KR102374601B1 - Ues of novel compounds for preventing, improving or treating amyotrophic lateral sclerosis - Google Patents

Abstract

The present invention relates to the use of a novel compound for the prevention, improvement or treatment of amyotrophic lateral sclerosis, and the present inventors have found that SOD1 aggregation is one of the important causes in ALS disease, and suppression of regulation of TDP-43 or intracellular stress suggested the possibility that aggregation of WT-SOD1 by sALS could be the cause. In addition, the present inventors have discovered a new compound, PRG-A-01 (SLC-B036), as an inhibitor of SOD1 aggregation and misfolding. The compound showed a protective effect against muscle weakness and movement disorders in ALS model mice. As a result of histological analysis, the nerves in the spinal cord were maintained by treatment with PRG-A-01 (SLC-B036). In addition, the present inventors obtained a more optimized drugable candidate compound (PRG-A-04). In conclusion, the compound of the present invention can be usefully utilized for development as a therapeutic agent for ALS disease.

Description

Use of novel compounds for preventing, improving or treating amyotrophic lateral sclerosis

The present invention relates to the use of the novel compound for the prevention, improvement or treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, in which motor neurons are selectively lost by cell death in the spinal cord. About 10-20% of patients show a genetic pattern (familial ALS; fALS), and the rest are classified as sporadic ALS (sALS). Some genes have been identified at ALS-associated loci in familial ALS, among which SOD1 is the first gene identified in fALS. It is speculated that fALS-related genes may also play a role in the pathogenesis of sALS, but the exact cause of sALS has not been elucidated so far. In addition, ALS is classified into typical ALS, ALS with dementia, and atypical ALS according to clinical symptoms. Indeed, mutations in SOD1 cause classic ALS, and C9orf72 is associated with ALS with dementia.

One of the important characteristics of ALS is that it is a progressive disease, in which neuronal death propagates through neural connections. In fact, Alzheimer's and Parkinson's have similar phenotypes. In relation to this feature, a prion-like propagation mechanism has been proposed, which is that a misfolded protein transforms a normal protein into an abnormal protein. Indeed, mutant amyloid beta (Aβ) can transform normal Aβ into abnormal Aβ. Recently, it has been reported that mutated or misfolded SOD1 can also be secreted and disseminated during disease progression. However, not many studies have been conducted on inhibitors of SOD1 aggregation and misfolding targeting ALS disease therapeutics.

Korean Patent Publication 10-2018-0081520 (2018.07.16)

The present invention relates to the use of the novel compound for the prevention, improvement or treatment of amyotrophic lateral sclerosis.

In order to achieve the above object, the present invention provides a compound represented by the following formula (1), a hydrate thereof, or a salt thereof.

[Formula 1]

In Formula 1,

이 단일결합일 때 X는 CH이고, R¹ 및 R²는 각각 다르며, (C1~C4)알콕시, 하이드록시 또는 (C1~C4)알킬카르복시에서 선택되며,

When this single bond, X is CH, R ¹ and R ² are each different and selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy,

이 이중결합일 때 X는 N이고, R¹ 및 R²는 각각 동일하거나 다를 수 있고, 수소, (C1~C4)알킬, (C1~C4)알콕시, 하이드록시, 할로, 나이트로, 시아노 또는 (C1~C4)알킬카르복시에서 선택됨.

When this double bond, X is N, R ¹ and R ² may each be the same or different, and may be hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4) selected from alkylcarboxy.

In addition, the present invention provides a pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis comprising a compound represented by the following formula (2), a hydrate thereof, or a salt thereof.

[Formula 2]

In Formula 2,

이 단일결합 또는 이중결합이며,

is a single bond or a double bond,

n is an integer from 0 to 1,

X is CH or N;

R ¹ and R ² may each be the same or different, and are selected from hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4)alkylcarboxy.

In addition, the present invention provides a health functional food composition for preventing or improving amyotrophic lateral sclerosis comprising the compound represented by Formula 2, a hydrate thereof, or a salt thereof.

The present invention relates to the use of a novel compound for the prevention, improvement or treatment of amyotrophic lateral sclerosis, and the present inventors have found that SOD1 aggregation is one of the important causes in ALS disease, and suppression of regulation of TDP-43 or intracellular stress suggested the possibility that aggregation of WT-SOD1 by sALS could be the cause. In addition, the present inventors have discovered a new compound, PRG-A-01 (SLC-B036), as an inhibitor of SOD1 aggregation and misfolding. The compound showed a protective effect against muscle weakness and movement disorders in ALS model mice. As a result of histological analysis, the nerves in the spinal cord were maintained by treatment with PRG-A-01 (SLC-B036). In addition, the present inventors obtained a more optimized drugable candidate compound (PRG-A-04). In conclusion, the compound of the present invention can be usefully utilized for development as a therapeutic agent for ALS disease.

1 shows the result that mutant SOD1 promotes WT (wild type; wild type; normal type)-SOD1 aggregation. A. The results show that mutant SOD1s induce WT-SOD1 aggregation. B. In vitro studies show that mutant SOD1s promote SOD1 aggregation. C. Results show that ER-stress induces WT-SOD1 aggregation. D. Results show that hypoxic conditions promote SOD1 aggregation and misfolding.
2A shows the results that mutant SOD1 promotes WT-SOD1 aggregation. Figure 2B shows the results that hypoxic conditions and Zn/Cu ion imbalance induce WT-SOD1 aggregation.
3 shows the results of screening compound inhibitors for misfolded SOD1 aggregation. A. A representative graph for compound screening is shown. Negative control (-; red line) was reacted without mutant SOD1, and positive control (+; blue line) was reacted with mutant SOD1 without compound. B. The results show that PRG-A-01 inhibits SOD1 aggregation. C. PRG-A-01 shows the results of showing a dose-dependent mutant SOD1 aggregation blocking effect.
4 shows the results of screening compound inhibitors for misfolded SOD1 aggregation. A. A schematic diagram of an ELISA system for screening compounds is shown. B. Effect of compounds on SOD1 expression is shown. C. The results of testing the effects of compounds on SOD1 aggregation using native gel analysis are shown. D. The chemical structure of PRG-A-01 is shown. E. PRG-A-01 showed no cytotoxicity.
5 shows the results that PRG-A-01 blocks SOD1 aggregation and misfolding. A. The results show that SOD1 aggregation induced by TDP-43 overexpression was inhibited by PRG-A-01 treatment. B. SOD1 aggregation induced through TDP-43 overexpression is reduced by PRG-A-01. C. The results show that PRG-A-01 blocks multimer formation of SOD1. D. Results show that PRG-A-01 reduces the misfold formation of SOD1. E. Misfolding SOD1 inhibitory effect according to the dose of PRG-A-01. F. PRG-A-01 shows that it inhibits misfolding of mutant SOD1.
6 shows the results that PRG-A-01 blocks SOD1 aggregation and misfolding. A. The results indicate that TDP-43 overexpression induces WT-SOD1 aggregation. B. The results show that the mutant SOD1 affects the intracellular localization of TDP43. C. The results show that PRG-A-01 blocks the aggregation of SOD1 induced by hypoxic stress.
7 is a result confirming the in vivo (in vivo) effect of PRG-A-01 in the ALS mouse model. A. A schematic diagram of the PRG-A-01 administration experiment is shown. B. Shows the results of grip strength measurement in the ALS mouse model (PRG-A-01 administration from 12 weeks of age). C. Represents a snapshot of the mouse within the video file. D. The results of histological analysis of the SOD1 ^{G93A - Tg} mouse spinal cord are shown. E. A brief schematic diagram of the mechanism of action of PRG-A-01 is shown.
8 shows the in vivo effect of PRG-A-01. A and B. The results of measuring the grip force of the forelimbs in male (A) and female (B) SOD1 ^{G93A - Tg} ALS model mice are shown. C. A schematic diagram of a behavioral experiment is shown. D. Kaplan-Meier survival curves of SOD1 ^{G93A - Tg} ALS model mice are shown. E. The results of pharmacokinetics (PK) analysis of PRG-A-01 are shown.
9 shows the optimization results of PRG-A-01. A and B. The results of measurement of cytotoxicity through MTT assay are shown. C. The results of undenatured gel analysis for SOD1 aggregation are shown. D. IF staining of SOD is shown. E. Chemical structure of PRG-A-04, an ALS treatment candidate compound.
10 shows the pharmacokinetic analysis results of PRG-A-02 (A), PRG-A-03 (B) and PRG-A-04 (C).
11 shows the effects of PRG-A compounds in ALS model mice. A. A snapshot of mice is shown. B. PRG-A-04 shows excellent effect. C. The Kaplan-Meier survival curve results of PRG-A-01, PRG-A-02 and PRG-A-03 are shown.

Accordingly, the present inventors determined that blocking the propagation of SOD1 protein or the interaction between WT-SOD1 and mutated/misfolded SOD1 could be a therapeutic agent targeting ALS disease, and completed the present invention. .

The present invention provides a compound represented by the following formula (1), a hydrate thereof, or a salt thereof.

[Formula 1]

In Formula 1,

이 단일결합일 때 X는 CH이고, R¹ 및 R²는 각각 다르며, (C1~C4)알콕시, 하이드록시 또는 (C1~C4)알킬카르복시에서 선택되며,

When this single bond, X is CH, R ¹ and R ² are each different and selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy,

이 이중결합일 때 X는 N이고, R¹ 및 R²는 각각 동일하거나 다를 수 있고, 수소, (C1~C4)알킬, (C1~C4)알콕시, 하이드록시, 할로, 나이트로, 시아노 또는 (C1~C4)알킬카르복시에서 선택됨.

When this double bond, X is N, R ¹ and R ² may each be the same or different, and may be hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4) selected from alkylcarboxy.

이 단일결합일 때 X는 CH이고, R¹ 및 R²는 각각 다르며, (C1~C4)알콕시, 하이드록시 또는 (C1~C4)알킬카르복시에서 선택되며,

이 이중결합일 때 X는 N이고, R¹ 및 R²는 각각 수소일 수 있으나, 이에 한정되는 것은 아니다.Preferably, the compound is

When this single bond, X is CH, R ¹ and R ² are each different and selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy,

In this double bond, X is N, and R ¹ and R ² may each be hydrogen, but is not limited thereto.

More preferably, the compound is (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3- (4-hydroxy-3-methoxyphenyl)propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen- 7-yl 3-(4-hydroxy-3-methoxyphenyl)propanoate; SNU-C4], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(pyridin-4-yl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(pyridin-4-yl)acrylate; SNU-C9], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4- acetoxy-3-methoxyphenyl)propanoate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl)propanoate; SNU-C15], but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis comprising a compound represented by the following formula (2), a hydrate thereof, or a salt thereof.

[Formula 2]

In Formula 2,

이 단일결합 또는 이중결합이며,

is a single bond or a double bond,

n is an integer from 0 to 1,

X is CH or N;

R ¹ and R ² may each be the same or different, and are selected from hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4)alkylcarboxy.

이 단일결합일 때 n은 1이고, X는 CH이고, R¹ 및 R²는 각각 동일하거나 다를 수 있고, (C1~C4)알콕시, 하이드록시 또는 (C1~C4)알킬카르복시에서 선택될 수 있으나, 이에 한정되는 것은 아니다.Preferably, the compound is

When this single bond, n is 1, X is CH, R ¹ and R ² may each be the same or different, and may be selected from (C1-C4) alkoxy, hydroxy, or (C1-C4) alkylcarboxy. , but is not limited thereto.

이 이중결합일 때 n은 0 내지 1의 정수, X는 CH 또는 N이고, R¹ 및 R²는 각각 동일하거나 다를 수 있고, 수소, (C1~C4)알콕시, 하이드록시, 할로, 나이트로 또는 (C1~C4)알킬카르복시에서 선택될 수 있으나, 이에 한정되는 것은 아니다.Preferably, the compound is

When this double bond, n is an integer of 0 to 1, X is CH or N, R ¹ and R ² may be the same or different, respectively, hydrogen, (C1-C4) alkoxy, hydroxy, halo, nitro or It may be selected from (C1~C4)alkylcarboxy, but is not limited thereto.

More preferably, the compound is (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(4-hydroxy-3-methoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g ]chromen-7-yl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate; PRG-A-01; SLC-B036], (S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H -pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7 ,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one; PRG-A-02], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-( 4-hydroxy-3-methoxyphenyl)propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7 -yl 3-(4-hydroxy-3-methoxyphenyl)propanoate; SNU-C4], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3,4-dimethoxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(3,4-dimethoxyphenyl)acrylate; SNU-C5], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(3,4 -dimethoxyphenyl) propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(3 ,4-dimethoxyphenyl)propanoate; SNU-C7], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(pyridin-4-yl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(pyridin-4-yl)acrylate; SNU-C9], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3-hydroxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(3-hydroxyphenyl)acrylate; SNU-C10], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(4-fluorophenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(4-fluorophenyl)acrylate; SNU-C11; PRG-A-03], (S,E)-7-((3-(4-fluorophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano [3,2-g]chromene-2-one[(S,E)-7-((3-(4-fluorophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H, 6H-pyrano[3,2-g]chromen-2-one; SNU-C13], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3-acetoxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(3-acetoxyphenyl)acrylate; SNU-C14], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4- acetoxy-3-methoxyphenyl)propanoate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl)propanoate; SNU-C15], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(4-acetoxy-3-methoxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen- 7-yl (E)-3-(4-acetoxy-3-methoxyphenyl)acrylate; SNU-C17], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3,4-difluorophenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7- yl (E)-3-(3,4-difluorophenyl)acrylate; SNU-C18] and (S,E)-7-((3-(3-methoxy-4-nitrophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H- pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-(3-methoxy-4-nitrophenyl)allyl)oxy)-8,8-dimethyl-7,8 -dihydro-2H,6H-pyrano[3,2-g]chromen-2-one; PRG-A-04], but is not limited thereto.

More preferably, the pharmaceutical composition can inhibit aggregation and misfolding of superoxide dismutase 1 (SOD1).

The pharmaceutical composition of the present invention may be prepared by using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, an expanding agent, a lubricant, and a lubricant. Alternatively, a solubilizing agent such as a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical formulation of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, sustained-release formulations of the active compound, etc. can The pharmaceutical composition of the present invention can be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered as The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health condition, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and concomitant drugs. Although not limited thereto, for example, for adults, when administered once to several times a day, the composition of the present invention can be administered at a dose of 0.01 ng/kg to 10 g/kg when administered once to several times a day .

In addition, the present invention provides a health functional food composition for preventing or improving amyotrophic lateral sclerosis comprising a compound represented by the following formula (2), a hydrate thereof, or a salt thereof.

[Formula 2]

In Formula 2,

이 단일결합 또는 이중결합이며,

is a single bond or a double bond,

n is an integer from 0 to 1,

X is CH or N;

R ¹ and R ² may each be the same or different, and are selected from hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4)alkylcarboxy.

이 단일결합일 때 n은 1이고, X는 CH이고, R¹ 및 R²는 각각 동일하거나 다를 수 있고, (C1~C4)알콕시, 하이드록시 또는 (C1~C4)알킬카르복시에서 선택될 수 있으나, 이에 한정되는 것은 아니다.Preferably, the compound is

When this single bond, n is 1, X is CH, R ¹ and R ² may each be the same or different, and may be selected from (C1-C4) alkoxy, hydroxy, or (C1-C4) alkylcarboxy. , but is not limited thereto.

이 이중결합일 때 n은 0 내지 1의 정수, X는 CH 또는 N이고, R¹ 및 R²는 각각 동일하거나 다를 수 있고, 수소, (C1~C4)알콕시, 하이드록시, 할로, 나이트로 또는 (C1~C4)알킬카르복시에서 선택될 수 있으나, 이에 한정되는 것은 아니다.Preferably, the compound is

When this double bond, n is an integer of 0 to 1, X is CH or N, R ¹ and R ² may be the same or different, respectively, hydrogen, (C1-C4) alkoxy, hydroxy, halo, nitro or It may be selected from (C1~C4)alkylcarboxy, but is not limited thereto.

More preferably, the compound is (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(4-hydroxy-3-methoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g ]chromen-7-yl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate; PRG-A-01; SLC-B036], (S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H -pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7 ,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one; PRG-A-02], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-( 4-hydroxy-3-methoxyphenyl)propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7 -yl 3-(4-hydroxy-3-methoxyphenyl)propanoate; SNU-C4], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3,4-dimethoxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(3,4-dimethoxyphenyl)acrylate; SNU-C5], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(3,4 -dimethoxyphenyl) propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(3 ,4-dimethoxyphenyl)propanoate; SNU-C7], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(pyridin-4-yl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(pyridin-4-yl)acrylate; SNU-C9], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3-hydroxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(3-hydroxyphenyl)acrylate; SNU-C10], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(4-fluorophenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(4-fluorophenyl)acrylate; SNU-C11; PRG-A-03], (S,E)-7-((3-(4-fluorophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano [3,2-g]chromene-2-one[(S,E)-7-((3-(4-fluorophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H, 6H-pyrano[3,2-g]chromen-2-one; SNU-C13], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3-acetoxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(3-acetoxyphenyl)acrylate; SNU-C14], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4- acetoxy-3-methoxyphenyl)propanoate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl)propanoate; SNU-C15], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(4-acetoxy-3-methoxyphenyl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen- 7-yl (E)-3-(4-acetoxy-3-methoxyphenyl)acrylate; SNU-C17], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(3,4-difluorophenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7- yl (E)-3-(3,4-difluorophenyl)acrylate; SNU-C18] and (S,E)-7-((3-(3-methoxy-4-nitrophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H- pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-(3-methoxy-4-nitrophenyl)allyl)oxy)-8,8-dimethyl-7,8 -dihydro-2H,6H-pyrano[3,2-g]chromen-2-one; PRG-A-04], but is not limited thereto.

The health functional food composition of the present invention may further include one or more additives selected from the group consisting of organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar and sorbitol. The organic acid may be, but is not limited to, citric acid, humic acid, adipic acid, lactic acid or malic acid, and the phosphate salt may be, but is not limited to, sodium phosphate, potassium phosphate, acid pyrophosphate or polyphosphate (polyphosphate), and antioxidant The agent may be a natural antioxidant such as, but not limited to, polyphenols, catechins, alpha-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid or phytic acid.

In another embodiment of the present invention, the health functional food, in addition to the active ingredient, various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as 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, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition according to an embodiment of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage.

According to an embodiment of the present invention, the dosage form of the health functional food is not limited thereto, but may be in the form of a solid, powder, granule, tablet, capsule, liquid or beverage.

In addition, the health functional food is not limited thereto, but confectionery, sugars, ice cream products, dairy products, meat products, fish meat products, tofu or jelly, edible oils and fats, noodles, teas, beverages, special nutritional foods, health supplements, seasoned foods , ice, ginseng products, pickled kimchi food, raisins, fruits, vegetables, dried fruits or vegetables, cut products, fruit juice, vegetable juice, mixed juices thereof, chips, noodles, processed livestock food, processed seafood, It can be used in the manufacture of foods such as milk processed food, fermented milk food, pulse food, grain food, microbial fermented food, confectionery and bread, seasonings, processed meat, acidic beverage, licorice, herbs, and the like.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

The following experimental examples are provided to provide experimental examples commonly applied to each embodiment according to the present invention.

< Experimental example >

1. Mouse

Experiments were performed in a facility approved by the Association for Laboratory Animal Care Assessment and Accreditation in accordance with the animal policy approved by Pusan National University. B6SJL-Tg (SOD1 ^G93A ) mice were purchased from Jackson Laboratory (Stock No: 002726). All mice were maintained under temperature and light-controlled conditions (20-23°C, light-dark cycle 12hr-12hr), and were provided with sterile food and water.

2. In vivo ( in in vivo ) drug treatment and histological analysis

SOD1 ^G93A Mice were administered vehicle (DMSO), SLC-B036 (10 mg/kg, 20 mg/kg) to 12-week-old or 14-week-old mice via intraperitoneal injection twice weekly for 6 weeks. Control mice were treated under the same conditions. For histological analysis, mice were sacrificed at 18 weeks. After dissection of the mouse, the spinal cord was fixed with 4% paraformaldehyde for 48 hours according to basic tissue processing procedures, and embedded in a paraffin block. The embedded tissue (cervical region of the spinal cord) was cut into 5 μm with a Leica microtome, and transferred to an adhesive-coated slide (Marienfeld laboratory glassware, Germany). After deparaffinization and rehydration, slides were stained with hematoxylin and eosin to determine the number of spinal nerves.

3. Measure your exercise performance

For the behavioral experiment, the grip strength was checked every 2 weeks using a grip strength meter. Mice were allowed to grasp a tension bar with their forelimbs, and then slowly pulled their tails until the bar was released. In order to confirm the motility, respiration, and quadriplegia of the mice, image analysis was performed at the end of administration (18-20 weeks).

4. Cell Culture and Reagents

HEK293 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37° C. and 5% CO ₂ in DMEM liquid medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. SK-N-SH cells were purchased from Korean Cell Line Bank (KCLB, Seoul, South Korea) and maintained in MEM medium containing 10% fetal bovine serum, 1% antibiotic, 25 mM HEPES, and 300 mg/L L-Glu. . Human fibroblasts (9 years old female) were purchased from Coriell Cell Repositories (New Jersey, USA) and maintained in EMEM containing 15% FBS, 2 mM Glutamine, and 26 mM HEPES without antibiotics. Thapsigargin (ER calcium scavenger: CAS 67526-95-8) was purchased from Calbiochem (Darmstadt, Germany). CoCl ₂ (hypoxia-inducer: C8661) was purchased from Sigma Aldrich (St, Louis, Mo, USA).

5. Compound screening

For compound screening, we applied an ELISA assay system. To select a binding inhibitor between WT-SOD1 and mutant SOD1, the present inventors used 0.5% paraformaldehyde (PFA) to immobilize the WT-SOD1 recombinant protein on a 96-well plate. The plate was dried, washed with phosphate-buffered saline (PBS), and the compounds at a final concentration of 50 μM were reacted, and then mutant SOD1-GST (A4V, G37R, G85R, G93A) proteins were added. After 2 hours of reaction, the 96-well plate was washed with PBS and blocked with 3% skim milk to prevent non-specific reaction. The plate was reacted with anti-GST antibody (diluted at 1:10,000) for 1 hour, and then reacted with anti-mouse IgG-HRP (diluted at 1:50,000) for 1 hour. After washing twice, the plate was reacted with a 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Calbiochem) for 30 minutes and a stop solution (1N H ₂ SO ₄ ) for 30 minutes. . Finally, the present inventors analyzed the values using an ELISA reader (absorbance 450 nm). Negative control (-; red line) was reacted without mutant SOD1, and positive control (+; blue line) was reacted with mutant SOD1 without compound.

6. Recombinant Protein

To prepare a recombinant protein, human SOD1 (WT, A4V, G37R, G85R, G93A) in Eco RI and HindIII sites of pGEX-TEV vector, an improved vector made by adding a TEV protease cleavage site to pGEX-4T1 (Invitrogen) was combined. Recombinant proteins were expressed as GST-fusion proteins in Escherichia coli; E. coli strain BL21 (DE3). The proteins were purified by glutathione-affinity chromatography.

7. western blot analyze

For SDS-PAGE, RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS and 10% sodium deoxycholate) and lysis buffer (50 mM Tris for Native-PAGE) -Cl, pH 7.5, 150 mM NaCl, 0.3% NP-40) was used to extract proteins from the cells. Samples were separated by SDS-PAGE or Native-PAGE and transferred to PVDF membrane. The blotted membrane was blocked with TBST buffer containing 3% skim milk for 1 hour, and reacted with specific antibodies. The reacted antibody was detected by ECL and X-ray film exposure. The antibodies used in the present invention are as follows: pan-SOD1 (GTX100554) was purchased from Genetex (California, USA). Misfolded SOD1-specific antibody (B8H10) was purchased from MediMabs (Montreal, Canada). Actin (sc-1616), GST (sc-138), and GFP (Green fluorescent protein; sc-8036) were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). TDP-43 antibody (10782-2-AP) was purchased from Proteintech (Rosemont, IL, USA). Anti-FLAG (Sigma; F3165) was purchased from Sigma Aldrich (St, Louis, Mo, USA), and HRP-conjugated goat anti-mouse, goat anti-rabbit and mouse anti-goat antibodies (Pierce, Thermo Fisher Scientific, Inc.) ., Rockford, IL, USA) was used as a secondary antibody.

8. Dot blot analyze

To detect misfolded SOD1 expression, cells transfected with the SOD1 vector were treated with the compound for 24 hours. After the reaction, the cells were lysed with a lysis buffer containing no surfactant, and then the cell lysate was immobilized on a nitrocellulose membrane using a Bio-Dot SF Microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA). In the case of the peptide reaction, the SOD1 recombinant protein was reacted with the compound for 1 hour, and then the samples were loaded onto the membrane. Each membrane was washed with TBS and blocked with 3% skim milk to prevent non-specific reaction. After blocking, the membrane was reacted with misfolded SOD1 or Actin antibody (1:8,000 in TBST containing 1% skim milk) for 30 minutes, and then a secondary antibody (goat anti-mouse IgG-horseradish peroxidase, 1% skim) 1:50,000 in milk blocking buffer) for 30 minutes. Antibodies reacted to proteins were detected by ECL and X-ray film exposure. Actin was used as a loading control.

9. immunofluorescence dyeing

Cells on the coverslip were washed with PBS, fixed with 4% PFA at room temperature for 30 minutes, and then infiltrated with 0.1% Triton X-100/PBS for 10 minutes. Cells were treated with blocking solution (anti-Human Antibody 1:500 dilution in PBS) for 1 h, then cells were treated with anti-pan SOD1 (1:400 dilution), misfolded SOD1 (B8H10; 1:200 dilution in blocking solution). dilution) and reacted overnight at 4°C. Finally, the cells were reacted with FITC and rhodamine-conjugated secondary antibody at 4°C for 6 hours. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI), and the endoplasmic reticulum (ER) was stained with ER-Tracker Red dye for 10 minutes. dyed. Cells were washed 3 times with PBS, and then coverslips were washed with mounting solution [H-5501; Vector Laboratories (Burlingame, CA, USA)] and analyzed by fluorescence microscopy (Zeiss).

10. Transfection of vectors

GFP-SOD1 (WT, G85R, G93A), non-tagged SOD1 (WT, A4V, G37R, G85R, G93A) and tdTomato-TDP43 expression vectors were purchased from Addgene (Cambridge, MA, USA). Transfection was performed using Jet-PEI reagent (JetPEI; Polyplus transfection, New York, NY, USA) according to the manufacturer's protocol. Briefly, the vector was mixed with JetPEI reagent in 150 mM NaCl buffer, and then the mixture was reacted for 15 minutes. The mixture was added to the cells in serum-free medium and allowed to react for 4 hours. After the reaction, the cells were replaced with a culture medium supplemented with 10% FBS.

11. Cell Viability Measurement

In order to check the cell viability, the cells were reacted with a 0.5 mg/ml MTT solution (475989; Merck, Darmstadt, Germany) at 37° C. for 4 hours. After removing the residual solution, washing with PBS, the precipitate was dissolved in 200 μl DMSO, and quantified by measuring the absorbance at 540 nm.

< Example >

The present inventors transfected (transfection) the mutant SOD1 into the cells expressing GFP-WT-SOD1. In the cytoplasm, WT-SOD1 was aggregated by the mutant SOD1 regardless of the mutation type ( FIGS. 1A and 2 ). In addition, as a result of confirming the interaction between WT-SOD1 protein and mutant recombinant SOD1 protein through Native-PAGE, it was found that WT-SOD1 was aggregated by treatment of mutant SOD1 (Fig. 1B; G93A-reaction was gel due to too large aggregation). did not move into mine). Next, the present inventors confirmed the aggregation of WT-SOD1 by cellular stress. Since ALS is a disease specific to motor neurons, and Ca ²⁺ is important not only for neuronal function but also for muscle contraction, we tested the effect of Thapsigargin (an ER calcium scavenger) on SOD1 aggregation. Treatment with Thapsigarigin certainly promoted the aggregation of WT-SOD1 as well as mutant SOD1s (Fig. 1C). In addition, the present inventors confirmed the aggregation of SOD1 and increase of misfolded SOD1 by CoCl ₂ (hypoxia-inducing agent) ( FIG. 1D ; red signal increased in CoCl ₂ -treated cells). On the other hand, SOD1 inhibited the enzyme activity. It is also known to use Cu/Zn ions for this purpose. Accordingly, the present inventors confirmed the effect of conditions lacking Cu/Zn ions on SOD1 aggregation on SOD1 aggregation. As shown in FIG. 2 , TPEN (Zn2+ chelator) and ATN-224 (Cu2+ chelator) induced aggregation of WT-SOD1. The above results support that SOD1 can be converted to an aggregated or misfolded form by cellular stress conditions without genetic mutation, and that misfolding of SOD1 can also be the cause of sporadic ALS.

If SOD1 aggregation is the cause of ALS disease, blocking SOD1 aggregation is one of the possible strategies for drug development. In fact, neutralizing antibodies against misfolded SOD1 showed a good effect in the SOD1 animal model. To prove this, the present inventors constructed a compound screening system based on ELISA. In order to discover a selective binding inhibitor for the binding of misfolded SOD1 to WT-SOD1, the present inventors immobilized WT-SOD1 on an ELISA plate and reacted with GST-tagged mutant SOD1s (A4V, G37R, G85R, G93A). (Fig. 4A). Previously, the present inventors performed a similar screening and obtained a compound library. Using the compound library, the present inventors measured the inhibitory effect of each compound (FIG. 3A). Since SOD1 removes superoxide after dimer formation, we removed too strong binding inhibitors (such as SLC-B035) and selected compounds specific for mutation (such as SLC-B040 or B036) (Fig. 3A) ). These compounds did not affect SOD1 expression (Fig. 4B). Next, the present inventors confirmed the effect of the selected compounds on SOD1 aggregation through undenatured gel analysis (FIG. 4C), and selected SLC-B036 as the first hit compound (FIG. 4D). Indeed, the compound was able to block the aggregation of mutant SOD1 in a dose-dependent manner ( FIGS. 3B and 3C ). Moreover, the compound showed no cytotoxic effect in normal human fibroblasts (Fig. 4E). Thereafter, SLC-B036 was renamed PRG-A-01.

Next, the present inventors confirmed the effect of the compounds on the aggregation of WT-SOD1. Recently, it has been reported that overexpression or deficiency of TDP-43 can induce SOD1 misfolding. The present inventors were also able to confirm the aggregation of WT-SOD1 by TDP-43 overexpression through immunofluorescence analysis ( FIGS. 5A and 6A ). In addition, SOD1 mutations increased cytoplasmic (F2 fraction) and insoluble (F4 fraction) TDP43 protein, suggesting that TDP-43 and SOD1 are associated with the pathogenesis of ALS (FIG. 6B). Treatment with PRG-A-01 was able to reduce the aggregation of SOD1 as well as TDP-43 ( FIG. 5A ). In addition, in the results of the undenatured gel analysis, it was confirmed that the aggregation of SOD1 and TDP43 was decreased by the treatment of PRG-A-01 ( FIG. 5B ). As a result of a cross-linking experiment using glutaraldehyde, the inhibitory effect of PRG-A-01 was more evident. The bands corresponding to the multimer size of SOD1 and TDP-43 formed through cross-linking were weakened by PRG-A-01 treatment (Fig. 5C). To find out if PRG-A-01 could block the misfold formation of SOD1, we performed dot blot analysis. Cell lysates transfected with WT- or mutant SOD1 were transferred to an aspirator and reacted with antibodies specific for misfolded SOD1. In the compound-treated cells, the band intensity clearly decreased ( FIG. 5D ). In addition, we were able to confirm a dose-dependent decrease in misfolded SOD1 by PRG-A-01 treatment in G93A-transfected cell lysates (Fig. 5E). When PRG-A-01 and SOD1-G93A were reacted, the antibody specific for misfolding did not recognize the mutant SOD1 (FIG. 5F), which indicates that PRG-A-01 changed the protein structure and misfolded SOD1 with WT- This suggests that it can be corrected in the form of SOD1. In addition, since the present inventors found that hypoxic stress can induce SOD1 misfolding, the effect of PRG-A-01 under hypoxic conditions was confirmed. Signal transduction of CoCl ₂ induced misfolding SOD1 was reduced by PRG-A-01 treatment ( FIG. 6C ), indicating that PRG-A-01 inhibited the induction of disease by SOD1 misfolding.

In order to confirm the in vivo effect of the compound, the present inventors administered PRG-A-01 ip twice a week to the SOD1 ^G93A-Tg mouse model ( FIG. 7A ). Treatment with PRG-A-01 was able to slow the decrease in muscle strength. Compared to wild-type mice, mice treated with PRG-A-01 maintained about 70% muscle strength ( FIG. 7B ). In contrast, vehicle-treated mice exhibited less than 40% muscle strength ( FIG. 7B ). In addition, despite the late treatment of PRG-A-01 (starting at 14 weeks of age), it had a good effect on muscle function ( FIGS. 8A and 8B ). In addition, PRG-A-01 showed a dose-dependent effect, and administration of 20 mg/kg was more effective than administration of 10 mg/kg ( FIGS. 8A and 8B ). Behavioral observations showed very impressive results, where despite 14-week-old dosing (phenotype started at 14-weeks of age), PRG-A-01 treated mice still had motor capacity at 17-weeks of age ( FIGS. 7C and 8C ). . The effect could be observed irrespective of gender. In contrast, vehicle-treated mice were unable to stand up and exhibited problems with autogenous breathing. As a result of histological analysis, the survival rate of cervical spinal nerves was high in PRG-A-01-treated mice ( FIG. 7D ). Conversely, most nerves disappeared in the vehicle-treated mouse spinal cord ( FIG. 7D ). However, PRG-A-01 only extended the lifespan by 10 days (Fig. 8D). In this regard, we predict that the half-life of PRG-A-01 may be very short in in vivo systems. Indeed, PRG-A-01 rapidly disappeared within 30 minutes in the body, but it disappeared in the blood too quickly to obtain pharmacokinetic (PK) data ( FIG. 8E ).

In order to overcome the rapid degradation, the present inventors prepared PRG-A-01 related derivatives and confirmed the effects of these compounds (Table 1). Among the newly synthesized compounds, PRG-A-02, PRG-A-03 and PRG-A-04 showed similar or superior effects to PRG-A-01. Indeed, these compounds did not show cytotoxicity in neurons (Fig. 9A) and normal fibroblasts (Fig. 9B). In addition, the present inventors confirmed the effect of the compounds on SOD1 aggregation through non-SDS-PAGE gel (FIG. 9C) and IF analysis (FIG. 9D). According to the cell-based assays and in vitro assays of the present invention, three compounds (PRG-A-02, PRG-A-03 and PRG-A-04) were selected and analyzed for PK. Since only PRG-A-04 showed an appropriate PK profile ( FIG. 10 ), we selected PRG-A-04 as a candidate compound for the treatment of ALS.

In the in vivo analysis, the present inventors were able to confirm good effects from the PRG-A-04 treatment group as well as the PRG-A-02 and PRG-A-03 treatment groups ( FIGS. 11A and 11B ). A more interesting result was that the lifespan of the ALS model mice increased according to the PK profile ( FIGS. 11C and 11D ). Although the present inventors could not obtain lifespan data of PRG-A-04, it can be predicted that the lifespan will be obviously extended compared to other compounds.

No. MW Western Blot Immunofluorscence ELISA in vivo Toxicity (MTT) PK study PRG -A-01
(SLC-B036) 422.43 Effect ○ Effect ○ Effect ○ Effect ○ no toxicity BA
very low PRG -A-02 408.44 Effect ○ Effect ○ Effect ○ Effect ○ no toxicity BA
Very Low (0.3%) SNU-C4 424.449 effect × effect × effect × - no toxicity - SNU-C5 436.46 effect × effect × Effect ○ - no toxicity - SNU-C7 438.476 effect × effect × effect × - no toxicity - SNU-C9 377.396 Effect ○ Effect ○ effect × - no toxicity - SNU-C10 392.407 Effect ○ Effect ○ Effect ○ - no toxicity - PRG -A-03
(SNU-C11) 394.398 Effect ○ Effect ○ Effect ○ Effect ○ no toxicity BA
Very low (3.7%) SNU-C13 380.415 effect × - - - - - SNU-C14 434.444 Effect ○ Effect ○ Effect ○ - no toxicity - SNU-C15 466.486 Effect ○ Effect ○ effect × - no toxicity - SNU-C17 464.47 Effect ○ Effect ○ - - - - SNU-C18 412.11 Effect ○ Effect ○ - - Toxic - PRG -A-04
(SNU-C19) 437.15 Effect ○ Effect ○ Effect ○ Effect ○ no toxicity BA=60.7 %

Chemical structures and NMR data of the compounds used in the present invention are as follows.

(S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g] chromen-2-one (PRG-A-02)

¹ H NMR: EW15731-164-P1D4 (400 MHz, CDCl ₃ )

δ 7.58 (d, J = 12 Hz, 1H), 7.16 (s, 1H), 6.90 - 6.86 (m, 3H), 6.78 (s, 1H), 6.51 (d, J =16 Hz, 1H), 6.22 ( d, J = 8.0 Hz, 1H), 6.14 - 6.10 (m, 1H), 5.65 (s, 1H), 4.32 - 4.30 (m, 1H), 4.22 - 4.20 (m, 1H), 3.91 (s, 3H) , 3.61 - 3.58 (m, 1H), 3.11 - 3.05 (m, 1H), 3.89 - 3.87 (m, 1H), 1.43 (s, 3H), 1.36 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl 3-(4-hydroxy-3-methoxyphenyl)propanoate (Compound 4; SNU-C4)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.59 (d, 1H), 7.06 (s, 1H), 6.82 - 6.73 (m, 2H), 6.64 - 6.57 (m, 2H), 6.24 (d, J = 9.5 Hz, 1H), 5.44 (br.s, 1H), 5.01 (t, J = 4.6 Hz, 1H), 3.79 (s, 3H), 3.09 (ddd, J = 17.2, 4.7, 1.2 Hz, 1H), 2.85 (app.t, J = 7.2 Hz, 2H), 2.70 - 2.59 (m, 3H), 1.31 (s, 3H), 1.29 (s, 3H).

HRMS: Calcd for C24H25O7 ⁺ [M+H]+ 425.1595, found 286.1652.

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(3,4-dimethoxyphenyl)acrylate (Compound 5; SNU-C5)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.67 - 7.55 (m, 2H), 7.18 (s, 1H), 7.08 (d, J = 8.2 Hz, 1H), 7.02 (s, 1H), 6.88 - 6.81 (m, 2H), 6.28 (d, J = 16.0 Hz, 1H), 6.24 (d, J = 9.4 Hz, 1H), 5.20 (app.t, J = 4.6 Hz, 1H), 3.91 (s, 3H) , 3.89 (s, 3H), 3.25 (dd, J = 17.3, 4.8 Hz, 1H), 2.94 (dd, J = 17.3, 4.5 Hz, 1H), 1.45 (s, 3H), 1.39 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl 3-(3,4-dimethoxyphenyl)propanoate (Compound 7; SNU -C7)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.59 (d, J = 9.6 Hz, 1H), 7.07 (s, 1H), 6.83 - 6.63 (m, 4H), 6.24 (d, J = 9.6 Hz, 1H) ), 5.03 (app.t, J = 4.8 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H), 3.11 (dd, J = 17.3, 4.6 Hz, 1H), 2.87 (t, J = 7.3 Hz, 2H), 2.69 (dd, J = 17.2, 4.6 Hz, 1H), 2.63 (app.td, J = 7.3, 1.9 Hz, 2H), 1.31 (s, 3H), 1.30 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(pyridin-4-yl)acrylate (Compound 9; SNU -C9)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 8.72 - 8.59 (m, 2H), 7.60 (d, 1H), 7.57 (d, 1H), 7.36 - 7.30 (m, 2H), 7.18 (s, 1H) , 6.83 (s, 1H), 6.58 (d, J = 16.0 Hz, 1H), 6.24 (d, J = 9.4 Hz, 1H), 5.21 (app.t, J = 4.6 Hz, 1H), 3.26 (ddd, J = 17.2, 4.8, 1.2 Hz, 1H), 3.00 - 2.89 (m, 1H), 1.44 (s, 3H), 1.39 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(3-hydroxyphenyl)acrylate (Compound 10; SNU -C10)

¹ H NMR (CD ₂ Cl ₂ , 400 MHz): δ ppm 7.64 - 7.55 (m, 2H), 7.25 (app.t, J = 7.9 Hz, 1H), 7.21 (s, 1H), 7.13 - 7.06 (m, 1H), 7.00 - 6.95 (m, 1H), 6.86 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 6.78 (s, 1H), 6.40 (d, J = 15.9 Hz, 1H), 6.18 (d) , J = 9.5 Hz, 1H), 5.18 (app.t, J = 4.8 Hz, 1H), 3.25 (ddd, J = 17.3, 4.9, 1.2 Hz, 1H), 2.94 (dd, J = 17.3, 4.7 Hz, 1H), 1.42 (s, 3H), 1.38 (s, 3H), 1.25 (brs, 1H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(4-fluorophenyl)acrylate (Compound 11; SNU -C11; PRG -A-03)

¹ H NMR (CD ₂ Cl ₂ , 400 MHz): δ ppm 7.66 - 7.55 (m, 2H), 7.55 - 7.46 (m, 2H), 7.18 (s, 1H), 7.11 - 7.01 (m, 2H), 6.76 ( s, 1H), 6.35 (d, J = 16.0 Hz, 1H), 6.16 (d, J = 9.5 Hz, 1H), 5.17 (app.t, J = 4.7 Hz, 1H), 3.23 (ddd, J = 17.3) , 4.8, 1.2 Hz, 1H), 2.92 (dd, J = 17.3, 4.7 Hz, 1H), 1.40 (s, 3H), 1.36 (s, 3H).

( S, E )-7-((3-(4- fluorophenyl ) allyl )oxy)-8,8- dimethyl -7,8- dihydro - 2H, 6H -pyrano[3,2-g]chromen-2-one (Compound 13; SNU-C13)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.57 (d, J = 9.5 Hz, 1H), 7.38 - 7.29 (m, 2H), 7.16 (s, 1H), 7.06 - 6.93 (m, 2H), 6.77 (s, 1H), 6.55 (d, J = 15.8 Hz, 1H), 6.25 - 6.13 (m, 2H), 4.33 (ddd, J = 12.8, 5.8, 1.5 Hz, 1H), 4.19 (ddd, J = 12.8) , 6.3, 1.4 Hz, 1H), 3.58 (dd, J = 7.4, 5.0 Hz, 1H), 3.08 (dd, J = 16.4, 5.0 Hz, 1H), 2.85 (ddd, J = 16.4, 7.4, 1.1 Hz, 1H), 1.42 (s, 3H), 1.35 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(3-acetoxyphenyl)acrylate (Compound 14; SNU -C14)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.63 (d, J = 16.0 Hz, 1H), 7.59 (d, J = 9.5 Hz, 1H), 7.42 - 7.33 (m, 2H), 7.25 - 7.22 (m , 1H), 7.17 (s, 1H), 7.11 (app.dt, J = 7.4, 2.1 Hz, 1H), 6.83 (s, 1H), 6.40 (d, J = 16.0 Hz, 1H), 6.24 (d, J = 9.5 Hz, 1H), 5.19 (app.t, J = 4.7 Hz, 1H), 3.24 (ddd, J = 17.2, 4.7, 1.2 Hz, 1H), 2.93 (dd, J = 17.2, 4.5 Hz, 1H) ), 2.30 (s, 3H), 1.43 (s, 3H), 1.39 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl 3-(4-acetoxy-3-methoxyphenyl)propanoate (Compound 15; SNU -C15)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.58 (d, J = 9.5, 0.8 Hz, 1H), 7.10 (s, 1H), 6.87 (dd, J = 7.9, 1.1 Hz, 1H), 6.77 (d , 1H), 6.74 - 6.65 (m, 2H), 6.23 (dd, J = 9.5, 2.2 Hz, 1H), 5.02 (app.t, J = 4.7 Hz, 1H), 3.74 (d, J = 1.2 Hz, 3H), 3.10 (ddd, J = 17.2, 4.8, 1.1 Hz, 1H), 2.90 (t, J = 7.5 Hz, 2H), 2.72 - 2.60 (m, 3H), 2.30 (d, J = 1.3 Hz, 3H) ), 1.31 (s, 3H), 1.30 (s, 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(4-acetoxy-3-methoxyphenyl)acrylate (Compound 17; SNU -C17)

¹ H NMR (CD ₂ Cl ₂ , 600 MHz): δ ppm 7.67 - 7.56 (m, 2H), 7.21 (s, 1H), 7.14 - 7.09 (m, 2H), 7.03 (d, J = 8.0 Hz, 1H) , 6.78 (s, 1H), 6.41 (d, J = 16.0 Hz, 1H), 6.19 (d, J = 9.5 Hz, 1H), 5.20 (app.t, J = 4.7 Hz, 1H), 3.82 (s, 3H), 3.26 (ddd, J = 17.3, 4.8, 1.2 Hz, 1H), 2.95 (dd, J = 17.3, 4.5 Hz, 1H), 2.27 (s, 3H), 1.43 (s, 3H), 1.38 (s) , 3H).

(S)-8,8- dimethyl -2- oxo -7,8- dihydro - 2H, 6H - pyrano[3,2-g]chromen -7- yl (E)-3-(3,4-difluorophenyl)acrylate (Compound 18; SNU -C18)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.62 - 7.53 (m, 2H), 7.32 (ddd, J = 11.1, 7.6, 2.2 Hz, 1H), 7.24 - 7.11 (m, 3H), 6.83 (s, 1H), 6.33 (d, J = 16.0, 1H), 6.24 (d, J = 9.4 Hz, 1H), 5.19 (app.t, J = 4.7 Hz, 1H), 3.25 (ddd, J = 17.4, 4.8, 1.1 Hz, 1H), 2.93 (dd, J = 17.4, 4.5 Hz, 1H), 1.43 (s, 3H), 1.39 (s, 3H).

( S, E )-7-((3-(3- methoxy -4- nitrophenyl ) allyl )oxy)-8,8- dimethyl -7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one (Compound 19; PRG -A-04)

¹ H NMR (CDCl ₃ , 400 MHz): δ ppm 7.85 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 9.5 Hz, 1H), 7.16 (s, 1H), 7.05 - 6.97 (m, 2H) ), 6.78 (s, 1H), 6.60 (app.dt, J = 15.9, 1.7 Hz, 1H), 6.40 (app.dt, J = 15.9, 5.5 Hz, 1H), 6.22 (d, J = 9.4 Hz, 1H), 4.39 (ddd, J = 13.6, 5.4, 1.7 Hz, 1H), 4.24 (ddd, J = 13.4, 5.5, 1.6 Hz, 1H), 3.97 (s, 3H), 3.59 (dd, J = 7.1, 4.9 Hz, 1H), 3.11 (dd, J = 16.7, 4.9 Hz, 1H), 2.87 (dd, J = 16.7, 7.2 Hz, 1H), 1.43 (s, 3H), 1.38 (s, 3H).

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

A compound represented by the following formula (1), a hydrate thereof, or a salt thereof:
[Formula 1]

In Formula 1,

When this single bond, X is CH, R ¹ and R ² are each different and selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy,

When this double bond, X is N, R ¹ and R ² may each be the same or different, and may be hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4) selected from alkylcarboxy. The method of claim 1, wherein the compound is

When this single bond, X is CH, R ¹ and R ² are each different and selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy,

In the case of this double bond, X is N, and R ¹ and R ² are each hydrogen. A compound, a hydrate thereof, or a salt thereof. The compound of claim 1, wherein the compound is (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3 -(4-hydroxy-3-methoxyphenyl)propanoate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen -7-yl 3-(4-hydroxy-3-methoxyphenyl)propanoate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2- g]chromen-7-yl (E)-3-(pyridin-4-yl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[ 3,2-g]chromen-7-yl (E)-3-(pyridin-4-yl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H, 6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl)propanoate [(S)-8,8-dimethyl-2-oxo-7 ,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl)propanoate] a compound selected from the group consisting of, its hydrate or its salt. A pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis comprising a compound represented by the following formula (2), a hydrate thereof, or a salt thereof:
[Formula 2]

In Formula 2,

is a single bond or a double bond,
n is an integer from 0 to 1,
X is CH or N;
R ¹ and R ² may each be the same or different, and are selected from hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4)alkylcarboxy. 5. The method of claim 4, wherein the compound is

When this is a single bond, n is 1, X is CH, R ¹ and R ² may each be the same or different, and are selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy. A pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis. 5. The method of claim 4, wherein the compound is

When this double bond, n is an integer of 0 to 1, X is CH or N, R ¹ and R ² may be the same or different, respectively, hydrogen, (C1-C4) alkoxy, hydroxy, halo, nitro or (C1-C4) A pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis, characterized in that it is selected from alkylcarboxy. 5. The method of claim 4, wherein the compound is (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl ( E)-3-(4-hydroxy-3-methoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2- g]chromen-7-yl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate], (S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl )oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-( 4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one], (S)-8, 8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-hydroxy-3-methoxyphenyl)propano ate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-hydroxy-3-methoxyphenyl) propanoate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-( 3,4-dimethoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(3,4-dimethoxyphenyl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen- 7-yl 3-(3,4-dimethoxyphenyl)propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g] chromen-7-yl 3-(3,4-dimethoxyphenyl)propanoate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g ]chromen-7-day (E) -3-(pyridin-4-yl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(pyridin-4-yl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g] Chromen-7-yl (E)-3-(3-hydroxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3, 2-g]chromen-7-yl (E)-3-(3-hydroxyphenyl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano [3,2-g]chromen-7-yl (E)-3-(4-fluorophenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H ,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(4-fluorophenyl)acrylate], (S,E)-7-((3-(4-fluorophenyl)allyl )oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-( 4-fluorophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one], (S)-8,8-dimethyl- 2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(3-acetoxyphenyl)acrylate [(S)- 8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(3-acetoxyphenyl)acrylate], (S) -8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl ) propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3 -methoxyphenyl)propanoate], (S) -8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(4-acetoxy-3 -Methoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(4-acetoxy-3-methoxyphenyl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromene- 7-yl (E)-3-(3,4-difluorophenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3, 2-g]chromen-7-yl (E)-3-(3,4-difluorophenyl)acrylate] and (S,E)-7-((3-(3-methoxy-4-nitrophenyl)allyl) Oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-(3) -methoxy-4-nitrophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one] A pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis. The amyotrophic lateral sclerosis according to any one of claims 4 to 7, wherein the pharmaceutical composition inhibits aggregation and misfolding of superoxide dismutase 1 (SOD1). A pharmaceutical composition for prophylaxis or treatment. A health functional food composition for preventing or improving amyotrophic lateral sclerosis comprising a compound represented by the following formula (2), a hydrate thereof, or a salt thereof:
[Formula 2]

In Formula 2,

is a single bond or a double bond,
n is an integer from 0 to 1,
X is CH or N;
R ¹ and R ² may each be the same or different, and are selected from hydrogen, (C1-C4)alkyl, (C1-C4)alkoxy, hydroxy, halo, nitro, cyano or (C1-C4)alkylcarboxy. 10. The method of claim 9, wherein the compound is

When this is a single bond, n is 1, X is CH, R ¹ and R ² may each be the same or different, and are selected from (C1-C4) alkoxy, hydroxy or (C1-C4) alkylcarboxy. A health functional food composition for preventing or improving amyotrophic lateral sclerosis. 10. The method of claim 9, wherein the compound is

When this double bond, n is an integer of 0 to 1, X is CH or N, R ¹ and R ² may be the same or different, respectively, hydrogen, (C1-C4) alkoxy, hydroxy, halo, nitro or (C1-C4) A health functional food composition for preventing or improving amyotrophic lateral sclerosis, characterized in that it is selected from alkylcarboxy. 10. The method of claim 9, wherein the compound is (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl ( E)-3-(4-hydroxy-3-methoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2- g]chromen-7-yl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate], (S,E)-7-((3-(4-hydroxy-3-methoxyphenyl)allyl )oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-( 4-hydroxy-3-methoxyphenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one], (S)-8, 8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-hydroxy-3-methoxyphenyl)propano ate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-hydroxy-3-methoxyphenyl) propanoate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-( 3,4-dimethoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E )-3-(3,4-dimethoxyphenyl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen- 7-yl 3-(3,4-dimethoxyphenyl)propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g] chromen-7-yl 3-(3,4-dimethoxyphenyl)propanoate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g ]chromen-7-day (E) -3-(pyridin-4-yl)acrylate[(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(pyridin-4-yl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g] Chromen-7-yl (E)-3-(3-hydroxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3, 2-g]chromen-7-yl (E)-3-(3-hydroxyphenyl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano [3,2-g]chromen-7-yl (E)-3-(4-fluorophenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H ,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(4-fluorophenyl)acrylate], (S,E)-7-((3-(4-fluorophenyl)allyl )oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-( 4-fluorophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one], (S)-8,8-dimethyl- 2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(3-acetoxyphenyl)acrylate [(S)- 8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(3-acetoxyphenyl)acrylate], (S) -8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3-methoxyphenyl ) propanoate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl 3-(4-acetoxy-3 -methoxyphenyl)propanoate], (S) -8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3-(4-acetoxy-3 -Methoxyphenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-7-yl (E)-3 -(4-acetoxy-3-methoxyphenyl)acrylate], (S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3,2-g]chromene- 7-yl (E)-3-(3,4-difluorophenyl)acrylate [(S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano[3, 2-g]chromen-7-yl (E)-3-(3,4-difluorophenyl)acrylate] and (S,E)-7-((3-(3-methoxy-4-nitrophenyl)allyl) Oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one[(S,E)-7-((3-(3) -methoxy-4-nitrophenyl)allyl)oxy)-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one] A health functional food composition for preventing or improving amyotrophic lateral sclerosis.

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