxy)-1-Iodo-9-methyltetradeca-1,7,9-trien-6-one (Compound 21)

[0245] Ba(OH)2·8H2O (7.42 g, 23.54 mmol, 1.2 equiv.) was heated under vacuum at 140 °C for 4 hours, then cooled to room temperature (rt), and a mixture of THF / H2O (40:1, 150 mL) was added. In the THF / H2O (40:1, 3 mL) Modulus 1 (compound 11 ) A solution of (6.79 g, 19.61 mmol, 1.0 equiv.) was added at room temperature (rt). After stirring for 1 hour, Module 2 (compound 12 ) (5.03 g, 19.61 mmol, 1.0 equiv.) was added, and the mixture was stirred at room temperature under dark conditions for 20 hours. Subsequently, 200 mL of saturated aqueous NH4Cl solution was added to quench the reaction. After separating the phases, the aqueous phase was extracted with EA (3 × 300 mL). The combined organic layer was dried with anhydrous MgSO4, filtered, and the solvent removed under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 10 cm, height 15 cm; eluent: ethyl acetate : hexane = 1 : 50) to extract the compound 21 It was obtained in the form of a yellow oil (7.32 g, yield 78%).

[0246] 1 1H NMR (500 MHz, Chloroform- d ) δ 7.16 (d, J = 15.9 Hz, 1H), 6.48 (dt,J = 14.3, 7.2 Hz, 1H), 6.06 (d, J = 15.9 Hz, 1H), 6.02 (dt, J = 14.3, 1.5 Hz, 1H), 5.99 (t, J = 2.3 Hz, 1H), 3.69 (p, J = 5.8 Hz, 1H), 2.55 (t, J = 7.3 Hz, 2H), 2.39 - 2.31 (m, 2H), 2.07 (td, J = 7.3, 1.4 Hz, 2H), 1.76 (d, J = 1.2 Hz, 3H), 1.75 - 1.70 (m, 2H), 1.44 (ddd, J = 7.4, 5.9, 3.2 Hz, 2H), 0.86 (s, 9H), 0.85 (t, J = 7.5 Hz, 3H), 0.02 (d, J = 8.0 Hz, 6H).

[0247] 13 C NMR (126 MHz, CDCl3) δ 200.35, 147.82, 145.93, 140.00, 134.33, 124.33, 77.41, 77.16, 76.91, 75.42, 73.10, 39.47, 36.59, 35.53, 30.09, 26.00, 23.05, 18.24, 12.58, 9.84, -4.33, -4.41.

[0248] HRMS (ESI): Calculated for C 21 H 37 IO2Si [M+Na] + : 499.1500, found: 499.1508.

[0249] TLC (ethyl acetate : hexane = 1 : 50) R f : 0.35 (UV, CAM).

[0250] Specific Rotation [α] D 25: -6.56 (c 0.5, MeOH).

[0251] Preparation of pentaenoate (compound 23)

[0252] compound 21 (4.77 g, 10 mmol, 1.0 equiv.) and modulus 3 (compound 13 All of ) (5.25 g, 15 mmol, 1.5 equiv.) was dissolved in degassed THF (3 mL each), and then, using a cannula at 23 °C, the degassed solution of AsPh3 (919 mg, 3 mmol, 0.3 equiv.) and Ag2O (127.4 mg, 0.55 mmol, 5.0 equiv.) in anhydrous THF (200 mL) was injected. Then, Pd(PPh3)4 (3.47 g, 3 mmol, 0.3 equiv.) was added all at once under an argon atmosphere. Degassed water (20 mL) was added to adjust the THF / H2O ratio to 10:1 (v / v). The flask was wrapped in aluminum foil to block light and protected, and then stirred at 23 °C for 3.5 hours. After the reaction was complete, THF was evaporated, and the resulting crude residue was purified by column chromatography (silica gel, diam. 5 cm, ht. 20 cm; eluent: ethyl acetate : hexane = 1 : 20) to obtain the Suzuki coupling product.

[0253] Camphorsulfonic acid (CSA) (727 mg, 3.13 mmol, 0.5 equiv.) was aliquoted and added to a solution of the Suzuki coupling product (3.24 g, 6.26 mmol, 1.0 equiv.) in MeOH (0.05 M). The mixture was stirred at 23 °C for 3 hours under light-blocking aluminum foil. The reaction mixture was diluted with EA and washed with a saturated aqueous solution of NaHCO3 (300 mL), water, and brine. The organic layer was dried with anhydrous MgSO4, filtered, and concentrated under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 5 cm, height 20 cm; eluent: ethyl acetate : hexane = 1:20) to obtain the compound 23 It was obtained in the form of a yellow oil (2.42 g, 60% yield over two steps).

[0254] 1 1H NMR (500 MHz, Chloroform- d ) δ 7.19 (t, J = 15.8 Hz, 2H), 6.37 (ddt, J = 14.5, 11.3, 1.5 Hz, 1H), 6.29 (d, J = 11.3 Hz, 1H), 6.08 (d, J = 15.9 Hz, 1H), 6.00 (t, J = 7.5 Hz, 1H), 5.86 (dt, J = 14.5, 7.1 Hz, 1H), 5.75 (d, J = 15.5 Hz, 1H), 3.64 (tt, J = 7.3, 5.0 Hz, 1H), 2.56 (t, J = 7.3 Hz, 2H), 2.37 (d, J = 7.1 Hz, 2H), 2.23 - 2.15 (m, 2H), 1.82 (d, J = 1.1 Hz, 3H), 1.77 (d, J= 1.2 Hz, 3H), 1.75 (d, J = 7.3 Hz, 2H), 1.56 - 1.49 (m, 2H), 1.46 (s, 9H), 0.94 (t, J = 7.4 Hz, 3H).

[0255] 13 C NMR (126 MHz, CDCl3) δ 200.74, 167.02, 148.34, 147.45, 139.06, 139.01, 138.86, 138.66, 138.13, 138.09, 135.24, 133.42, 131.90, 127.41, 126.69, 124.78, 118.44, 80.17, 77.41, 77.16, 76.91, 72.97, 72.83, 40.34, 39.72, 36.61, 35.61, 32.77, 30.11, 30.00, 28.33, 23.82, 22.07, 20.39, 12.59, 12.58, 10.10, 10.07.

[0256] HRMS (ESI): Calculated for C 25 H 38 O4[M+Na] + : 499.1500, found: 499.1508.

[0257] TLC (ethyl acetate:hexane = 1:50) R f : 0.35 (UV, CAM).

[0258] Specific Rotation [α] D 25 : -10.66 (c 0.5, MeOH).

[0259] Preparation of Macrolactone (Compound 25)

[0260] Compound in CH2Cl2 (36 mL, 0.05 M) 23 A solution of (721 mg, 1.79 mmol) was cooled to 0 ℃ and light was blocked with aluminum foil. After cooling, Trifluoroacetic acid (TFA) (7.2 mL) was added dropwise. The mixture was stirred at 0 °C for 4 hours in the absence of light. Afterward, the reaction mixture was diluted with EA and washed with saturated pH 2.8 buffer solution, water, and brine. The organic layer was dried with anhydrous MgSO4, filtered, and concentrated under vacuum. The resulting crude residue was used directly in the next reaction without further purification by column chromatography.

[0261] The above crude residue was dissolved in THF (45 mL) to a concentration of 0.04 M, and the solution was mixed with a solution of triethylamine (750 μL, 5.37 mmol, 3.0 equiv.), and then a solution of 2,4,6-trichlorobenzoyl chloride (TCBC) (420 μL, 2.69 mmol, 1.5 equiv.) was added at room temperature. The mixture was stirred for 3 hours at room temperature while blocking light with aluminum foil. The crude mixed anhydride produced above was diluted with toluene using a syringe, and then added to a solution of 4-(dimethylamino)pyridine (DMAP) dissolved in toluene (0.01 M, 134 mL) over 2 hours using a syringe pump. Subsequently, the syringe was further washed with toluene (10 mL). After stirring for an additional hour, the reaction mixture was poured into a saturated NaHCO3 solution (200 mL), extracted with EA (3 × 200 mL), washed with brine (200 mL), dried with anhydrous MgSO4, and concentrated under reduced pressure. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 5 cm, height 20 cm; eluent: ethyl acetate : hexane = 1:9) to obtain the compound 25It was obtained as a white solid (266.4 mg, 60% yield over two steps).

[0262] 1 1H NMR (500 MHz, Chloroform- d ) δ 7.18 (dd, J = 15.3, 0.7 Hz, 1H), 7.10 (dd, J = 15.7, 0.7 Hz, 1H), 6.14 (ddd, J = 14.8, 11.0, 1.5 Hz, 1H), 6.01 (dd, J = 10.4, 5.7 Hz, 1H), 5.93 (d, J = 11.2 Hz, 1H), 5.80 (d, J = 15.3 Hz, 1H), 5.61 (d, J = 15.6 Hz, 1H), 5.49 (ddd, J = 15.1, 10.2, 5.3 Hz, 1H), 4.98 (dtd, J = 8.8, 5.3, 3.6 Hz, 1H), 2.76 (ddd, J = 15.2, 10.6, 5.1 Hz, 1H), 2.38 - 2.33 (m, 1H), 2.30 (d, J = 2.2 Hz, 1H), 2.28 (dd, J = 8.0, 1.6 Hz, 1H), 2.26 - 2.20 (m, 1H), 2.21 - 2.16 (m, 1H), 2.10 - 2.01 (m, 1H), 1.92 - 1.80 (m, 2H), 1.77 (d, J = 1.1 Hz, 3H), 1.73 - 1.63 (m, 1H), 1.47 (t, J = 1.1 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H).

[0263] 13C NMR (126 MHz, CDCl3) δ 200.01, 168.06, 151.65, 146.53, 142.47, 141.62, 137.40, 136.24, 131.09, 127.07, 124.22, 115.21, 77.41, 77.16, 76.91, 75.19, 41.76, 34.00, 26.24, 23.67, 12.32, 12.11, 10.70.

[0264] HRMS (ESI): Calculated for C 21 H 28 O3[M+H] + : 329.2112, found: 329.2122.

[0265] TLC (ethyl actate:hexane = 1:8) R f : 0.4 (UV, CAM).

[0266] Specific Rotation [α] D 25 : -212.4 (c 0.6, MeOH).

[0267] 3-Dehydroxy-5- epi Preparation of -collinoketone (compound 2)

[0268] Under a purged argon atmosphere in a glove box, the compound 25 (170 mg, 0.518 mmol, 1.0 equiv.) was added to a 30 mL microwave tube containing 15 mL of toluene. The tube was sealed with a cap and removed from the glove box. Subsequently, the reaction was carried out in a microwave reactor at 200 °C for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed under vacuum. The resulting crude residue was purified by flash column chromatography (silica gel, diameter 2.5 cm, height 8 cm; eluent: ethyl acetate : hexane = 2:8) to obtain the compound 2 was obtained as a white solid (107 mg, 63% yield).

[0269] 1 H NMR (500 MHz, Chloroform- d ) δ 5.93 (d, J = 11.3 Hz, 1H), 5.47 (dt, J = 9.1, 1.9 Hz, 1H), 5.24 (dd, J = 11.4, 9.5 Hz, 1H), 5.12 (d, J = 10.1 Hz, 1H), 4.23 (dddd, J = 11.1, 7.5, 5.4, 2.3 Hz, 1H), 2.88 (t, J = 10.7 Hz, 1H), 2.81 (t, J = 9.5 Hz, 1H), 2.47 (dp, J = 13.6, 2.0 Hz, 1H), 2.42 - 2.36 (m, 1H), 2.36 - 2.31 (m, 1H), 2.23 (qd, J = 11.3, 3.7 Hz, 1H), 2.12 (ddt, J = 12.3, 5.7, 2.9 Hz, 1H), 2.00 (q, J = 5.0, 3.6 Hz, 1H), 1.96 (dt, J = 14.9, 2.6 Hz, 1H), 1.83 (s, 3H), 1.77 - 1.72 (m, 1H), 1.71 (d, J = 7.4 Hz, 1H), 1.67 (dd, J = 9.7, 6.4 Hz, 2H), 1.59 (ddd, J = 14.0, 7.3, 5.3 Hz, 1H), 1.47 (d, J = 1.3 Hz, 3H), 0.99 (t, J = 7.5 Hz, 3H).

[0270] 13C NMR (126 MHz, CDCl3) δ 210.66, 174.19, 138.05, 135.66, 134.58, 131.38, 124.42, 123.47, 78.48, 77.41, 77.16, 76.90, 55.10, 47.08, 45.41, 43.03, 42.10, 31.23, 31.10, 28.13, 26.15, 24.46, 13.07, 9.76.

[0271] HRMS (ESI): Calculated for C 21 H 28 O3[M+H] + : 329.2112, found: 329.2121.

[0272] TLC (ethyl actate:hexane = 3:7) R f : 0.4 (UV, CAM).

[0273] Specific Rotation [α] D 25 : -47.475 (c 0.4, MeOH).

[0274] manufactured compound 2 The XRD analysis and NOE-based stereochemical results are shown in Fig. 1. The thermal ellipsoids shown in Fig. 1 (thermal ellipsoids indicated with 50% probability) visually represent the relative spatial arrangement of atoms in the compound, while the NOE correlation lines represent the stereochemical arrangement based on the distances between interacting atoms. Through XRD analysis, the compound 2 The structure of has been clearly identified, and the compound 2The crystallographic data were deposited at the Cambridge Crystallographic Data Center (Deposit No.: CCDC 2391862). XRD analysis revealed an (S)-structure at the C5 position and a Z-structure of the C6-C7 olefin, which was found to be opposite to the stereochemical configuration previously observed in collinoketones. Furthermore, Nuclear Overhauser Effect (NOE) analysis of the compound 2 The relative positions of hydrogen atoms on the tricyclic framework were determined. Based on NOE correlation lines, it was confirmed that 5-H, 18-H, 14-H, and 16-H are located at the top of the tricyclic group, and 10-H, 12-H, 17-H, and 19-H are located at the bottom.

[0275] [Experimental Example]

[0276] 1. Experimental procedure for evaluating therapeutic efficacy

[0277] (1) Peptide preparation

[0278] Aβ42 synthesis

[0279] Aβ42 was synthesized through Fmoc solid-phase peptide synthesis (Fmoc-SPPS) and purification according to a previously reported method [Anti-amyloidogenic Approach to Access Amyloid-β (1-42) in Fmoc Solid-Phase Synthesis. Bulletin of the Korean Chemical Society. 2015;36(8):2147-2149.].

[0280] Aβ 42 The Flamma 552 cover

[0281] After synthesizing Aβ42, 2.2 mmol of Flamma 552 carboxylic acid, 2.2 mmol of DIC (diisopopylcarbodiimide), and 1.0 M Oxyma Aβ 42Add to and react at room temperature for 48 hours. Flamma 552-labeled Aβ 42 I got .

[0282] Synthesis of Tau R3

[0283] tau(306-336) R3, Ac-VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ-NH2 was synthesized in-house by Fmoc-SPPS. For C-terminal amidation, 5 eq. of Fmoc-Rink amide linker, 5 eq. of DIC, and 5 eq. of Oxyma were dissolved in DMF (N,N-dimethylformamide) and added to polystyrene AM-NH2 resin swollen with DMF. After activation in a shaker for 2 hours, amino acids were sequentially coupled using an automated peptide synthesizer (Liberty Blue, CEM, USA). 1.0 M Oxyma and 1.0 M DIC were used as amino acid coupling agents. Between each coupling step, the N-terminal Fmoc protecting group was removed by 20% piperidine (1:4, piperidine / DMF, v / v). Finally, N-terminal acetylation was performed by adding a mixture of 40 eq. of acetic anhydride and 4 eq. of DIEA (N,N-diisopropylethylamine) dissolved in an appropriate amount of DMF, and this reaction was carried out in a shaker for 3 hours. After synthesis, the final peptide was cleaved from the resin by 92.5% TFA (92.5:2.5:2.5:2.5, TFA / TIS / deionized water / DODT(3,6-Dioxa-1,8-octanedithiol), v / v / v / v), and this process was carried out in a shaker for 4 hours. TFA was removed through a rotary evaporator, and the peptide was separated and obtained by centrifugation (3000 rpm, 15 min, twice) after cold ether precipitation.

[0284] Peptide Analysis and Purification

[0285] To analyze and purify the synthesized peptides, reverse-phase high-performance liquid chromatography (RP-HPLC) (1220; Agilent) and electrospray ionization mass spectroscopy (ESI-MS) (6120; Agilent) were performed using a binary gradient of solvent A (0.1% TFA in deionized water) and solvent B (0.09% TFA in acetonitrile (ACN)). A Kinetex 5 μm C18 (150 × 4.6 mm) column was used for analysis, and a Phenomenex jupiter 10 μm C18 (250 × 21.2 mm) column was used for purification. UV detection was performed at 230 nm.

[0286] (2) Thioflavin T (ThT) Fluorescence Assay

[0287] Aβ 42 Inhibition and disaggregation

[0288] Aβ 42 3-dehydroxy-5- for aggregations / aggregates epi ThT fluorescence analysis was performed to evaluate the inhibitory / dissociative effects of -collinoketone (Compound 2). Autosynthesized Aβ 42 After dissolving in DMSO (4 mM, 100% DMSO), dilute 100-fold with deionized water to obtain 40 μM Aβ 42 Stock (1% DMSO) was prepared. 3-dehydroxy-5- epi -collinoketone (Compound 2) was dissolved in DMSO (10 mM, 100% DMSO) and then diluted with deionized water to prepare 1 mM (20% DMSO). 3-dehydroxy-5- epiTo evaluate the Aβ aggregation inhibitory ability of -collinoketone (compound 2), 20 μM of Aβ 42 500 μM of 3-dehydroxy-5- epi It was incubated with -collinoketone (Compound 2) at 37 °C for one day in a final DMSO concentration of 10.5% DMSO in deionized water. Aβ 42 In the fibril dissociation analysis, 40 μM Aβ 42 Pre-aggregated Aβ by incubating stock (1% DMSO) at 37°C for one day 42 Fibrils were obtained. Subsequently, 500 μM of 3-dehydroxy-5- epi -collinoketone (compound 2) pre-aggregated Aβ 42 The fibrils were treated and incubated at 37°C for an additional day.

[0289] Tau K18 inhibition and disaggregation

[0290] 3-dehydroxy-5- on Tau K18 aggregations / aggregates epi ThT fluorescence analysis was performed to evaluate the inhibitory / dissociative effects of -collinoketone (Compound 2). Tau K18 was dissolved in PBS (1 mg / mL), and 3-dehydroxy-5- epi -collinoketone (Compound 2) was dissolved in DMSO (10 mM, 100% DMSO). To induce tau agglutination, 2 mg / mL heparin and 10 mM DTT were added to the tau protein to final concentrations of 0.1 mg / mL and 100 μM, respectively. 3-dehydroxy-5- epi To evaluate the Tau K18 aggregation inhibitory ability of -collinoketone (compound 2), 35 μM of Tau K18 and 1000 μM of 3-dehydroxy-5- epiIt was incubated with -collinoketone (Compound 2) at 37 °C for 3 days in a final DMSO concentration of 10% DMSO in PBS. For the Tau K18 aggregate dissociation assay, Tau K18 was incubated at 37 °C for 3 days to obtain pre-aggregated Tau K18. Subsequently, 1000 μM of 3-dehydroxy-5- epi -collinoketone (compound 2) was treated to pre-aggregated Tau K18 and incubated at 37°C for an additional 3 days.

[0291] Tau Repeat domain (R3) inhibition and disaggregation

[0292] 3-dehydroxy-5- on aggregation / aggregation of Tau repeat domain 3 (R3) epi ThT fluorescence analysis was performed to evaluate the inhibitory / dissociative effects of -collinoketone (compound 2). R3 was dissolved in PBS (70 μM), and 3-dehydroxy-5- epi -collinoketone (Compound 2) was dissolved in DMSO (10 mM, 100% DMSO). 3-dehydroxy-5- epi To evaluate the R3 aggregation inhibitory ability of -collinoketone (compound 2), 35 μM of R3 was mixed with 1000 μM of 3-dehydroxy-5- epi It was incubated with -collinoketone (Compound 2) at 37 °C for 3 days in a final DMSO concentration of 10% DMSO in PBS. For the R3 aggregate dissociation assay, pre-aggregated R3 was obtained by incubating R3 at 37 °C for 3 days. Subsequently, 1000 μM of 3-dehydroxy-5- epi -collinoketone (compound 2) was treated to pre-aggregated R3 and incubated at 37°C for an additional 3 days.

[0293] After incubation, 25 μL of each sample was loaded three times onto a half-area 96-well black plate (3694, Corning Inc., USA), and 75 μL of ThT solution (5 μM in 50 mM glycin buffer, pH 8.5) was added to each well. The fluorescence intensity of ThT bound to β-sheets was measured at 450 nm (excitation) and 485 nm (emission) using a microplate reader (Infinite M200 Pro, TECAN, USA).

[0294] (3) Preparation of A3 (amyloid aggregate assay)

[0295] Aβ containing a cysteine ​​residue at the C-terminus 42 The peptide was synthesized using an automated microwave peptide synthesizer (Liberty Blue, CEM) according to a previously reported method [Immobilized amyloid hexamer fragments to map active sites of amyloid-targeting chemicals. ACS Chemical Neuroscience 2023, 14, 1, 9-18]. The synthesized peptide was dissolved in DMSO and then diluted with binding buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, 10 mM EDTA, pH 7.2) to prepare a 50 μg / mL peptide solution. A volume of 100 μL of the peptide solution was dispensed into each well of a maleimide-activated microplate, and then shaken at room temperature for 2 hours to perform immobilization for the preparation of A3 (amyloid aggregate assay). To block unreacted maleimide groups, 200 μL of cysteine ​​solution (10 μg / mL in binding buffer) was added and incubated at room temperature for 1 hour with shaking.

[0296] Subsequently, Flamma552-labeled Aβ 42After dissolving in DMSO, the solution was diluted with deionized water to a final concentration of 10 μM (10% DMSO) and incubated at 37 °C for 8 hours. Fluorescence intensity was measured at 555 / 580 nm (excitation / emission) to establish fluorescence reference values ​​for each well. 3-dehydroxy-5- epi -collinoketone (Compound 2) (500 μM in binding buffer) was dispensed into each well and incubated at room temperature for 24 hours with shaking. After incubation, fluorescence intensity was measured again at 555 / 580 nm (excitation / emission). Through the difference between fluorescence intensity before and after incubation, 3-dehydroxy-5- epi The change in fluorescence intensity due to treatment with -collinoketone (compound 2) was calculated. Between each step, the plate was washed three times with 200 μL of wash buffer (0.1 M sodium phosphate, 0.15 M sodium chloride, and 0.05% Tween-20, pH 7.2).

[0297] (4) Blood-Brain Barrier (BBB) ​​Permeability Test

[0298] The Parallel artificial membrane permeability assay (PAMPA) was performed using the PAMPA-BBB kit (BioAssay Systems, PMBBB-096) according to the manufacturer's instructions. 3-dehydroxy-5- in DMSO epiStock solutions were prepared by dissolving -collinoketone (Compound 2), promazine hydrochloride (high permeability control), and diclofenac (low permeability control), respectively, and then diluted with PBS (pH 7.4) to a final concentration of 500 μM. The polyvinylidene difluoride (PVDF) membranes in each well of the donor plate were coated with 5 μL of BBB lipid solution. Then, 3-dehydroxy-5- epi 200 μL each of 200 μL of 2-collinoketone (Compound 2) and the control were added, and 300 μL of PBS was filled into each well of the acceptor plate. A donor plate was placed on top of the acceptor plate to form a sandwich configuration. A separate tube was prepared for equilibrium standards. The sandwich plate and equilibrium standards were incubated at room temperature for 18 hours without stirring. After transferring 100 μL each of the equilibrium standards, acceptor, and donor solutions to a UV plate, the absorbance spectra of the solutions were measured using a plate reader (SpectraMax M2e) at 10 nm intervals from 200 nm to 500 nm. Based on the measured data, the effective permeability (Pe) of each sample was calculated using the data analysis equation specified in the BioAssay Systems manufacturer's instructions.

[0299] (5) Animal preparation

[0300] 5XFAD Alzheimer's disease model mice (strain name: B6SJL-Tg(APPSwFILon,PSEN1*M146L*L286V)6799Vas / Mmjax) and wild-type mice (C57BL / 6 x SJL) were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). 5XFAD mice express amyloid precursor protein (APP) mutations, Sweden mutation (K670N / M671L), Florida mutation (I716V), London mutation (V717I), and presenilin (PSEN1) mutation (M146L / L286V).

[0301] PS19 mice (strain name: B6;C3-Tg(Prnp-MAPT*P301S)PS19Vle / J) and wild-type mice (C57BL / 6 x C3H) were obtained from Jackson Laboratory (Bar Harbor, Maine, USA). PS19 mice express the MAPT P301S mutation.

[0302] Heterozygous transgenic mice were maintained through crosses with wild-type mice, and the genotypes of all mice were confirmed by tail DNA PCR analysis according to standard PCR conditions at Jackson Laboratory. Seven-week-old male ICR mice were obtained from Orient Bio Inc. (South Korea).

[0303] All mice were housed in the laboratory and allowed free access to water and feed under controlled temperature, humidity, and a 12-hour light-dark cycle. All animal experiments were performed in accordance with the National Institutes of Health (NIH) guidelines on the care and use of laboratory animals, and the experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University Center for Laboratory Animals (Republic of Korea, IACUC-A-202404-1837-01).

[0304] (6) Drug administration

[0305] 5XFAD mouse model

[0306] In vivo efficacy was evaluated using 6.4-month-old female 5XFAD mice and littermate wildtype mice. 3-dehydroxy-5- epi collinoketone (compound 2, 30 mg / kg / day) and vehicle (10% DMSO, 10% Tween80 in PBS) were administered orally (oral gavage, PO) daily for 5 days, according to mouse body weight. One day after the completion of drug administration, all mice (approximately 6.5 months old) were anesthetized and sacrificed.

[0307] PS19 mouse model

[0308] In vivo efficacy was evaluated using 7.5-month-old female PS19 mice and littermate wildtype mice. 3-dehydroxy-5- epicollinoketone (compound 2, 30 mg / kg / day) and vehicle (10% DMSO, 10% Tween80 in PBS) were administered orally (oral gavage, PO) twice a week for 3 weeks, according to mouse body weight. One day after the completion of drug administration, all mice (approximately 8.5 months old) were anesthetized and sacrificed.

[0309] (7) Thioflavin S (ThS) staining

[0310] After perfusing mice with 0.9% physiological saline, the mice's brains were excised, and each hemisphere was fixed in 4% paraformaldehyde (PFA) (pH 7.4) at 4°C for 24 hours. The fixed brain tissue was immersed in a 30% sucrose solution for 2 days, and then sectioned to a thickness of 25 μm using a cryostat (CM1860, Leica, Germany). The brain sections were attached to glass slides, and antigen retrieval was performed by immersing the slides in 1% sodium dodecyl sulfate (SDS). Non-specific binding was blocked by incubating in PBS-T (Phsophate Buffered Saline with Tween 20) containing 5% horse serum at room temperature for 1 hour. To detect Aβ plaques, brain sections were stained with 1.5 mM thioflavin S (ThS, in 50% ethanol) for 7 minutes and de-stained with 80% ethanol and PBS for 1 minute each. The stained brain tissue samples were imaged using a fluorescence microscope (DM-2500, Leica), and the number of Aβ plaques in the hippocampus was analyzed using Image J software (NIH).

[0311] (8) Construction of a Tau repeat domain 3 (R3)-injected acute AD mouse model

[0312] Prior to the behavioral study, purified R3 was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), sonicated for 15 minutes to dissolve aggregates, and the peptides were homogenized by completely removing HFIP through nitrogen gas evaporation. To prepare pre-aggregated oligomer-rich R3 aggregates, HFIP-treated R3 was incubated at 37 °C for 12 hours, and then 3-dehydroxy-5- epi Samples were further incubated at 37 °C for 6 hours with or without treatment with -collinoketone (Compound 2). Three samples were prepared as follows: vehicle (10% DMSO in PBS), 3-dehydroxy-5- epi R3 aggregates untreated with -collinoketone (compound 2) (10% DMSO in PBS), and 3-dehydroxy-5- epi R3 aggregates treated with β-collinoketone (Compound 2) (10% DMSO in PBS). 5 μL of each cultured R3 sample was injected into the intraventricular (ICV) region of ICR mice (male, 7 weeks old, n=9 / group). The procedure was performed in accordance with the reference [Intracerebroventricular injection of amyloid-βpeptides in normal mice to acutely induce Alzheimer-like cognitive deficits. Journal of visualized experiments: JoVE, (109), p.53308.].

[0313] (9) Immunoblotting

[0314] Brain Lysis

[0315] Cortical and hippocampal regions were collected from the brains of wild-type, 5XFAD, and PS19 mice used in the experiment. Frozen brain tissues were homogenized in RIPA buffer (Radioimmunoprecipitation assay buffer) supplemented with a protease and phosphatase inhibitor cocktail, and then incubated on ice for 30 minutes. The lysate was centrifuged at 14,000 rpm at 4°C for 30 minutes. The supernatant was collected, and the concentration of the lysate was quantified using a BCA (bicinchoninic acid) assay kit.

[0316] Dot Blot Analysis

[0317] 5 μg of brain lysate was spotted onto a nitrocellulose (NC) membrane and completely dried. The membrane was blocked with TBS-T (Tris Buffered Saline with 0.1% Tween 20) containing 5% skim milk, and then treated with primary antibodies overnight at 4°C. To visualize total Aβ and oligomeric Aβ, anti-Aβ 6E10 antibody (1:1000) and anti-oligomer A11 antibody (1:1000) were used as primary antibodies. To visualize oligomeric Tau, anti-tau oligomer T22 antibody (1:1000) was used as primary antibodies. After overnight incubation, the membrane was incubated with HRP-conjugated IgG antibody (horseradish peroxidase conjugated IgG antibody) for 1 hour and developed using ECL (enhanced chemiluminescence) solution (Thermo Fisher Scientific). All washing steps were performed three times for 5 minutes each using TBS-T.

[0318] Western Blot Analysis

[0319] 20 μg of brain lysate was loaded onto an SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) gel, and proteins separated by electrophoresis were transferred to a nitrocellulose (NC) membrane. The blotted membrane was blocked with TBS-T containing 5% skim milk and treated with the primary antibody overnight at 4°C. Subsequently, proteins were detected using an HRP-conjugated secondary antibody and developed using ECL solution. Protein expression levels in each mouse group were quantified using Image J, normalized against β-actin, and plotted using Prism 9 software.

[0320] (10) Y-maze test

[0321] The Y-maze test was performed to evaluate changes in cognitive function through short-term spatial working memory (based on voluntary alternation) and exploratory activity (based on the total number of arm entries). The Y-maze consists of a Y-shaped maze (length 40 cm, width 10 cm, height 12 cm) with three symmetrical arms forming a 120° angle with each other. Each mouse was placed on one arm and allowed to move freely through the maze for a total of 5 minutes. The number of entries for each arm and the number of consecutive entries into three different arms—i.e., the number of triads—were recorded, and based on this, the alternation percentage was calculated as follows: Alternation percentage (%) = Number of triads / (Total number of arm entries - 2) × 100. An entry was considered to have occurred when all four paws passed through the central circular area and entered the corresponding arm.

[0322] 2. Results

[0323] Amyloid beta plaques, which cause problems in the brain, are formed when Aβ monomers bind to each other. First, the monomers repeatedly combine to form oligomers, and as these oligomers further aggregate, they gradually form more complex structures. If this process continues, amyloid beta fibrils are eventually formed, and as these fibrils accumulate, they develop into Aβ plaques.

[0324] Aβ 42 Inhibitory and dissociative effects of 3-dehydroxy-5-epi-collinoketone on aggregation (in vitro)

[0325] 3-dehydroxy-5- epi To evaluate the potential of 3-dehydroxy-5-epi-collinoketone (2) for treating Alzheimer's disease (AD), a series of in vitro analyses were performed. The focus was on investigating the ability to inhibit aggregation from a preventive perspective and the ability to dissociate already formed aggregates from a therapeutic perspective. Considering the process in which Aβ monomers aggregate to form oligomers and gradually develop into fibrils and plaques, Thioflavin-T (ThT) fluorescence assay and A3 aggregate assay were used to confirm the action of 3-dehydroxy-5-epi-collinoketone (2).

[0326] ThT is a fluorescent chemical that inserts into the β-sheet structure of fibrils to quantify fibril formation. An increase in the red-shifted intensity of ThT fluorescence indicates increased fibril production.

[0327] First, to evaluate the inhibitory effect on Aβ fibril formation, 3-dehydroxy-5-epi-collinoketone (2) was added to one experimental group and nothing was added to the control group while culturing the synthesized Aβ for 24 hours. Afterward, the ThT fluorescence intensity was measured and showed a tendency to decrease by 29.21% compared to the control group (Fig. 2a), which suggests that the compound has an effect of inhibiting Aβ fibril formation.

[0328] In addition, to evaluate the ability to dissociate Aβ fibrils, Aβ was cultured for 24 hours to form sufficiently aggregated Aβ fibrils, and then 3-dehydroxy-5-epi-collinoketone (2) was treated in the same manner. ThT fluorescence analysis showed a tendency to decrease by 22.10% compared to the control group (Fig. 2b), which means that the compound has the ability to dissociate already aggregated Aβ fibrils.

[0329] Subsequently, the effect of dissociating Aβ oligomers was further confirmed through an A3 aggregate assay. To this end, Aβ was immobilized on the bottom of a microplate, and then fluorescently labeled Aβ was added to induce the formation of oligomers (Fig. 2c). Afterward, fluorescence intensity was analyzed after treatment with 3-dehydroxy-5-epi-collinoketone (2), and a significant decrease of 51.50% was observed compared to the control group (Fig. 2d). This suggests that the compound also has a strong effect on dissociating Aβ oligomers.

[0330] In summary, the above results suggest that 3-dehydroxy-5-epi-collinoketone (2) not only inhibits Aβ aggregation but also has the ability to effectively dissociate already formed Aβ fibrils and oligomers, and may inhibit pathological mechanisms associated with Aβ accumulation.

[0331] Aβ 42 Inhibitory and dissociative effects of 3-dehydroxy-5-epi-collinoketone on aggregation (in vivo)

[0332] To evaluate the in vivo therapeutic efficacy of 3-dehydroxy-5-epi-collinoketone (2) against Aβ, experiments were performed using 6.4-month-old female 5XFAD transgenic mice with APP and PSEN1 mutations.

[0333] First, blood-brain barrier (BBB) ​​permeability tests were performed to evaluate whether the drug could be effectively delivered into the brain. PAMPA-BBB analysis confirmed that 3-dehydroxy-5-epi-collinoketone (2) crosses the BBB at a level similar to promazine hydrochloride, which is known to have high brain permeability (Fig. 3a).

[0334] Subsequently, in vivo experiments were conducted on 5XFAD mice via oral administration. The experimental group was orally administered 3-dehydroxy-5-epi-collinoketone (2) (30 mg / kg / day) for 5 days, while the control group was orally administered vehicle (10% DMSO, 10% Tween80 in PBS) for the same period. Additionally, age-matched wild-type mice were included as a control group. After administration was completed, all mice were sacrificed, brains were extracted, and the degree of Aβ accumulation was evaluated through chemofluorescence staining and protein immunoblot (Western blot) analysis (Fig. 3b).

[0335] Thioflavin S (ThS) staining was performed to visualize Aβ plaques inside the mouse brain as green fluorescence (Fig. 3c). Quantitative analysis of Aβ plaques showed that in the brains of 5XFAD mice administered 3-dehydroxy-5-epi-collinoketone (2), compared to the control group, a decrease of 25.29% (P = 0.0957) was observed in the cortex region and a decrease of 18.86% (P = 0.0273) was observed in the hippocampus region (Figs. 3d, 3f). In particular, Aβ plaques were found to be significantly reduced (18.86%, P < 0.05) in the hippocampus region (Fig. 3f).

[0336] In addition, to quantify the concentrations of total Aβ and oligomeric Aβ in RIPA soluble brain lysates, dot blot analysis was performed using antibodies 6E10 (anti-Aβ, detects all forms of Aβ) and A11 (anti-oligomeric Aβ, detects oligomeric Aβ). As a result, in brain lysates of 5XFAD mice administered 3-dehydroxy-5-epi-collinoketone (2), the total soluble Aβ concentration in both the cortex and hippocampus was significantly reduced compared to the control group (cortex: 27.43%, P < 0.05; hippocampus: 18.70%, P < 0.05) (Fig. 3e, 3g). Although the Aβ oligomeric concentration showed a decreasing trend in both the cortex and hippocampus, this decrease did not reach a statistically significant level.

[0337] Based on the above results, it was confirmed that 3-dehydroxy-5-epi-collinoketone (2) has high BBB permeability and exhibits pharmacological effects that inhibit Aβ accumulation, suggesting that it has potential as a treatment for neurodegenerative diseases.

[0338] Inhibitory and dissociative effects of 3-dehydroxy-5-epi-collinoketone (2) on tau and tau repeat domain 3 (in vitro)

[0339] 3-dehydroxy-5- epi -collinoketone ( 2To evaluate the potential for dual-targeting therapy targeting both Aβ and tau simultaneously, Thioflavin-T (ThT) analysis was performed to evaluate the inhibitory and dissociative effects of tau aggregation. Tau protein forms a β-sheet structure during the process of aggregating into paired helical filaments (PHF), and this aggregation is known to be a major pathological feature of neurodegenerative diseases. Accordingly, the inhibitory and dissociative effects of 3-dehydroxy-5-epi-collinoketone (2) were confirmed using a tau K18 fragment composed of a microtubule-binding domain (MTBD) (Fig. 4a).

[0340] First, tau K18 (35 μM) was cultured with 3-dehydroxy-5-epi-collinoketone (1000 μM) at 37 °C for 3 days, and then ThT fluorescence intensity was measured. As a result, it was confirmed that tau K18 fibril formation was significantly inhibited by 68.87% (P < 0.0001) in the group treated with 3-dehydroxy-5-epi-collinoketone (2) (Fig. 4b). In addition, to evaluate the tau aggregation dissociation ability of 3-dehydroxy-5-epi-collinoketone (2), pre-aggregated tau K18 fibrils were obtained and cultured for an additional 3 days under the same conditions. ThT fluorescence analysis showed that treatment with 3-dehydroxy-5-epi-collinoketone (2) significantly reduced tau K18 fibrils by 50.85% (P < 0.0001) compared to the untreated control group during the same total culture period (Fig. 4c), and reduced them by 41.36% compared to pre-aggregated fibrils before treatment. This suggests that 3-dehydroxy-5-epi-collinoketone (2) not only effectively inhibits tau K18 aggregation but also has the ability to dissociate already aggregated tau K18 fibrils.

[0341] As shown in Fig. 4a, the MTBD of tau protein consists of four repeat domains: R1, R2, R3, and R4, and R3 is particularly reported to be associated with neurotoxicity. Accordingly, the inhibitory and dissociative effects of 3-dehydroxy-5-epi-collinoketone (2) on R3 aggregation were analyzed. When R3 (35 μM) was cultured with 3-dehydroxy-5-epi-collinoketone (1000 μM) at 37 °C for 3 days, the ThT fluorescence intensity was measured, and it was confirmed that R3 fibril formation was significantly inhibited by 76.78% (P < 0.0001) (Fig. 4d). In addition, after obtaining pre-aggregated R3 fibrils, 3-dehydroxy-5-epi-collinoketone (2) was treated and cultured for 3 days, and as a result, ThT fluorescence intensity was significantly reduced by 61.64% (P < 0.0001) compared to the untreated control (Fig. 4e). This means that 3-dehydroxy-5-epi-collinoketone (2) effectively inhibits tau R3 aggregation and has the ability to dissociate already formed tau aggregates.

[0342] Based on the above results, it was confirmed that 3-dehydroxy-5-epi-collinoketone (2) not only effectively inhibits the aggregation of tau proteins but also has the ability to dissociate already formed tau aggregates. In particular, it exhibits strong aggregation inhibition and dissociation effects even on the highly neurotoxic tau R3 domain, suggesting potential as a dual-action therapeutic candidate that targets both Aβ and tau simultaneously.

[0343] Inhibitory and dissociative effects of 3-dehydroxy-5-epi-collinoketone (2) on tau and tau repeat domain 3 (in vivo)

[0344] To determine whether the therapeutic effect of R3 could lead to cognitive improvement, a Y-maze test was performed using an R3-injected mouse model with Alzheimer's-like cognitive impairment to investigate 3-dehydroxy-5- on spatial task and hippocampal memory abilities. epi The efficacy of -collinoketone (2) was evaluated.

[0345] Considering that the oligomer form is more toxic than the fibril form and is more closely associated with cognitive impairment, the pre-aggregation period to generate an abundance of the oligomer form was shortened from 3 days to 12 hours. Subsequently, the therapeutic activity was evaluated by dividing the samples into a 3-dehydroxy-5-epi-collinoketone (2) treatment group and a non-treatment group and culturing them for an additional 6 hours. After 18 hours of culturing, the presence of aggregates was confirmed by ThT fluorescence analysis, and it was found that the ThT fluorescence intensity decreased by 83.75% (P < 0.0001) in the samples co-cultured with 3-dehydroxy-5-epi-collinoketone (2) (Fig. 5d). In addition, the oligomer concentration was evaluated by dot blot analysis using the T22 antibody (anti-tau oligomer), and it was confirmed that the oligomer concentration decreased by 32.61% in the 3-dehydroxy-5-epi-collinoketone (2) treatment group (Fig. 5e).

[0346] Subsequently, 5 μL of each cultured R3 sample was injected into the intraventricular (ICV) region of ICR mice (male, 7 weeks old, n=9 / group) to conduct a Y-maze experiment (Fig. 5a). The R3 injection group showed a significantly reduced number of spontaneous changes (change value: 49.92%) compared to the vehicle group (change value: 68.60%), which was a result reflecting cognitive decline. On the other hand, the R3 injection group treated with 3-dehydroxy-5-epi-collinoketone (2) showed a tendency for cognitive function to recover to a change level (change value: 65.07%) close to that of the vehicle group (Fig. 5b). The total number of arm entries in all experimental groups was similar, confirming that there was no difference in exploratory activity (Fig. 5c).

[0347] Since tau K18 and R3 are core fragments of the tau protein, experiments were conducted using a PS19 transgenic mouse model with a MAPT P301S mutation to confirm the therapeutic effect of 3-dehydroxy-5-epi-collinoketone (2) on the tau protein in vivo. 3-dehydroxy-5-epi-collinoketone (2) was orally administered to 6.5-month-old female PS19 mice at a dose of 30 mg / kg / day for 3 weeks, and wild-type (WT) and PS19 mice administered with vehicle (10% DMSO, 10% Tween 80 in PBS) were used as controls (Fig. 5f). After administration, all mice were sacrificed and protein immunoblot analysis was performed.

[0348] Increased tauopathy is closely associated with synaptic dysfunction, and the postsynaptic marker postsynaptic density protein 95 (PSD95) and the presynaptic marker synaptophysin are known to be downregulated in the presence of aggregated tau protein. Accordingly, to determine whether administration of 3-dehydroxy-5-epi-collinoketone (2) could alleviate synaptic loss in the hippocampus, the expression levels of PSD95 and synaptophysin were analyzed. As a result, in the 3-dehydroxy-5-epi-collinoketone (2) administration group, PSD95 (7.60%) in the hippocampus region was lower compared to the vehicle-treated PS19 control group. P = 0.0954) and synaptophysin (10.41%, P The expression levels of < 0.05 were all found to increase (Fig. 5g), suggesting the possibility of improving cognitive function in neurodegenerative diseases through synaptic protective effects.

[0349] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A 3-dehydroxycollinoketone compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] Claim 2 The 3-dehydroxycolinoketone compound of claim 1, wherein the 3-dehydroxycolinoketone compound is represented by the following Chemical Formula 2, a 3-dehydroxycolinoketone compound or a pharmaceutically acceptable salt thereof.[Chemical Formula 2] Claim 3 A method for preparing a 3-dehydroxycholinoketone compound represented by Chemical Formula 1 using Module 1 represented by Chemical Formula 11, Module 2 represented by Chemical Formula 12, and Module 3 represented by Chemical Formula 13. [Chemical Formula 1] [Chemical Formula 11] [Chemical Formula 12] [Chemical Formula 13] In the above formula, R1 to R6 are each independently C1-C6 alkyl; and TBS is tert -butyldimethylsilyl( tert -butyldimethylsilyl) Claim 4 A method for preparing a 3-dehydroxycolinoketone compound according to claim 3, comprising: 1) reacting the module 1 of Formula 11 and the module 2 of Formula 12 to prepare a compound of Formula 21; 2) reacting the compound of Formula 21 with the module 3 of Formula 13 to prepare a compound of Formula 22; 3) deprotecting the compound of Formula 22 to prepare a compound of Formula 23; 4) hydrolyzing the compound of Formula 23 to prepare a compound of Formula 24; 5) preparing a compound of Formula 25 from the compound of Formula 24; and 6) performing a transannular [6+4] cycloaddition reaction on the compound of Formula 25 to prepare a 3-dehydroxycolinoketone compound of Formula 2; [Formula 2] [Chemical Formula 21] [Chemical Formula 22] [Chemical Formula 23] [Chemical Formula 24] [Chemical Formula 25] Claim 5 A method for preparing a 3-dehydroxycholinoketone compound, wherein step 1) above is a Horner-Wadsworth-Emmons (HWE) olefinization reaction in the presence of a base. Claim 6 A method for preparing a 3-dehydroxycholinoketone compound according to claim 5, wherein the base is selected from the group consisting of barium hydroxide (Ba(OH)2), barium hydroxide octahydrate (Ba(OH)2·8H2O), sodium hydride (NaH), and potassium t-butoxide (KOtBu). Claim 7 A method for preparing a 3-dehydroxycholinoketone compound according to claim 4, wherein step 2) is performed in the presence of a palladium catalyst, a base, and a ligand. Claim 8 In claim 7, the palladium catalyst is one or more selected from the group consisting of Pd(PPh3)4, Pd(OAc)2, PdCl2(dppf)CH2Cl2, Pd(PPh3)2Cl2, Pd2(dba)3, PdCl2(CH3CN)2, Pd[P(t-Bu)3]2, PdCl2((P(o-tolyl)3)2, PdCl2(dppf), PdCl2(dppe), PdCl2(dppp), and PdCl2(TPP); and the base is silver oxide (Ag2O), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), potassium carbonate (K2CO3), cesium carbonate (Cs2CO3), sodium carbonate (Na2CO3), potassium fluoride (KF), cesium fluoride (CsF), sodium hydroxide (NaOH), One or more selected from the group consisting of potassium hydroxide (KOH), sodium methoxide (CH3ONa), sodium t-butoxide (tBuONa), cesium acetate (CsOAc), potassium acetate (KOAc), triethylamine, and diisopropylethylamine; and the ligand is one or more selected from triphenyl arsine, triphenylphosphine (PPh3), tri(o-tolyl)phosphine, tri(t-butyl)phosphine, tri(cyclohexyl)phosphine, 1,1'-bis(diphenylphosphino)ferrocene (DPPF), 1,2-bis(diphenylphosphino)ethane (DPPE), 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (rac-BINAP), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthen (XantPhos), A method for preparing a 3-dehydroxycholinoketone compound, one or more selected from the group consisting of 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (SPhos) and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos). Claim 9 A method for preparing a 3-dehydroxycholinoketone compound, wherein, in claim 4, step 3) is performed in the presence of camphorsulfonic acid (CSA). Claim 10 A method for preparing a 3-dehydroxycholinoketone compound, wherein, in paragraph 4, step 4) is performed under acidic conditions. Claim 11 A method for preparing a 3-dehydroxycholinoketone compound, wherein step 5) above is to perform a Yamaguchi esterification reaction in the presence of a base, 2,4,6-trichlorobenzoyl chloride, and 4-(dimethylamino)pyridine (4-(dimethylamino)pyridine, DMAP). Claim 12 A method for preparing a 3-dehydroxycholinoketone compound according to claim 11, wherein the base is one or more selected from the group consisting of triethylamine, pyridine, N-methylimidazole, potassium carbonate (K2CO3), cesium carbonate (Cs2CO3), sodium carbonate (Na2CO3), DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) and sodium hydride (NaH). Claim 13 A method for preparing a 3-dehydroxycolinoketone compound according to claim 4, wherein the module 1 of Formula 11 is prepared by comprising: a) a step of preparing a compound of Formula 11-b by reacting a compound of Formula 11-a with a hydrozirconation and iodination reaction; and b) a step of preparing Module 1 of Formula 11 by reacting the compound of Formula 11-b with a phosphonate compound of Formula 11-c under a C1-C10 alkyllithium. [Formula 11] [Chemical Formula 11-a] [Chemical Formula 11-b] [Chemical Formula 11-c] In the above equation, R1, R2, and R a Each is independently a C1-C6 alkyl. Claim 14 In claim 4, module 2 of the above chemical formula 12 comprises: c) reacting the compound of the following chemical formula 12-a with isobutenylmagnesium bromide, and then tert -Butyldimethylsilyl chloride ( tert A method for preparing a 3-dehydroxycolinoketone compound, comprising the steps of: reacting with -butyldimethylsilyl chloride (TBSCl) to prepare a compound of Formula 12-b; and d) selectively allylic oxidation of the compound of Formula 12-b in the presence of SeO2 to prepare Module 2 of Formula 12. [Formula 12] [Chemical Formula 12-a] [Chemical Formula 12-b] In the above equation, TBS is tert -butyldimethylsilyl( tert -butyldimethylsilyl) Claim 15 In claim 4, module 3 of the above formula 13 comprises: e) a step of preparing a compound of formula 13-b by zirconium-catalyzed carboalumination and iodination reaction of the compound of formula 13-a; f) a step of preparing a compound of formula 13-c by oxidizing the compound of formula 13-b in the presence of manganese oxide; g) a step of preparing a compound of formula 13-e by Wittig reaction of the compound of formula 13-c with the compound of formula 13-d; and h) a step of reacting a compound of Formula 13-e with a triC1-C10 alkylborate in the presence of C1-C10 alkyllithium, and then reacting it with a diol compound of Formula 13-f to produce Module 3 of Formula 13; a method for preparing a 3-dehydroxycolinoketone compound, comprising the steps of: [Formula 13] [Chemical Formula 13-a] [Chemical Formula 13-b] [Chemical Formula 13-c] [Chemical Formula 13-d] [Chemical Formula 13-e] [Chemical Formula 13-f] In the above formula, R3 to R6 are each independently C1-C6 alkyl. Claim 16 A pharmaceutical composition for the treatment and prevention of neurodegenerative diseases containing, as an active ingredient, a 3-dehydroxycholinoketone compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof. Claim 17 In paragraph 16, the above pharmaceutical composition further comprises an excipient and a carrier. Claim 18 In paragraph 16, the above-mentioned neurodegenerative brain diseases are Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, Amyotrophic Lateral Sclerosis (ALS), mild cognitive impairment, amyloidosis, multiple system atrophy, multiple sclerosis, tauopathies, Pick's disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, and Lewy body dementia. A pharmaceutical composition selected from the group consisting of dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia. Claim 19 A method for the prevention or treatment of a neurodegenerative disease comprising the step of administering a 3-dehydroxycholinoketone compound or a pharmaceutically acceptable salt according to claim 1 or 2, or a pharmaceutical composition according to claim 16, to an individual who has developed or is at risk of developing a neurodegenerative disease. Claim 20 A health functional food composition for the prevention or improvement of neurodegenerative diseases containing a 3-dehydroxycholinoketone compound according to claim 1 or 2 or a food-grade acceptable salt thereof as an active ingredient.

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