WO2023085645A1 - Novel ramalin derivative and use thereof for prevention or treatment of neurodegenerative diseases - Google Patents

Abstract

The present invention relates to a ramalin derivative and use thereof for the prevention or treatment of neurodegenerative diseases and, more specifically, to use of a ramalin derivative for the prevention or treatment of neurodegenerative diseases, in which a derivative obtained by substituting a hydroxyl group of a phenyl ring of ramalin with a methyl group or a fluorine atom exhibits inhibitory activity against BACE-1 and antioxidant and anti-inflammatory activities without toxicity.

Description

Novel Ramalin derivatives and their use for prevention or treatment of degenerative brain diseases

The present invention relates to a ramalin derivative and its use for the prevention or treatment of degenerative brain diseases, and more particularly, a derivative obtained by replacing a hydroxyl group of a phenyl ring of ramalin with a methyl group or a fluorine atom is effective against BACE-1. It relates to a novel ramalin derivative used for preventing or treating degenerative brain diseases by exhibiting inhibitory activity, antioxidant and anti-inflammatory activity without toxicity, and a use thereof for preventing or treating degenerative brain diseases.

In 2016, the number of dementia patients worldwide was 23.8 million, which is increasing every year due to population aging and population growth. Thus, care and support for people with dementia have far-reaching implications for families, the health system, and society as a whole (Nichols, E. et al., Lancet Neurol., 2019, 18, 88-106; Etters, L. et al., J. Am. Assoc. Nurse Parct., 2008, 20, 423-428). Dementia is known to be associated with reduced life expectancy, making it the 5th leading cause of death in developed countries (Dumurgier, J. et al., Lancet Healthy Longev., 2021, 2, e449-e450). In addition, Alzheimer's Disease (hereinafter referred to as AD), which accounts for more than half of these deaths, is the most common cause of dementia. Patients diagnosed with Alzheimer's disease experience progressively worse symptoms, including memory impairment, speech impairment, visuospatial disturbances, and mental disorders including delusions and hallucinations. In addition, the pathogenesis of Alzheimer's disease is diverse and complex. Low concentration of acetylcholine (ACh) in the synaptic cleft, aggregation and accumulation of β-amyloid (hereinafter referred to as Aβ) peptide, entanglement of the microtubule-associated protein tau, and oxidative stress are considered to be the main causes of Alzheimer's disease. It was also observed that Aβ aggregation and accumulation in the brain could be the result of increased Aβ production, decreased Aβ protease activity, or altered Aβ transport across the BBB (blood-brain barrier) (Mucke, L. Alzheimer's disease. Nature , 2019, 461, 895-897). In addition, β-site APP cleavage enzyme 1 (BACE-1) associated with increased production of Aβ is considered a target for the prevention and treatment of Alzheimer's disease (Vassar, R. Bace 1. J. Mol. Neurosci., 2004, 23, 105- 113). Aβ is also produced through sequential cleavage of amyloid precursor protein (APP) by BACE-1 and BACE-2, and BACE-1 is a key enzyme initiating this process (Roberson, E. D. et al., Science, 2006, 314, 781-784). Experimental evidence based on studies in human APP transgenic mice suggests that memory deficits can be prevented by deletion of the BACE-1 gene (Oakley, H. et al., J. Neurosci., 2006, 26; 10129-10140). Reportedly, Aβ aggregates can act as pathogenic mediators and induce oxidative stress, neuroinflammation, and BACE-1 expression. This further aggravates AD (Tamagno, E. et al., Free Radic. Biol. Med., 2006, 41, 202-212). In addition to Aβ accumulation through BACE-1, oxidative stress and inflammation can enhance Aβ production. In a study of the association between Aβ formation and inflammation, plaques were observed in the brains of monkeys with chronic inflammation (Philippens, I. H. et al., J. Alzheimer's Dis., 2017, 55, 101-113). Therefore, given that inflammation increases the production of reactive oxygen species (ROS) that cause oxidative damage to lipids, proteins, and nucleic acids, reducing inflammation based on the use of anti-inflammatory drugs can significantly lower the prevalence of Alzheimer's disease ( McGeer, E. G. et al., Exp. Gerontol., 1998, 33, 371-378). In addition, NF-κB associated with inflammation is involved in the expression of BACE-1 regulated by various mechanisms (Roßner, S. et al., Prog. Neurobiol., 2006, 79, 95-111). Thus, BACE-1, inflammation and ROS are all associated with Aβ formation from the onset of Alzheimer's disease to the post-onset period.

From another point of view, oxidative stress by ROS appears like the development of Alzheimer's disease, and it has been reported to be the cause of early onset and exacerbation of Alzheimer's disease (Smith, M. A. et al., Biochim. Biophys. Acta Mol. Basis Dis. , 2000, 1502, 139-144). Mitochondrial ROS produced under normal conditions are balanced by antioxidants and related enzymes. However, dysfunction due to oxidative damage in this regard results in high levels of ROS production and release of apoptosis-inducing proteins (Dumont, M. et al., Free Radic Biol Med., 2011, 51, 1014-1026; Wang , X. et al., Biochim. Biophys. Acta Mol. Basis Dis., 2014, 1842, 1240-1247; Takuma, K. et al., J. Pharmacol. Sci., 2005, 97, 312-316). In addition, mitochondrial endoplasmic reticulum dysfunction due to oxidative stress is UPR (Unfolded protein response) (Smith, M. A. et al., Biochim. Biophys. Acta Mol. Basis Dis., 2000, 1502, 139-144; Oakley, H. et al. , J. Neurosci., 2006, 26, 10129-10140; Konno, T. et al., Cells, 2021, 10, 233) can protect cells from accumulation of toxic proteins by inducing a cellular stress response, but , can lead to neurodegeneration (Wang, X. et al., Biochim. Biophys. Acta Mol. Basis Dis., 2014, 1842, 1240-1247; Hoozemans, J. J. M. et al., Acta Neuropathol., 2005, 110 , 165-172).

Alzheimer's disease exhibits neuropathological features of extracellular beta-amyloid (β-amyloid, Aβ) plaques and intracellular nerve fiber tangles. Internal proteolytic cleavage of amyloid precursor protein (APP), which produces beta-amyloid, involves the sequential action of two proteases, β-secretase and γ-secretase. β-secretase mediates the first step in beta-amyloid production by β-cleavage of APP, which releases a large soluble extracellular fragment (APPβ). It is known that the C-terminal fragment (CTF) of APP (APP-CTF, C99) is cleaved by gamma secretase at several positions to generate the pathogenic species Aβ42 and Aβ40.

In addition, these beta-amyloids are produced by continuous cleavage of APP mediated by BACE1 (beta-site APP-cleaving enzyme 1, beta-secretase-1) and presenilin/γ-secretase. The production and aggregation of beta-amyloid play an important role in triggering complex pathological events that cause neurodegeneration, entanglement of nerve fibers, inflammation, and loss of neurons.

In addition, it has been reported that BACE1 knockout mice show complete absence of beta amyloid production without obvious side effects (Luo Y et al., Neurobiol Dis. Vol. 14(1), pp. 81-88, 2003), and adult Alzheimer's disease. It has been reported that when BACE1 is deleted in a mouse model, amyloid deposition is eliminated and synaptic function is restored (Xiangyou Hu et al., J. Exp. Med. Vol. 215, No. 3, pp. 927-940, 2018), inhibition of BACE1 has become a target for the development of new therapeutic agents for the treatment of Alzheimer's disease.

Meanwhile, Alzheimer's disease has a complex pathophysiology including pathological protein aggregation, neurotransmission disorders, increased oxidative stress, and microglia-mediated neuroinflammation. Therapeutics targeting only one of the molecular targets associated with AD have been reported. For example, therapeutics with high BACE-1 inhibitory activity can successfully inhibit and eliminate Aβ production, but cannot restore cognitive function. Therefore, when developing a treatment for Alzheimer's disease, it is important to focus on multi-target drugs rather than single-target drugs. Ramalin, which has antioxidant, BACE-1 inhibitory and anti-inflammatory activities, can serve as a promising therapeutic agent with multiple activities that can address the complex nature of Alzheimer's disease. However, its application is limited due to low stability in an aqueous environment and cytotoxicity at high concentrations.

Because of its various disorders, no cure has been established so far for AD. In addition, the pathogenesis of the disease is still unclear and not fully understood, and there are no approved drugs available to treat or halt its progression. Therefore, to solve this problem, it is necessary to develop a drug that exhibits various physiological activities such as antioxidant, anti-inflammatory, and BACE-1 inhibitory activity that can be used as an AD treatment rather than targeting a specific mechanism of action.

On the other hand, ramalin isolated from Ramalina terebrata, an Antarctic lichen, exerts strong antioxidant and antibacterial effects (Paudel, B. et al., Z. Naturforsch. C., 2010, 65, 34-38; Paudel, B. et al., Phytomedicine, 2011, 18, 1285-1290). In previous studies, various physiological activities of Ramalin were investigated until it was developed as an oxidative disease treatment, anti-aging functional food, and whitening and wrinkle-improving functional cosmetics (Yim, J. H. et al., WO2010053327A2, May 14, 2010). In addition, anti-inflammatory (Park, B. et al., Biosci. Biotechnol. Biochem., 2015, 79, 539-552) and reduction of lipid accumulation (Kim, B. et al., Yonsei Med. J. , 2018, 59, 85-91) and anti-hepatic fibrosis (Kim, M. K. et al., Bioche. Biophys. Res. Commun., 2018, 504, 25-33) activity. Antioxidant and anti-inflammatory effects as well as BACE-1 inhibitory effects were confirmed, suggesting potential as a treatment for Alzheimer's disease.

Accordingly, the present inventors have made diligent efforts to solve the above problems, and as a result, a compound of molecular formula C11H16N3O4 isolated from the Antarctic lichen Ramalina terebrata (ramalin, γ-glutamyl-N'-(2-hydroxyphenyl)hydrazide) ), a derivative obtained by binding a substituent such as a methyl group or a fluorine atom to the phenyl ring of ) exhibits inhibitory activity against BACE-1, which causes Aβ accumulation based on structural transformation, and removes active oxygen, which is important for the prevention and treatment of Alzheimer's disease. It was confirmed that it can act as a therapeutic agent for Alzheimer's disease because it shows activity against various targets related to Alzheimer's disease without toxicity, such as showing antioxidant activity that plays a role and inhibitory effect of inflammation that exacerbates Alzheimer's disease, and the present invention was completed.

Summary of Invention

An object of the present invention is to provide a novel derivative obtained by replacing a functional group (phenolic hydroxyl group) of a phenyl ring of a ramalin compound isolated from the Antarctic lichen Ramalina terebrata with an alkyl group or a halogen atom and a method for preparing the same. there is.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases containing a ramalin derivative as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving degenerative brain diseases containing a ramalin derivative as an active ingredient.

In order to achieve the above object, the present invention provides a ramalin derivative represented by Formula 1:

[Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy.

(a) obtaining a compound of Formula 13 by performing a coupling reaction between 1-benzyl-N-benzyloxycarbonyl-L-glutamic acid of Formula 11 as a starting material and phenylhydrazine of Formula 12; and (b) deprotecting the benzyl group (Bn) and benzyloxycarbonyl group (Cbz) of the glutamic acid hydrate of Formula 13 and then recrystallizing the obtained crude product to obtain a Ramalin derivative of Formula 1. Provided is a method for preparing the ramalin derivative comprising:

[Formula 1]

[Formula 11]

[Formula 12]

[Formula 13]

In Formulas 1, 12 and 13, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano, provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₃ , R ₄ and R ₅ are hydrogen atoms and R ₂ is hydroxy; is excluded.

The present invention also provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing the ramalin derivative represented by Chemical Formula 1 as an active ingredient.

The present invention also provides a food composition for preventing or alleviating degenerative brain diseases containing the ramalin derivative represented by Chemical Formula 1 as an active ingredient.

The present invention also provides a method for treating or preventing degenerative brain disease comprising the step of administering to a subject a pharmaceutical composition for preventing or treating degenerative brain disease comprising the ramalin derivative represented by Chemical Formula 1 as an active ingredient. provides

The present invention also provides a use of a composition for preventing or treating degenerative brain diseases comprising the ramalin derivative represented by Formula 1 as an active ingredient.

The present invention also provides a use in the manufacture of a drug for preventing or treating degenerative brain diseases comprising the ramalin derivative represented by Formula 1 as an active ingredient.

1 is a diagram showing a preferred embodiment of the ramalin derivative of the present invention.

2 is a diagram showing a ¹ H NMR (400 MHz) spectrum of RA-2Me, a ramalin derivative of the present invention.

3 is a diagram showing ^{a 13} C NMR (100 MHz) spectrum of RA-2Me, a ramalin derivative of the present invention.

4 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-2Me of the present invention.

5 is a diagram showing the COZY spectrum of the Ramalin derivative RA-2Me of the present invention.

6 is a diagram showing a ¹ H NMR (400 MHz) spectrum of RA-3Me, a ramalin derivative of the present invention.

7 is a diagram showing ^{a 13} C NMR (100 MHz) spectrum of RA-3Me, a ramalin derivative of the present invention.

8 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-3Me of the present invention.

9 is a diagram showing a ¹ H NMR (400 MHz) spectrum of RA-4Me, a ramalin derivative of the present invention.

10 is a diagram showing ^{a 13} C NMR (100 MHz) spectrum of RA-4Me, a ramalin derivative of the present invention.

11 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-4Me of the present invention.

12 is a diagram showing a ¹ H NMR (400 MHz) spectrum of RA-25Me, a ramalin derivative of the present invention.

13 is a diagram showing ^{a 13} C NMR (100 MHz) spectrum of RA-25Me, a ramalin derivative of the present invention.

14 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-25Me of the present invention.

15 is a diagram showing a ¹ H NMR (400 MHz) spectrum of RA-34Me, a ramalin derivative of the present invention.

16 is a diagram showing ^{a 13} C NMR (100 MHz) spectrum of RA-34Me, a ramalin derivative of the present invention.

17 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-34Me of the present invention.

18 is a diagram showing the COZY spectrum of the Ramalin derivative RA-34Me of the present invention.

19 is a diagram showing the HSQC spectrum of the Ramalin derivative RA-34Me of the present invention.

20 is a diagram showing a ¹ H NMR (400 MHz) spectrum of the Ramalin derivative RA-2F of the present invention.

21 is a diagram showing a ¹³ C NMR (100 MHz) spectrum of the Ramalin derivative RA-2F of the present invention.

22 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-2F of the present invention.

23 is a diagram showing the HSQC spectrum of the Ramalin derivative RA-2F of the present invention.

24 is a diagram showing a ¹ H NMR (400 MHz) spectrum of the Ramalin derivative RA-4F of the present invention.

25 is a diagram showing a ¹³ C NMR (100 MHz) spectrum of the Ramalin derivative RA-4F of the present invention.

26 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-4F of the present invention.

27 is a diagram showing the ¹ H NMR (400 MHz) spectrum of the Ramalin derivative RA-24F of the present invention.

28 is a diagram showing a ¹³ C NMR (100 MHz) spectrum of the Ramalin derivative RA-24F of the present invention.

29 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-24F of the present invention.

30 is a diagram showing the HSQC spectrum of the Ramalin derivative RA-24F of the present invention.

31 is a diagram showing a ¹ H NMR (400 MHz) spectrum of RA-PF, a ramalin derivative of the present invention.

32 is a diagram showing a ¹³ C NMR (100 MHz) spectrum of the Ramalin derivative RA-PF of the present invention.

33 is a diagram showing the DEPT spectrum of the Ramalin derivative RA-PF of the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, the phenyl ring of ramalin (ramalin, γ-glutamyl-N'-(2-hydroxyphenyl)hydrazide), a compound of molecular formula C ₁₁ H ₁₆ N ₃ O ₄ isolated from the Antarctic lichen Ramalina terebrata A derivative obtained by replacing the functional group (phenolic hydroxyl group) with a methyl group or a fluorine atom shows an inhibitory activity against BACE-1, which causes Aβ accumulation based on structural transformation, and plays an important role in the prevention and treatment of Alzheimer's disease by removing active oxygen It was found that it can act as a therapeutic agent for Alzheimer's disease because it shows activity against various targets related to Alzheimer's disease without toxicity, such as antioxidant activity that causes Alzheimer's disease and inhibition of inflammation that exacerbates Alzheimer's disease.

Accordingly, in one aspect, the present invention relates to a ramalin derivative represented by Chemical Formula 1.

[Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy.

In the present invention, it is preferable that each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ in Formula 1 is independently a hydrogen atom, C _{1 -C 6} alkyl or halogen.

Preferred examples of the ramalin derivative according to the present invention may be selected from Chemical Formulas 2 to 10, and are shown in FIG. 1.

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

In another aspect of the present invention, (a) performing a coupling reaction between 1-benzyl-N-benzyloxycarbonyl-L-glutamic acid of Formula 11 as a starting material and phenylhydrazine of Formula 12 to obtain a compound of Formula 13 ; and (b) deprotecting the benzyl group (Bn) and benzyloxycarbonyl group (Cbz) of the glutamic acid hydrate of Formula 13 and then recrystallizing the obtained crude product to obtain a Ramalin derivative of Formula 1. It relates to a method for preparing the ramalin derivative.

[Formula 1]

[Formula 11]

[Formula 12]

[Formula 13]

In Formulas 1, 12 and 13, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano, provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₃ , R ₄ and R ₅ are hydrogen atoms and R ₂ is hydroxy; is excluded.

In the present invention, the step (a) is performed by adding triethylamine (TEA) to a solution of 1-benzyl-N-benzyloxycarbonyl-L-glutamic acid dissolved in dichloromethane (DCM) at a temperature of -5 ° C. ) and ethylchloroformate (ECF) were sequentially added, the reaction mixture was stirred for 2 hours, and phenylhydrazine HCl salt was added while maintaining the temperature to perform a coupling reaction.

In the present invention, the deprotection in step (b) may be performed in an atmosphere of Pd/C and H ₂ gas.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases containing a ramalin derivative represented by Formula 1 as an active ingredient.

[Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy.

In the present invention, it is preferable that each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ in Formula 1 is independently a hydrogen atom, C _{1 -C 6} alkyl or halogen.

Preferred examples of Ramalin derivatives according to the present invention may be selected from Formulas 2 to 10, as discussed above.

In the present invention, the degenerative brain disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease and dementia, and particularly preferably Alzheimer's disease.

In the present invention, BACE1 expression can be inhibited.

In the present invention, the composition containing the ramalin derivative may additionally contain an appropriate pharmaceutically acceptable carrier, excipient or diluent according to a conventional method. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate. , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition containing the ramalin derivative of the present invention is a powder, pill, granule, capsule, suspension, internal solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solution, suspension, freeze-dried agent and suppository according to a conventional method. It may have any one formulation selected from.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 60, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method, and the dosage is determined by the patient's health condition, weight, age, gender, The range varies depending on diet, excretion rate, severity of disease, drug form, administration time, administration route and administration period, but can be appropriately selected by those skilled in the art. However, for desirable effects, the compound of the present invention is preferably administered at 0.001 to 1000 mg/kg per day, preferably at 0.01 to 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of Alzheimer's.

In another aspect, the present invention relates to a food composition for preventing or alleviating degenerative brain diseases containing a ramalin derivative represented by Chemical Formula 1 as an active ingredient.

[Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy.

In the present invention, it is preferable that each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ in Formula 1 is independently a hydrogen atom, C _{1 -C 6} alkyl or halogen.

Preferred examples of Ramalin derivatives according to the present invention may be selected from Formulas 2 to 10, as discussed above.

In the present invention, the degenerative brain disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease and dementia, and particularly preferably Alzheimer's disease.

In the present invention, the food composition containing the ramalin derivative may further include additives, excipients, or flavoring agents acceptable in food science. The food of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. The health functional food of the above type can be prepared in various forms according to conventional methods known in the art. For example, as a health food, the ramalin derivative of the present invention can be prepared in the form of tea, juice, or drink to be consumed, or granulated, encapsulated, or powdered to be consumed. In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruits, canned fruits, jams, marmalades, etc.), fish, meat and their processed foods (e.g. ham, sausage corned beef, etc.), Bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort food, It can be prepared by adding the ramalin derivative of the present invention to frozen foods, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).

The health functional food also includes various forms such as functional foods, nutritional supplements, health foods, food additives, etc. as food compositions, and various forms such as Ramalin mentioned above are prepared according to conventional methods known in the art. Manufactured in the form of tea, juice, or drink, or granulated, encapsulated, or powdered, or added to beverages, fruits and processed foods, fish, meat and its processed foods, breads, noodles, and seasonings with these compounds or extracts It can be provided by manufacturing.

In another aspect, the present invention relates to a method for preventing or treating a degenerative brain disease comprising administering a composition containing a ramalin derivative represented by Chemical Formula 1 as an active ingredient.

In another aspect, the present invention relates to the use of a composition containing the ramalin derivative represented by Chemical Formula 1 as an active ingredient for the prevention or treatment of degenerative brain diseases.

In the present invention, specifically, the inhibitory activity of Ramalin derivatives on BACE-1, which causes Aβ accumulation, is evaluated, and the antioxidant effect that plays an important role in the prevention and treatment of Alzheimer's disease by removing active oxygen and the aggravation of Alzheimer's disease The anti-inflammatory effect was investigated, and as a result, the inhibitory activity of the Ramalin derivatives on BACE-1 tended to decrease in a concentration-dependent manner. In addition, ramalin derivatives showed antioxidant activity similar to that of ramalin, and showed anti-inflammatory effects without toxicity in RAW 264.7 cells stimulated with LPS. Accordingly, it was confirmed that ramalin derivatives, like ramalin, show activity against various targets related to Alzheimer's disease, and therefore can act as a therapeutic agent for Alzheimer's disease.

In addition, in another aspect of the present invention, treatment or treatment of degenerative brain disease comprising the step of administering to a subject a pharmaceutical composition for preventing or treating degenerative brain disease comprising a ramalin derivative represented by Chemical Formula 1 as an active ingredient; It relates to a preventive method, uses of a composition for the prevention or treatment of degenerative brain diseases comprising a ramalin derivative represented by Formula 1 as an active ingredient, and a composition for preventing or treating degenerative brain diseases comprising a ramalin derivative represented by Formula 1 as an active ingredient It relates to use in the manufacture of a prophylactic or therapeutic medicament.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[Example]

All solvents and reagents were obtained from commercial suppliers Merck (Darmstadt, Germany) or TCI (Tokyo, Japan) and used without further purification. All glasses were completely dried in a drying oven (60° C.) or flame and cooled under dry argon vapor immediately before use. Filters were purchased from a commercial supplier, GE Healthcare (GF/F, 0.7 μm, Whatman, UK). All reactions were performed in an inert atmosphere of argon. Solvents and liquid reagents were transferred using a syringe. The organic extract was dried with Na ₂ SO ₄ as a drying agent and concentrated under reduced pressure using a rotary evaporator (Eyela, Tokyo, Japan). Residual solvent was removed under high vacuum (Vacuubrand RZ 2.5, Wertheim, Germany, 1 x 10 ^-2 mbar). Accurate mass spectra were obtained using an AB Sciex Triple TOF 4600 (Framingham, MA, USA) instrument with interface in direct injection mode. NMR data were obtained using a mixture of D ₂ O (with 0.01 mg/mL DSS)-acetone- d6 (6:1 v/v) or DMSO-d6 on a Jeol JNM ECP-400 spectrometer (Jeol Ltd., Tokyo, Japan). ) were collected from An internal reference or residual solvent signal as solvent was used to reference [D ₂ O (with DSS) - acetone - d ₆ : d H 0.00 / d C 29.8, DMSO - d 6 : d H 2.50 / d C 39.5] . Peak splitting patterns were abbreviated as m (multiplet), s (singlet), d (doublet) and t (triplet), respectively. A microplate reader (Thermo Scientific Inc., San Diego, CA, USA) and a multimode plate reader (Multistkan ^TM GO, Thermo Scientific, Waltham, MA, USA) were used for absorbance analysis.

Example 1: Synthesis and identification of Ramalin and its derivatives

Synthesis of p-Glu-Hyd analogues

A 250 mL round bottom flask equipped with a magnetic stir bar was charged with 1-benzyl-N-Cbz-L-glutamic acid (2.0 g, 5.39 mmol) in DCM (50 mL) solvent. The reaction mixture was cooled to -5 °C and TEA (1.2 eq, 6.47 mmol, 902 μL) was added slowly. After 10 min, ECF (1.2 eq, 6.47 mmol, 615 μL) was added dropwise to the mixture over 1 h. The reaction mixture was stirred at -5 °C for 4 hours. Another 100mL pear flask was charged with phenyl hydrazine HCl salt (1.2eq, 6.47mmol) and TEA (1.2eq, 6.47mmol, 902μL) and then slowly added to the main reaction flask at -5°C for 1 hour. When the hydrazine addition was complete, the reaction mixture was warmed to room temperature and stirred for 16 hours. After completion of the reaction, the organic layer was washed with distilled water, 1N HCl, 0.5N NaHCO ₃ , and distilled water in order to separate the layers and recover the organic layer. The organic phase was dried over Na ₂ SO ₄ and concentrated on a rotary evaporator. Purification was performed by recrystallization from ethyl acetate/n-hexane (1:5).

Synthesis and identification of Ramalin derivatives

A 500 mL round bottom flask equipped with a magnetic stir bar was charged with the appropriate p-Glu-Hyd analogue (4.5 mmol), palladium on carbon (10% by weight) in 200 mL of MeOH. The mixture was stirred for 16 hours under a hydrogen atmosphere (1 atm, hydrogen balloon). Upon completion, the reaction mixture was filtered through glass microfiber filter paper (0.7 μm). The filtrate was concentrated on a rotary evaporator. Purification was performed by recrystallization from MeOH/ethyl acetate (1:5). ¹ H NMR data of the Ramalin derivatives of Chemical Formulas 2 to 10 are as follows, and ¹ H NMR (400 NHz), ¹³ C NMR (100 MHz), DEPT, and HSQC spectra of each compound are shown in FIGS. 2 to 33 shown

N ⁵ -(o-tolylamino)-L-glutamine (RA-2Me). From Benzyl-N-Cbz-L-glutamic acid; 0.94 g, 70%, white solid; ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 7.17 (m, 2H, PhH), 6.90 (td, J = 7.6, 1.2, 1H, PhH), 6.84 (dd , J = 8.0, 0.8, 1H, PhH), 3.81 (t, J = 6.0, 1H, H-2), 2.56 (m, 2H, H-4), 2.22 (s, 3H, CH ₃ ), 2.21 ( m, 2H, H-3); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 174.8, 174.0, 145.2, 131.0, 127.4, 123.9, 121.3, 112.2, 54.5, 29.9, 26.5, 16.5; HRESIMS m/z 252.1352 [M + H] ⁺ (calcd for C ₁₂ H ₁₈ N ₃ O ₃ , 252.1348).

N ⁵ -(m-tolylamino)-L-glutamine (RA-3Me). From Benzyl-N-Cbz-L-glutamic acid; 0.97 g, 72%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 7.19 (t, J = 7.6, 1H, PhH), 6.79 (d, J = 7.6, 1H, PhH), 6.72 (s, 1H, PhH), 6.69 (d, J = 8.0, 1H, PhH), 3.80 (t, J = 6.4, 1H, H-2), 2.54 (m, 2H , H-4), 2.27 (s, 3H, CH ₃ ), 2.20 (m, 2H, H-3); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 175.0, 174.1, 147.7, 140.3, 129.8, 122.3, 114.2, 110.9, 54.5, 29.9, 26.5, 20.9; HRESIMS m/z 252.1367 [M + H] ⁺ (calcd for C ₁₂ H ₁₈ N ₃ O ₃ , 252.1348).

N ⁵ -(p-tolylamino)-L-glutamine (RA-4Me). From Benzyl-N-Cbz-L-glutamic acid; 0.90 g, 67%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 7.15 (d, J = 8.4, 2H, PhH), 6.82 (d, J = 8.0, 2H, PhH), 3.79 (t, J = 6.4, 1H, H-2), 2.52 (m, 2H, H-4), 2.25 (s, 3H, CH ₃ ), 2.18 (m, 2H, H-3); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 174.9, 174.1, 145.1, 131.5, 130.2, 114.1, 54.5, 29.9, 26.5, 19.9; HRESIMS m/z 252.1367 [M + H] ⁺ (calcd for C ₁₂ H ₁₈ N ₃ O ₃ , 252.1348).

N ⁵ -((2,5-dimethylphenyl)amino)-L-glutamine (RA-25Me). From Benzyl-N-Cbz-L-glutamic acid; 1.12 g, 78%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 6.89 (d, J = 7.6, 1H, PhH), 6.57 (d, J = 7.6, 1H, PhH), 6.52 (s, 1H, PhH), 3.67 (t, J = 6.4, 1H, H-2), 2.43 (m, 2H, H-4), 2.10 (s, 3H, CH ₃ ), 2.06 (m, 2H, H-3), 2.03 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 174.4, 173.7, 144.9, 137.1, 130.7, 121.5, 120.4, 112.5, 54.2, 29.6, 26.3, 20.4, 15.8; HRESIMS m/z 266.1504 [M + H] ⁺ (calcd for C ₁₃ H ₂₀ N ₃ O ₃ , 266.1505).

N ⁵ -((3,4-dimethylphenyl)amino)-L-glutamine (RA-34Me). From Benzyl-N-Cbz-L-glutamic acid; 1.01 g, 70%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 7.06 (d, J = 8.0, 1H, PhH), 6.71 (d, J = 2.4, 1H, PhH), 6.64 (dd, J = 8.0, 2.4, 1H, PhH), 3.80 (t, J = 6.4, 1H, H-2), 2.53 (m, 2H, H-4), 2.20 (m, 2H, H-3), 2.19 (s, 3H, CH ₃ ). 2.15 (s, 3H, CH ₃ ); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 174.7, 173.9, 145.7, 138.3, 130.6, 129.9, 115.3, 111.4, 54.5, 29.9, 26.6, 19.4, 18.3; HRESIMS m/z 266.1508 [M + H] ⁺ (calcd for C ₁₃ H ₂₀ N ₃ O ₃ , 266.1505).

N ⁵ -((2-fluorophenyl)amino)-L-glutamine (RA-2F). From Benzyl-N-Cbz-L-glutamic acid; 0.85 g, 62%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 7.15-7.08 (m, 2H, PhH), 6.97-6.90 (m, 2H , PhH), 3.80 (t, J = 6.0, 1H, H-2), 2.55 (m, 2H, H-4), 2.20 (m, 2H, H-3); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 175.0, 174.0, 151.5, 135.3, 125.2, 121.7, 115.7, 114.9, 54.5, 29.9, 26.5; HRESIMS m/z 256.1059 [M + H] ⁺ (calcd for C ₁₁ H ₁₅ FN ₃ O ₃ , 256.1097).

N ⁵ -((4-fluorophenyl)amino)-L-glutamine (RA-4F). From Benzyl-N-Cbz-L-glutamic acid; 0.90 g, 65%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 7.09-7.04 (m, 2H, PhH), 6.92-6.88 (m, 2H , PhH), 3.82 (td, J = 6.0, 1.2, 1H, H-2), 2.54 (m, 2H, H-4), 2.21 (m, 2H, H-3); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 175.0, 174.1, 158.0 143.7, 116.1, 115.2, 54.4, 29.8, 26.4; HRESIMS m/z 256.1106 [M + H] ⁺ (calcd for C ₁₁ H ₁₅ FN ₃ O ₃ , 256.1097).

N ⁵ -((2,4-difluorophenyl)amino)-L-glutamine (RA-24F). From Benzyl-N-Cbz-L-glutamic acid; 1.10 g, 75%, white solid, ¹ H NMR (400 MHz, D ₂ O/Acetone- d ₆ (6/1)): δ 6.99 (m, 1H, PhH), 6.94 (m, 1H, PhH), 6.89 (m, 1H, PhH), 3.79 (t, J = 6.4, 1H, H-2), 2.52 (m, 2H, H-4), 2.18 (m, 2H, H-3); ¹³ C NMR (100 MHz D ₂ O/Acetone- d ₆ (6/1)): δ 175.1, 174.1, 157.1, 151.2, 131.8, 115.8, 111.4, 104.3, 54.5, 29.8, 26.4; HRESIMS m/z 274.0963 [M + H] ⁺ (calcd for C ₁₁ H ₁₄ F ₂ N ₃ O ₃ , 274.1003).

N ⁵ -((perfluorophenyl)amino)-L-glutamine (RA-PF). From Benzyl-N-Cbz-L-glutamic acid; 1.39 g, 79%, white solid, ¹ H NMR (400 MHz, DMSO- d ₆ ): δ 3.20 (t, J = 6.4, 1H, H-2), 2.27 (m, 2H, H-4), 1.85 (m, 2H, H-3); ¹³ C NMR (100 MHz DMSO- d ₆ ): δ 172.0, 169.5, 137.3 (m, 4C), 133.5 (m, 1C), 124.8 (m, 1C), 53.5, 29.6, 26.9; HRESIMS m/z 328.0725 [M + H] ⁺ (calcd for C ₁₁ H ₁₄ F ₂ N ₃ O ₃ , 328.0721).

Blois, MS et al. The DPPH radical scavenging activity of Ramalin and its derivatives was evaluated according to the method (Blois, MS, Nature , 1958, 181 , 1199-1200). Briefly, 150 μL of Ramalin, derivatives and BHA at concentrations of 10, 5, 2.5, and 1 μM in MeOH were mixed with 50 μL of 0.1 mM DPPH dissolved in MeOH and then left in the dark at room temperature for 30 min. The mixture was then measured at 540 nm.

Ramalin was successfully synthesized according to a method developed in a previous study (Yim, JH et al., WO201308959A1, March 26, 2013). The synthesis was performed by coupling phenylhydrazine to the starting material, 1-benzyl-N-Cbz-L-glutamic acid. After lowering the temperature to -5°C, triethylamine (TEA) and ethyl chloroformate (ECF) were sequentially added to a solution in which the starting material was dissolved in dichloromethane (DCM). Then, the reaction mixture was stirred for about 2 hours, and the coupling reaction was completed by adding hydrazine HCl salt while maintaining the temperature. Then, after deprotection of benzyl and Cbz groups was performed using Pd/C and H ₂ gas, the crude product was recrystallized to obtain ramalin. Similar reaction conditions were used to obtain ramalin derivatives (Scheme 1).

[Scheme 1]

Reportedly, Ramalin is stable at room temperature (i.e., 25° C.) when in dry solid form. However, it is unstable in aqueous solution. This is considered a disadvantage (Pagire, SH et al., Bioorg. Med. Chem. Lett., 2018, 28, 529-532). Therefore, to improve stability and BBB penetration, the structure was modified by replacing the phenolic hydroxyl group at position 2 with a methyl group or a fluorine atom and changing the position. Thus, several derivatives were obtained. Specifically, RA-2Me, RA-3Me, and RA-4Me were synthesized by exchanging the position of RA-25Me and RA-34Me, which have two methyl groups in the electron-donating methyl group and the phenyl ring. It was synthesized by introducing an extra methyl group. RA-2F, RA-4F, RA-24F and RA-pentafluoride (PF) were also successfully synthesized to investigate the effect of electron-withdrawing fluorine atoms as opposed to methyl groups. In addition, given that ramalin and its derivatives are low molecular weight antioxidants, they have a high possibility of penetrating the BBB (Gilgun-Sherki, Y. et al., Neuropharmacology, 2001, 40, 959-975). Alzheimer's-related therapies must penetrate the BBB to reach their targets. Therefore, in order to estimate the BBB permeability, the lipophilicity, number of hydrogen bonds, polar surface area (PSA), and molecular weight (MW) of Ramalin and its derivatives were investigated (Table 1) (Rankovic, Z., J. Med. Chem ., 2015, 58, 2584-2608). The average MW of drugs acting on the central nervous system introduced between 1983 and 2002 is about 310 Da, and a MW < 450 Da has been proposed as a requirement for these drugs (Rankovic, Z., J. Med. Chem., 2015, 58, 2584-2608). Considering that Ramalin and its derivatives are water-soluble low-molecular-weight compounds and have a balance between lipophilicity and hydrophilicity, their effect on AlogP exhibiting lipophilicity was not significant. It is also considered favorable in terms of BBB penetration when considering PSA values (90 Å ² < PSA < 140 Å ² ) (Rankovic, Z., J. Med. Chem., 2015, 58, 2584-2608; Waring, MJ, Bioorg. Med. Chem. Lett., 2009, 19, 2844-2851).

Example 2: Antioxidant effect of ramalin and its derivatives

The in vitro antioxidant effects of Ramalin (free radical scavenging ability, Fe ³⁺ reducing power, superoxide anion scavenging ability, tyrosinase inhibitory effect) were confirmed in previous studies (Paudel, B. et al., Phytomedicine, 2011, 18, 1285-1290). The antioxidant effect of the newly synthesized ramalin derivatives was confirmed through a DPPH assay (2,2-diphenyl-1-picryl-hydrazyl-hydrate assay) (Table 1). Among the tested derivatives, the antioxidant effect of RA-4Me was the most similar to that of Ramalin. In addition, the antioxidant effect of RA-PF was relatively high. IC50 values are shown, but further experiments are needed to determine whether antioxidant activity is reduced in the absence of phenyl protons.

Example 3: Analysis of anti-inflammatory activity of Ramalin and its derivatives

cell culture

Macrophage-like mouse cell line RAW 264.7 (KCLB No. 40071; Korean Cell Line Bank, Seoul, Korea) was cultured in 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Burlington, ON, Canada) and 5% CO ₂ at 37°C. were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1% (w/v) antibiotic-antifungal solution (Invitrogen, Grand Island, NY, USA).

Cytotoxicity assay

Cytotoxicity was determined by MTT (Amresco, Solon, OH, USA) colorimetric assay. RAW 264.7 cells were seeded in a 96-well plate at a density of 2×10 ⁵ cells/mL and incubated for 24 hours in the presence of various concentrations of Ramalin and its derivatives. After incubation with the test substance, MTT solution (5 μL of 5 mg/mL in PBS) was added to the wells and incubated at 37° C. for 4 hours. Then, 100 μL of fresh DMSO was treated to dissolve the crystals for 10 minutes, and the cells were detected with a microplate reader (Thermo Scientific Inc., San Diego, CA, USA) and absorbance was measured at 570 nm. Relative cell viability was calculated by comparing the absorbance of the untreated control. All experiments were performed in triplicate.

Measurement of Nitric Oxide Production

Nitrite accumulation was used as an indicator of NO production in the medium and nitrite levels were measured using Griess reagent (1% sulfanilamide, 0.1% N-(1-napatyl)-ethylenediamine dihydrochloride and 5% phosphoric acid). In order to measure the amount of nitrite, 1×10 ⁶ cells/mL were inoculated in a 96-well plate, and the concentration of Ramalin and its derivatives was displayed at 37° C. for 1 hour, followed by stimulation with 0.5 μg/mL. LPS (0.5 μg/mL, Sigma-Aldrich, CA, USA) for 24 h in a final volume of 200 μL. Then, 100 μL of cell culture supernatant was mixed with 100 μL of Griess reagent in a 96-well plate. A standard curve was generated using sodium nitrite and the concentration of nitrite was determined by absorbance at 540 nm using a microplate reader. All determinations were performed in triplicate.

Anti-inflammatory activity of Ramalin and its derivatives

The importance of inflammation in the pathogenesis of Alzheimer's disease has been recognized. Inflammation has also been shown to play a role in Alzheimer's disease pathology and exacerbate the disease (Heppner, F. L. et al., Nat. Rev. Neurosci., 2015, 16, 358-372). Accordingly, the cytotoxicity of Ramalin and its derivatives was investigated, and the anti-inflammatory activity was confirmed by measuring the nitric oxide concentration. Ramalin was found to be slightly cytotoxic at maximum concentration (FIG. 2). Conversely, the derivative showed no cytotoxic effect in these cells. In addition, Ramalin exhibited NO inhibitory activity in a concentration-dependent manner. Its derivatives, including RA-25Me, RA-34Me, RA-2F, RA-4F, RA-24F and RA-PF, also showed concentration-dependent NO inhibition, whereas RA-2Me, RA-3Me and RA-4Me showed weak anti-inflammatory show effect. Also, among these derivatives, RA-24F showed a stronger anti-inflammatory effect than Ramalin without cytotoxicity.

Example 4: Analysis of β-secretase (BACE-1) inhibitory activity of Ramalin and its derivatives

BACE-1 inhibition assay was performed using the β-Secretase FRET kit (BACE-1, Thermo Fisher Scientific, San Diego, CA, USA) according to the manufacturer's instructions. The assay was performed according to the manufacturer's protocol as previously described. Stocks of Ramalin and its derivatives were prepared (20 mM) in deionized distilled water (DDW). Samples were further diluted in assay buffer and a black 96-well microplate was mixed with 10 μL of BACE-1 substrate. The reaction was then started by adding 10 μL of 3xBACE-1 enzyme to each well. Plates were incubated for 60 minutes at room temperature in the dark. After incubation, the reaction was stopped by adding 10 μL of 2.5 mM sodium acetate to each well. Finally, a multi-well spectrofluorometer instrument was used on a multimode plate reader (Multistkan ^TM GO, Thermo Scientific, Waltham, MA, USA) at an excitation wavelength of 545 nm and an emission wavelength of 585 nm. The half-maximal inhibitory concentration (IC ₅₀ ) was calculated by plotting the obtained relative fluorescence units per hour (RFU/h) against the logarithm of the inhibitor concentration. All determinations were performed in triplicate.

Slight BACE-1 expression can lead to accumulation of Aβ that causes Alzheimer's disease, which can be fatal given that Alzheimer's disease is a chronic disease. Thus, BACE-1 inhibition is a promising target for AD inhibition. The BACE-1 inhibitory activity of Ramalin and its derivatives was confirmed using PanVera ^® BACE-1 Fluorescence Resonance Energy Transfer Assay (FRET) kit (P2985, Madison, WI, USA) (Niu, Y. et al., Chem. Biol. Drug Des., 2012, 80, 775-780). The corresponding IC ₅₀ values of Ramalin and its synthesized derivatives are shown in Table 2. Experiments were performed in triplicate each for all compounds. Thus, except for RA-24F, Ramalin and all other derivatives were observed to exhibit IC50 values at micromolar concentrations. Specifically, Ramalin's BACE-1 inhibitory activity was 17.66 ± 2.74 μM, and RA-25Me showed the highest activity among the derivatives, 9.81 ± 1.21 μM.

Alzheimer's disease has a complex pathophysiology that includes pathological protein aggregation, impaired neurotransmission, increased oxidative stress, and microglia-mediated neuroinflammation. Therapeutics targeting only one of the molecular targets associated with AD have been reported. For example, therapeutics with high BACE-1 inhibitory activity can successfully inhibit and eliminate Aβ production, but cannot restore cognitive function. Therefore, when developing a treatment for Alzheimer's disease, it is important to focus on multi-target drugs rather than single-target drugs. Ramalin, which has antioxidant, BACE-1 inhibitory and anti-inflammatory activities, can serve as a promising therapeutic agent with multiple activities that can address the complex nature of Alzheimer's disease. However, its application is limited due to low stability in an aqueous environment and cytotoxicity at high concentrations. RA-25Me and RA-34Me showed activities similar to Ramalin, highlighting the possibility of improving these disadvantages. Therefore, improvements in physical properties and activity of Ramalin through the synthesis of additional derivatives may be applied to the treatment and prevention of Alzheimer's disease in the future.

Antioxidant, BACE-1 inhibitory and anti-inflammatory activities of Ramalin and its derivatives were evaluated. Nine derivatives of ramalin were synthesized in relatively high yields (62-79%) under conditions similar to those used for the synthesis of ramalin. The newly synthesized derivatives with methyl or fluoro atoms showed similar or better antioxidant effects than Ramalin. In addition, the antioxidant effect of the derivative differs depending on the location of the methyl group, and the derivative having an F atom showed a relatively low antioxidant effect. Characteristically, RA-2Me showed a higher antioxidant effect than Ramalin. In addition, most of the synthesized derivatives, except for RA-2Me, RA-3Me, and RA-4Me, inhibited NO in a concentration-dependent manner without cytotoxicity, and specifically, RA-24F showed a higher anti-inflammatory effect than Ramalin. No cytotoxicity. In addition, except for RA-24F, Ramalin and its derivatives exhibited BACE-1 inhibitory activity in a concentration-dependent manner. Among them, RA-25Me showed the highest BACE-1 inhibitory activity at a concentration of 9.81 ± 1.21 μM.

No clear electron effect is observed in aromatic systems composed of electron-tron withdrawing or donating groups. However, when RA-2Me and RA-2F were compared, RA-2Me showed excellent antioxidant effect and RA-2F showed excellent anti-inflammatory effect. Also, the BACE-1 inhibitory activity of both substances showed similar results. Among the synthesized derivatives, RA-25Me and RA-34Me showed antioxidant, anti-inflammatory, and BACE-1 inhibitory activities similar to Ramalin, but were superior to Ramalin in terms of cytotoxicity and stability. Conversely, RA-24F showed high anti-inflammatory activity, but no BACE-1 inhibitory activity could be confirmed.

Intensive efforts have been made to develop small molecule BACE-1 inhibitors with sufficient inhibitory activity to penetrate the BBB and reach the brain (Coimbra, J. R. et al., Front. Chem., 2018, 6, 178). Therapies targeting BACE-1 inhibition, favored by big pharma, can reduce Aβ levels, but no improvement in cognitive function has been observed in Alzheimer's disease patients (Voytyuk, I. et al., Biol. Psychiatry, 2018, 83 , 320-327). This suggests that early diagnosis and treatment of Alzheimer's disease is important. Recent studies have reported the development of small molecules with a multi-target approach centered on BACE-1 considering the complexity of Alzheimer's disease (Prati, F. et al., J. Med. Chem., 2018, 61, 619- 637). Examples of such molecules are coumarin derivatives, inhibitors of BACE-1 and AChE (Piazzi, L. et al., Bioorg. Med. Chem. Lett., 2008, 18, 423-426) and inhibitors of BACE-1 and GSK-3β. Curcumin derivatives (Di Martino, R. M. C. et al., J. Med. Chem., 2016, 59, 531-544) are inhibitors. Therefore, compounds with multi-target activity are in the spotlight in that drugs exhibiting only BACE-1 inhibitory activity have limitations. In addition, memoquin, which exhibits inhibitory activities and antioxidant effects on BACE-1 and AChE, is a suitable example of a multi-target compound (Capurro, V. et al., PLoS One, 2013, 8, e56870). Spatial and episodic memory deficits in mice with scopolamine-induced amnesia resolved after treatment with memoquine. Although further confirmation is needed in this regard, antioxidant effects are required for substantial cognitive improvement as well as elimination and inhibition of Aβ production in AD. Prior to Alzheimer's disease onset through Aβ accumulation, ROS-induced oxidative damage accelerates Alzheimer's disease induction. In addition, ROS causes oxidative damage to nucleic acids, lipids, and proteins related to the induction of Alzheimer's disease, and these damaging substances were initially identified in the brains of Alzheimer's disease patients (Choi, D. Y. et al., Brain Res. Bull., 2012, 87, 144-153). Reportedly, oxidative damage induces neuroinflammation and mediates BACE-1 activity (Zameer, S. et al., Neurotoxicology, 2019, 70, 122-134; Reddy, P. H., J. Neurochem., 2006 , 96, 1-13). Aβ generated by BACE-1 can in turn induce ROS production, further exacerbating the symptoms of AD (Tamagno, E. et al., Free Radic. Biol. Med., 2006, 41, 202-212; Reddy , P. H. et al., Trends Mol. Med., 2008, 14, 45-53; Lin, M. T. et al., Nature, 2006, 443, 787-795). Therefore, Ramalin, a powerful antioxidant, has considerable potential as a multi-target treatment for Alzheimer's disease based on its various physiological activities. However, further studies on additional AD inducer-related activities and derivatives are needed. Therefore, based on the results presented here, the synthesis of derivatives with improved activity should continue.

Ramalin derivatives according to the present invention have inhibitory activity against BACE-1, which induces Aβ accumulation, and exhibit antioxidant effects that play an important role in preventing and treating Alzheimer's disease by removing active oxygen. In addition, it exhibits an anti-inflammatory effect that exacerbates Alzheimer's disease, and shows an anti-inflammatory effect without toxicity in RAW 264.7 cells stimulated with LPS. As such, ramalin derivatives are active against various targets related to Alzheimer's disease, so they can act as a therapeutic agent for Alzheimer's disease. can

Having described specific parts of the present invention in detail above, it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Alzheimer's disease is a complex condition that includes pathological protein aggregation, neurotransmission disorders, increased oxidative stress, and microglia-mediated neuroinflammation. have menstruation Therapeutics targeting only one of the molecular targets associated with AD have been reported. For example, therapeutics with high BACE-1 inhibitory activity can successfully inhibit and eliminate Aβ production, but cannot restore cognitive function. Therefore, when developing a treatment for Alzheimer's disease, it is important to focus on multi-target drugs rather than single-target drugs. Ramalin, which has antioxidant, BACE-1 inhibitory and anti-inflammatory activities, can serve as a promising therapeutic agent with multiple activities that can address the complex nature of Alzheimer's disease. However, its application is limited due to low stability in an aqueous environment and cytotoxicity at high concentrations. RA-25Me and RA-34Me showed activities similar to Ramalin, highlighting the possibility of improving these disadvantages. Therefore, improvements in physical properties and activity of Ramalin through the synthesis of additional derivatives may be applied to the treatment and prevention of Alzheimer's disease in the future.

Antioxidant, BACE-1 inhibitory and anti-inflammatory activities of Ramalin and its derivatives were evaluated. Nine derivatives of ramalin were synthesized in relatively high yields (62-79%) under conditions similar to those used for the synthesis of ramalin. The newly synthesized derivatives with methyl or fluoro atoms showed similar or better antioxidant effects than Ramalin. In addition, the antioxidant effect of the derivative differs depending on the location of the methyl group, and the derivative having an F atom showed a relatively low antioxidant effect. Characteristically, RA-2Me showed a higher antioxidant effect than Ramalin. In addition, most of the synthesized derivatives, except for RA-2Me, RA-3Me, and RA-4Me, inhibited NO in a concentration-dependent manner without cytotoxicity, and specifically, RA-24F showed a higher anti-inflammatory effect than Ramalin. No cytotoxicity. In addition, except for RA-24F, Ramalin and its derivatives exhibited BACE-1 inhibitory activity in a concentration-dependent manner. Among them, RA-25Me showed the highest BACE-1 inhibitory activity at a concentration of 9.81 ± 1.21 μM.

No clear electron effect is observed in aromatic systems composed of electron-tron withdrawing or donating groups. However, when RA-2Me and RA-2F were compared, RA-2Me showed excellent antioxidant effect and RA-2F showed excellent anti-inflammatory effect. Also, the BACE-1 inhibitory activity of both substances showed similar results. Among the synthesized derivatives, RA-25Me and RA-34Me showed antioxidant, anti-inflammatory, and BACE-1 inhibitory activities similar to Ramalin, but were superior to Ramalin in terms of cytotoxicity and stability. Conversely, RA-24F showed high anti-inflammatory activity, but no BACE-1 inhibitory activity could be confirmed.

Intensive efforts have been made to develop small molecule BACE-1 inhibitors with sufficient inhibitory activity to penetrate the BBB and reach the brain (Coimbra, J. R. et al., Front. Chem., 2018, 6, 178). Therapies targeting BACE-1 inhibition, favored by big pharma, can reduce Aβ levels, but no improvement in cognitive function has been observed in Alzheimer's disease patients (Voytyuk, I. et al., Biol. Psychiatry, 2018, 83 , 320-327). This suggests that early diagnosis and treatment of Alzheimer's disease is important. Recent studies have reported the development of small molecules with a multi-target approach centered on BACE-1 considering the complexity of Alzheimer's disease (Prati, F. et al., J. Med. Chem., 2018, 61, 619- 637). Examples of such molecules are coumarin derivatives, inhibitors of BACE-1 and AChE (Piazzi, L. et al., Bioorg. Med. Chem. Lett., 2008, 18, 423-426) and inhibitors of BACE-1 and GSK-3β. Curcumin derivatives (Di Martino, R. M. C. et al., J. Med. Chem., 2016, 59, 531-544) are inhibitors. Therefore, compounds with multi-target activity are in the spotlight in that drugs exhibiting only BACE-1 inhibitory activity have limitations. In addition, memoquin, which exhibits inhibitory activities and antioxidant effects on BACE-1 and AChE, is a suitable example of a multi-target compound (Capurro, V. et al., PLoS One, 2013, 8, e56870). Spatial and episodic memory deficits in mice with scopolamine-induced amnesia resolved after treatment with memoquine. Although further confirmation is needed in this regard, antioxidant effects are required for substantial cognitive improvement as well as elimination and inhibition of Aβ production in AD. Prior to Alzheimer's disease onset through Aβ accumulation, ROS-induced oxidative damage accelerates Alzheimer's disease induction. In addition, ROS causes oxidative damage to nucleic acids, lipids, and proteins related to the induction of Alzheimer's disease, and these damaging substances were initially identified in the brains of Alzheimer's disease patients (Choi, D. Y. et al., Brain Res. Bull., 2012, 87, 144-153). Reportedly, oxidative damage induces neuroinflammation and mediates BACE-1 activity (Zameer, S. et al., Neurotoxicology, 2019, 70, 122-134; Reddy, P. H., J. Neurochem., 2006 , 96, 1-13). Aβ generated by BACE-1 can in turn induce ROS production, further exacerbating the symptoms of AD (Tamagno, E. et al., Free Radic. Biol. Med., 2006, 41, 202-212; Reddy , P. H. et al., Trends Mol. Med., 2008, 14, 45-53; Lin, M. T. et al., Nature, 2006, 443, 787-795). Therefore, Ramalin, a powerful antioxidant, has considerable potential as a multi-target treatment for Alzheimer's disease based on its various physiological activities. However, further studies on additional AD inducer-related activities and derivatives are needed. Therefore, based on the results presented here, the synthesis of derivatives with improved activity should continue.

Claims (18)

Ramalin derivatives characterized by being represented by Formula 1: [Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy. The Ramalin derivative according to claim 1, wherein each of R ¹ , R ² , R ³ , R ⁴ and R ⁵ in Formula 1 is independently a hydrogen atom, C _{1 -C 6} alkyl or halogen. The Ramalin derivative according to claim 1, characterized in that it is selected from Formulas 2 to 10: [Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

A method for preparing the ramalin derivative of claim 1 comprising the following steps: (a) obtaining a compound of Formula 13 by performing a coupling reaction between 1-benzyl-N-benzyloxycarbonyl-L-glutamic acid of Formula 11 as a starting material and phenylhydrazine of Formula 12; and (b) deprotecting the benzyl group (Bn) and benzyloxycarbonyl group (Cbz) of the glutamic acid hydrate of Formula 13 and then recrystallizing the obtained crude product to obtain the Ramalin derivative of Formula 1. [Formula 1]

[Formula 11]

[Formula 12]

[Formula 13]

In Formulas 1, 12 and 13, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano, provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₃ , R ₄ and R ₅ are hydrogen atoms and R ₂ is hydroxy; is excluded. The method of claim 4, wherein step (a) is performed in a solution of 1-benzyl-N-benzyloxycarbonyl-L-glutamic acid dissolved in dichloromethane (DCM) at -5 ° C. TEA) and ethylchloroformate (ECF) were sequentially added, the reaction mixture was stirred for 2 hours, and phenylhydrazine HCl salt was added while maintaining the temperature to perform a coupling reaction. Method for preparing derivatives. 5. The method of claim 4, wherein the deprotection in step (b) is performed in an atmosphere of Pd/C and H ₂ gas. A pharmaceutical composition for preventing or treating degenerative brain diseases containing a ramalin derivative represented by Formula 1 as an active ingredient: [Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy. The method of claim 7, wherein in Formula 1, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently a hydrogen atom, C _1- C ₆ Alkyl or halogen, characterized in that prevention or treatment of degenerative brain disease pharmaceutical composition for use. The pharmaceutical composition for preventing or treating degenerative brain diseases according to claim 7, wherein the composition is selected from Formulas 2 to 10: [Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

The method of claim 7, wherein the degenerative brain disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease, and dementia. composition. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 7, wherein the degenerative brain disease is Alzheimer's disease. The pharmaceutical composition for the prevention or treatment of degenerative brain diseases according to claim 7, wherein the BACE1 expression is inhibited. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 7, further comprising a pharmaceutically acceptable carrier, excipient or diluent. A food composition for preventing or alleviating degenerative brain diseases containing a ramalin derivative represented by Formula 1 as an active ingredient: [Formula 1]

In Formula 1, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, C _1- C ₆ alkyl, C _3- C ₆ cycloalkyl, halogen, hydroxy, carboxyl, amino or cyano , provided that R ¹ , R ² , R ³ , R ⁴ and R ⁵ are both hydrogen atoms or R ₁ , R ₂ , R ₃ and R ₄ are hydrogen atoms and R ₅ is hydroxy. The method of claim 14, wherein in Formula 1, R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently a hydrogen atom, C _1- C ₆ Alkyl or halogen to prevent or improve degenerative brain diseases, characterized in that food composition for use. [Claim 15] The food composition for preventing or improving degenerative brain diseases according to claim 14, which is selected from Formulas 2 to 10: [Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

The food for preventing or improving degenerative brain disease according to claim 14, wherein the degenerative brain disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease and dementia. composition. [Claim 15] The food composition for preventing or improving degenerative brain disease according to claim 14, further comprising a food-acceptable additive, excipient or flavoring agent.

Images