KR20210131912A - Novel alpha-synuclein radioligands and diagnostic composition of neurodegenerative diseases comprising the same - Google Patents

Abstract

The present invention relates to a novel alpha-synuclein radioligand and a composition for diagnosing neurodegenerative diseases comprising the same. The novel alpha-synuclein radioligand compound of the present invention has excellent selectivity and affinity for alpha-synuclein, thereby having an effect of being able to be usefully used for early detection of degenerative brain diseases such as Parkinson's disease.

Description

Novel alpha-synuclein radioligands and compositions for diagnosing neurodegenerative diseases comprising the same

It relates to a novel alpha-synuclein radioligand and a composition for diagnosing neurodegenerative diseases comprising the same.

Recently, as the aging phenomenon due to the increase in life expectancy has emerged as a social problem, the importance of research on degenerative brain disease is being emphasized along with the increase in the elderly population. Degenerative brain disease is known to be caused by the death of nerve cells due to neurodegeneration due to aging and genetic or environmental factors, resulting in protein aggregation, and impairments in motor function, memory, and cognition due to degenerative nerves. Degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and Lou Gehrig's disease in consideration of the main symptoms and brain regions affected.

Positron emission tomography (PET) is a very useful method for diagnosing various brain diseases non-invasively. In particular, in the case of degenerative brain diseases that lack effective therapeutic agents, the development of PET radiopharmaceuticals to increase the efficacy of initial treatment through early diagnosis is being actively carried out. In the case of Alzheimer's disease, a typical degenerative brain disease, three radiopharmaceuticals for PET imaging of beta-amyloid plaques, known as causative substances, have been approved by the Ministry of Food and Drug Safety and are being actively used in clinical practice. However, the development of radiopharmaceuticals for the diagnosis of degenerative brain diseases in which the Lewy body, including Parkinson's disease, is an important causative material is currently in its infancy, and the development of new compounds is urgently required.

Lewy bodies are abnormal protein aggregates that develop in the nerve cells of patients with Parkinson's disease, Lewy body dementia, and other disorders. Lewy bodies can be judged under a microscope of brain tissue and are observed as spherical materials that are replaced at the locations of other components. There are two morphological types of Lewy bodies: typical brainstem Lewy bodies and cortical Lewy bodies. Typical Lewy bodies are eosinophilic cytoplasmic inclusions consisting of a dense core surrounded by bundles of radioactive nerve fibers, the major structural component of alpha synuclein. Conversely, cortical Lewy bodies have characteristics distinguishing them from Lewy body dementia, but mainly include Peek's disease and cortical base degeneration, as well as enlarged neurons in Tau disease patients.

Alpha-synuclein, one of the major constituents of Lewy bodies, is a type of protein abundant in the human brain. mainly found Presynaptic terminals release chemical messengers called neurotransmitters from compartments known as synaptic vesicles, and the release of these substances relays signals between nerve cells, allowing the brain to function normally. Within these structures, alpha-synuclein clusters the synaptic ER, plays a role in maintaining the supply of synaptic vesicles at the presynaptic terminal, and regulates the release of dopamine, a neurotransmitter that controls the initiation and cessation of voluntary and involuntary movements. plays a role

Parkinson's disease is characterized by progressive loss of dopaminergic neurons, the presence of proteinaceous neurons composed of Lewy bodies, neurites, and alpha-synuclein aggregates. Alpha-synuclein fibrous aggregates are a major component of Lewy bodies and Lewy neurites. A recent study suggests that the profibrotic oligomer of alpha-synuclein may be a key contributor to the progression of Parkinson's disease.

Various radioligands acting on alpha-synuclein have been reported, and the representative limitations of the currently reported alpha-synuclein ligands are a lack of selectivity and high fat solubility.

First, the abnormal protein aggregates that may exist in the brain tissue of degenerative brain disease patients are typically beta-amyloid plaques, neurofibrillary tangles, and three types of lewy bodies. there is Each of these protein aggregates has a beta sheet structure, so that one compound can bind to several types of protein aggregates. Therefore, there is a need to develop a ligand that selectively acts only on Lewy bodies for accurate diagnosis through the search for causative substances.

Next, although adequate fat solubility is essential for passage of the blood-brain barrier (BBB), high fat solubility is a hindrance in terms of quality of PET images. Therefore, proper fat solubility of the alpha-synuclein ligand is required to improve the degree of passage of the blood vessel-brain barrier and the quality of PET images.

In a previous paper (J. Med. Chem. 2015, 58, 6002-6017), 3-(benzylidine) indolin-2-one derivatives, or pharmaceutically acceptable salts thereof, as well as methods and stereoscopic methods for their preparation It is disclosed that the isomer has a diagnostic effect on degenerative brain diseases such as Parkinson's disease.

On the other hand, the present invention has a diagnostic effect on degenerative brain diseases such as Parkinson's disease by binding a specific compound to α-synuclein. There is a definite difference in the molecular structure in that one double bond of -diene is transformed into an aromatic heterocyclic compound, or a substituent bonded to the nitrogen of indolinone is replaced with another substituent. It is different from the contents of the previous paper (J. Med. Chem. 2015, 58, 6002-6017).

In the field of diagnostic research using radiopharmaceuticals, the study of radioligands for imaging of alpha-synuclein is one of the most challenging topics. Although early detection of degenerative brain disease is still impractical, there are several promising therapeutics for the treatment of Alzheimer's disease that target beta-amyloid, Tau, and neuronal inflammation, but there is no treatment for Parkinson's disease. the current situation.

Here, the development of an alpha-synuclein ligand is currently underway to utilize the Lewy body as a characteristic of Parkinson's disease and to detect the Lewy body without surgical operation. The development of a novel alpha-synuclein ligand having

An object of one aspect of the present invention is to provide an indolinone-based compound having good reactivity with alpha-synuclein and good selectivity for alpha-synuclein for diagnosis and early detection of degenerative brain diseases such as Parkinson's disease.

In order to achieve the above object,

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

[Formula 1]

In Formula 1,

X is C, O, or S,

when X is -C-, -C- is _{joined together with R 1} to form phenyl;

R ₁ is =S, or

when X is -C-, R ₁ is joined together with -C- to form phenyl;

R ₂ is an unsubstituted or substituted C _2-10 straight or branched chain alkenyl, or an unsubstituted or substituted 5 to 6 membered hetero atom including one or more heteroatoms selected from the group consisting of N, O and S aryl,

wherein said substituted alkenyl and substituted heteroaryl are substituted with unsubstituted phenyl or phenyl substituted with one or more substituents selected from the group consisting of cyano and nitro; and

R ₃ is unsubstituted or substituted _{phenylC 1-3} alkyl, unsubstituted or substituted phenoxycarbonyl or unsubstituted or substituted phenylsulfonyl,

The substituted phenylalkyl, substituted phenoxycarbonyl and substituted phenylsulfonyl are halogen, hydroxy, C _1-5 straight or branched chain alkyl, unsubstituted or substituted C _1-5 straight chain with one or more halogens. or one or more substituents selected from the group consisting of branched chain alkoxy.

In addition, the present invention provides a composition for diagnosing neurodegenerative diseases comprising the compound of Formula 1, a stereoisomer or a pharmaceutically acceptable salt thereof, as an active ingredient.

In addition, the present invention comprises the steps of administering the diagnostic composition to a subject; and

Including; measuring a signal for alpha-synuclein;

Provided is a method for providing information for diagnosing neurodegenerative diseases.

The novel alpha-synuclein radioligand compound of the present invention has excellent selectivity and affinity for alpha-synuclein, and thus can be usefully used for early detection of degenerative brain diseases such as Parkinson's disease.

1 is a graph showing _{the binding affinity (IC 50} ) of a comparative compound for alpha-synuclein fibers.
Figure 2 is a graph showing the results of analysis of the binding affinity of the compounds of the present invention in a competitive condition with Thio-T.

Hereinafter, the present invention will be described in detail.

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

[Formula 1]

In Formula 1,

X is C, O, or S,

when X is -C-, -C- is _{joined together with R 1} to form phenyl;

R ₁ is =S, or

when X is -C-, R ₁ is joined together with -C- to form phenyl;

R ₂ is an unsubstituted or substituted C _2-10 straight or branched chain alkenyl, or an unsubstituted or substituted 5 to 6 membered hetero atom including one or more heteroatoms selected from the group consisting of N, O and S aryl,

wherein said substituted alkenyl and substituted heteroaryl are substituted with unsubstituted phenyl or phenyl substituted with one or more substituents selected from the group consisting of cyano and nitro; and

R ₃ is unsubstituted or substituted _{phenylC 1-3} alkyl, unsubstituted or substituted phenoxycarbonyl or unsubstituted or substituted phenylsulfonyl,

The substituted phenylalkyl, substituted phenoxycarbonyl and substituted phenylsulfonyl are halogen, hydroxy, C _1-5 straight or branched chain alkyl, unsubstituted or substituted C _1-5 straight chain with one or more halogens. or one or more substituents selected from the group consisting of branched chain alkoxy.

In another aspect,

Wherein R ₂ is unsubstituted or substituted C _2-4 straight or branched chain alkenyl, or unsubstituted or substituted 5 to 6 atoms including one or more heteroatoms selected from the group consisting of N, O and S is a heteroaryl of

wherein said substituted alkenyl and substituted heteroaryl are substituted with unsubstituted phenyl, or phenyl substituted with cyano or nitro; and

wherein R ₃ is unsubstituted or substituted benzyl, phenoxycarbonyl or phenylsulfonyl,

The substituted benzyl is substituted with one or more substituents selected from the group consisting _{of halogen, unsubstituted or C 1-5 straight or branched chain alkoxy substituted with one or more halogens.}

In another aspect,

wherein X is O or S; and

Wherein R ₁ is =S.

In another aspect,

wherein X is -C-, and R ₁ are joined to form phenyl; and

R ₂ is a substituted 5-6 membered heteroaryl containing one or more heteroatoms selected from the group consisting of N, O and S, wherein the substituted heteroaryl is substituted with phenyl.

In another aspect,

wherein X is -C-, and R ₁ are joined to form phenyl; and

R ₂ is a substituted C _2-4 straight-chain or branched alkenyl, and the substituted alkenyl is substituted with phenyl substituted with cyano or nitro.

In another aspect,

The compound is any one compound selected from the group consisting of the following compounds, a stereoisomer or a pharmaceutically acceptable salt thereof:

(1) (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxothiazolidine -4-one;

(2) (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxoxazolidine -4-one;

(3) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylfuran-2-yl)methylene)indolin-2-one;

(4) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one;

(5) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one;

(6) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((4-phenylthiophen-2-yl)methylene)indolin-2-one;

(7) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one;

(8) 4-((E)-3-((Z)-1-(4-(2-fluoroethoxy)benzyl)-2-oxoindolin-3-ylidene)prop-1-ene -1-yl)benzonitrile;

(9) (Z)-1-(2-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one;

(10) (Z)-1-(3-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one;

(11) (Z)-1-(4-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one;

(12) phenyl (Z)-3-((E)-3-(4-nitrophenyl)allylidene)-2-oxoindoline-1-carboxylate; and

(13) 3-(3-(4-nitrophenyl)allylidene)-1-(phenylsulfonyl)indolin-2-one.

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. get it from Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Late, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, a precipitate formed by dissolving the derivative of Formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and adding an organic or inorganic acid It can be prepared by filtration and drying, or by distilling the solvent and excess acid under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, bases can be used to prepare pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

Furthermore, the present invention includes not only the compound represented by Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers, hydrates, and the like, which can be prepared therefrom.

The term “hydrate” refers to a compound of the invention comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolec μLar forces. or salts thereof. The hydrate of the compound represented by Formula 1 of the present invention may include a stoichiometric or non-stoichiometric amount of water that is bound by non-covalent intermolecular forces. The hydrate may contain 1 equivalent or more, preferably, 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by Formula 1 of the present invention, an isomer thereof, or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

The term “solvate” means a compound of the invention or a salt thereof which contains either a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor include solvents that are volatile, non-toxic, and/or suitable for administration to humans.

The term “isomer” refers to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but differs structurally or sterically. Such isomers include structural isomers such as tautomers, stereoisomers such as R or S isomers having an asymmetric carbon center, geometric isomers (trans, cis), and optical isomers (enantiomers). All these isomers and mixtures thereof are also included within the scope of the present invention.

Another aspect of the present invention provides a composition for diagnosing neurodegenerative diseases comprising the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the neurodegenerative disease is Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (DLB), Alzheimer's disease Lewy body variant (LBVAD), multiple system atrophy (MSA), Alzheimer's disease, Pick's disease or Tau disease, preferably Parkinson's disease.

Since the compound of the present invention has excellent binding affinity to alpha-synuclein, it is useful for diagnosing neurodegenerative diseases, particularly for diagnosing diseases related to Lewy bodies including alpha-synuclein or alpha-synuclein aggregates. is used sparingly

In one embodiment of the present invention, the compound of the present invention exhibits excellent binding affinity to alpha-synuclein, and in particular, as it exhibits superior binding affinity compared to the conventional comparative compound, it can be usefully used for diagnosing neurodegenerative diseases. It was confirmed that there is (see FIG. 2).

Another aspect of the present invention comprises the steps of administering the composition for diagnosis of claim 7 to a subject; and measuring a signal for alpha-synuclein; provides a method for providing information for diagnosing neurodegenerative diseases, including.

Since the compound of the present invention has excellent binding affinity to alpha-synuclein, it is useful for diagnosing neurodegenerative diseases, particularly for diagnosing diseases related to Lewy bodies including alpha-synuclein or alpha-synuclein aggregates. is used sparingly

The signal measurement step is to measure and image the signal through a positron emission tomography (PET) device.

The signal is a signal due to the binding of the compound of the present invention and alpha-synuclein or a Lewy body including an aggregate of alpha-synuclein, and through this, information on the diagnosis of a neurodegenerative disease is provided.

The neurodegenerative disease is Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (DLB), Lewy body variant of Alzheimer's disease (LBVAD), multiple system atrophy (MSA), Alzheimer's disease, Pick's disease or tau disease, preferably Parkinson's disease.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Example 1> (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxotia Jolidin-4-one

Step 1: Preparation of 4-(2-fluoroethoxy)benzaldehyde

4-Hydroxybenzaldehyde ( 1 ), 1-bromo-2-fluoroethane (2.0 eq), K ₂ CO ₃ (2.2 eq) was added to DMF and stirred at 100° C. for 5 hours. Thereafter, the mixture was concentrated in vacuo, and the resulting solid was _{dissolved in CH 2} Cl ₂ , washed with water, and then _{water was removed using MgSO 4} and concentrated in vacuo to obtain the title compound.

Step 2: Preparation of (Z)-4-(2-fluoroethoxy)benzaldehyde oxime

Put the compound of step 1 in 0.7 ml of MeOH, dissolve hydroxylamine hydrochloride (1.2 eq) and sodium hydroxide (1.2 eq) in 0.7 ml of water, then add to reflux and stir for 4 hours. Then, cold water is added, and the product is _{extracted with CH 2} Cl ₂ , washed with water, saturated NaHCO ₃ , and NaCl, _{water is removed using MgSO 4} , and concentrated in vacuo to obtain the title compound as a white solid.

Step 3: Preparation of (4-(2-fluoroethoxy)phenyl)methanamine

Lithium aluminum hydride (2.0 ep) dissolved in 1.4 ml of THF is added dropwise to the compound of step 2 for 5 minutes, followed by stirring at room temperature for 3 hours and reflux for 1 hour. Then, 40% aqueous NaOH was added, extracted with _{CH 2} Cl _{2 , water was removed using MgSO 4} , and concentrated in vacuo to obtain the title compound as a white solid.

Step 4: Preparation of 1-(2-fluoroethoxy)-4-(isothiocyanatomethyl)benzene

Thiophosgene (1.1 eq) was added dropwise at 0 °C to THF in which the compound prepared in step 3 and triethylamine (3.0 eq) were dissolved, stirred for 30 minutes, and then stirred at room temperature for 30 minutes. Then, water (5ml) and diethyl ether (10ml) were added, the organic layer was extracted, washed with brine, _{water was removed using NaSO 4} , and then concentrated at 40°C. Thereafter, it is separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1) to obtain the title compound.

Step 5: Preparation of 3-(4-(2-fluoroethoxy)benzyl)-2-thioxothiazolidin-4-one

Dissolve the compound of step 4 in 1.5ml of CH ₂ Cl ₂ , add methyl thioglycolate (1.0 eq) and triethylamine (1.0 eq), and stir at room temperature for 2 hours. Then, 0.7ml of water is added, and the organic layer is washed twice _{with water, water is removed using NaSO 4} , and separated by column chromatography (hexane: CH ₂ Cl ₂ = 2: 1) to obtain the title compound.

Step 6: (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxothiazolidine Preparation of -4-one

The compound of step 5, 4-nitro cinnamaldehyde ( 7 , 1.0 eq), and piperidine (0.8 eq) were dissolved in 1.2 ml of EtOH and stirred at reflux for 1 hour. Then water is added and acetic anhydride is added until pH 3-4 to give the title compound as a brown solid.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.25 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 11.4 Hz, 1H), 7.51 (d, J = 15.0 Hz, 1H), 7.37 (dd, J = 15.2, 11.5 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 5.15 (s, 2H), 4.78 - 4.73 (m, 1H), 4.70 - 4.66 (m, 1H), 4.25 - 4.20 (m, 1H), 4.19 - 4.16 (m, 1H).

<Example 2> (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxoxa Jolidin-4-one

Step 1: Preparation of 3-(4-(2-fluoroethoxy)benzyl)-2-thioxoxazolidin-4-one

After dissolving 4-fluorophenyl isothiocyanate in 1.5 ml of toluene, methyl glycolate (2.0 eq), N,N-diisopropylethylamine (2.0 eq), lithium perchlorate (2.0 eq) were added thereto, and refluxed 18 time to stir. After _{extraction with CH 2} Cl ₂ , washed with water, and separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1), 3-(4-(2-fluoroethoxy)benzyl)- 2-thioxoxazolidin-4-one ( 8 ) is obtained.

Step 2: (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxoxazolidine Preparation of -4-one

3-(4-(2-fluoroethoxy)benzyl)-2-thioxoxazolidin-4-one ( 8 ), 4-nitro cinnamaldehyde ( 7 , 1.0 eq), piperidine (2.8 eq) was dissolved in 0.3 ml of EtOH and stirred at reflux for 2 hours. Then, water was added, and acetic anhydride was added until the pH was 3-4, and (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3- (4-nitrophenyl)allylidene)-2-thioxoxazolidin-4-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.23 (d, J = 8.9 Hz, 2H), 7.97 (d, J = 8.9 Hz, 2H), 7.52 - 7.42 (m, 2H), 7.34 - 7.29 ( m, 2H), 6.94 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 10.5 Hz, 1H), 4.90 (d, J = 7.8 Hz, 2H), 4.78 - 4.75 (m, 1H), 4.70 - 4.66 (m, 1H), 4.25 - 4.22 (m, 1H), 4.20 - 4.17 (m, 1H).

<Example 3> (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylfuran-2-yl)methylene)indolin-2-one

Step 1: Preparation of (Z)-3-((5-phenylfuran-2-yl)methylene)indolin-2-one

Dissolve 2-oxindole ( 9 ) and 5-phenyl-2-furaaldehyde ( 10 , 1.0 eq) in 5.6 ml of acetic acid and 0.11 ml of 37% HCl, followed by stirring at reflux for 3 hours. After that, 50 ml of water is added at room temperature and the resulting solid is filtered. Then column chromatography (CH ₂ Cl ₂ : EtOAc = 100: 5, v/v) to give (Z)-3-((5-phenylfuran-2-yl)methylene)indolin-2-one ( 11 ).

Step 2: Preparation of (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylfuran-2-yl)methylene)indolin-2-one

(Z)-3-((5-phenylfuran-2-yl)methylene) indolin-2-one (11 ) was dissolved in 1.4ml DMF, and then 60% NaH (1.5 eq) was added at 0°C. After 15 minutes, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene ( 12 , 1.2 eq) was added and stirred for 24 hours. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using NaSO 4} , and column chromatography (hexane: CH ₂ Cl ₂ : EtOAc) = 10: 10: 3, v/v/v) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylfuran-2-yl)methylene ) to obtain an indolin-2-one.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.55 (dd, J = 7.8, 1.2 Hz, 1H), 7.94 (dd, J = 8.5, 1.2 Hz, 2H), 7.61 - 7.55 (m, 2H), 7.51 (s, 1H), 7.49 (d, J = 3.7 Hz, 1H), 7.48 - 7.44 (m, 1H), 7.38 (d, J = 3.7 Hz, 1H), 7.32 - 7.27 (m, 3H), 7.18 (td, J = 7.6, 1.1 Hz, 1H), 7.02 (d, J = 7.7 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 4.94 (s, 2H), 4.77 - 4.72 (m, 1H), 4.69 - 4.63 (m, 1H), 4.23 - 4.18 (m, 1H), 4.18 - 4.13 (m, 1H).

<Example 4> (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one

Step 1: Preparation of (Z)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one

Dissolve 2-oxindole ( 9 ), 5-phenylthiophene-2-carboxaldehyde ( 13 , 1.0 eq) in 1.9ml acetic acid and 0.04ml 37% HCl and stir at reflux for 3 hours. After that, 19 ml of water is added at room temperature and the resulting solid is filtered. Then column chromatography (CH ₂ Cl ₂ : EtOAc = 100:5, v/v) to give (Z)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one ( 14 ) as an orange solid.

Step 2: Preparation of (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one

(Z)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one ( 14 ) was dissolved in 0.8ml DMF, and then 60% NaH (1.5 eq) was added at 0°C. After 15 minutes, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene ( 12 , 1.2 eq) was added and stirred for 1 hour. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using MgSO 4} , and column chromatography (hexane: CH ₂ Cl ₂ : EtOAc) = 10: 10: 1, v/v/v) as an orange solid (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylthiophene-2) -yl)methylene)indolin-2-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.20 (d, J = 0.7 Hz, 1H), 7.94 (dd, J = 3.9, 0.7 Hz, 1H), 7.86 (dd, J = 8.3, 1.2 Hz, 1H), 7.80 (dd, J = 8.3, 1.3 Hz, 2H), 7.78 - 7.73 (m, 1H), 7.69 (d, J = 3.9 Hz, 1H), 7.49 (dd, J = 8.4, 7.1 Hz, 3H) ), 7.44 - 7.37 (m, 1H), 7.30 (d, J = 8.7 Hz, 3H), 7.22 (td, J = 7.7, 1.2 Hz, 1H), 7.05 (td, J = 7.5, 1.0 Hz, 1H) , 7.00 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 8.8 Hz, 3H), 4.95 (d, J = 9.6 Hz, 3H), 4.76 - 4.72 (m, 1H), 4.69 - 4.64 ( m, 1H), 4.24 - 4.18 (m, 1H), 4.18 - 4.13 (m, 1H).

<Example 5> (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one

Step 1: Preparation of 5-phenyl-2-pyridine-carboxaldehyde

5-Bromo-2-pyridine carboxaldehyde ( 17 ), phenylboronic acid ( 18 , 1.2 eq), K ₂ CO ₃ (3.0 eq), Pd(PPh ₃ ) ₄ (0.04 eq) were prepared in two parts under argon gas. Place in a round bottom flask and degas. 10ml each of dioxane and water was added to the flask using a syringe and stirred at 100°C for 17 hours. After that, the mixture was cooled to room temperature, extracted with EtOAc, and water was removed using _{MgSO 4 .} After separation by column chromatography (10% EtOAc in hexane) to obtain 5-phenyl-2-pyridine-carboxaldehyde (15) as a pale orange solid

Step 2: Preparation of (Z)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one

2-oxindole ( 9 ), 5-phenyl-2-pyridine-carboxaldehyde ( 15 , 1.0 eq) was dissolved in 1.3ml acetic acid and 0.03ml 37% HCl and stirred at reflux for 3 hours. Then, add 13 ml of water at room temperature and filter the generated solid. Then column chromatography (CH ₂ Cl ₂ : EtOAc = 100: 10, v/v) to give (Z)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one ( 16 ) as an orange solid.

Step 3: Preparation of (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one

(Z)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one ( 16 ) was dissolved in 0.7ml DMF, and then 60% NaH (1.5 eq) was added at 0°C. After 15 minutes, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene ( 12 , 1.2 eq) was added and stirred for 30 minutes. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using MgSO 4} , and column chromatography (hexane: CH ₂ Cl ₂ : EtOAc) = 10: 10: 2, v/v/v) as an orange solid (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylpyridine-2- 1) methylene) indolin-2-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 9.27 (d, J = 2.4 Hz, 1H), 9.19 (dd, J = 7.7, 1.2 Hz, 1H), 8.28 (dd, J = 8.1, 2.5 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.89 (dd, J = 7.1, 1.6 Hz, 2H), 7.79 (s, 1H), 7.55 (t, J = 7.6 Hz, 2H), 7.48 ( td, J = 7.2, 1.4 Hz, 1H), 7.31 (d, J = 8.7 Hz, 3H), 7.06 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.93 ( d, J = 8.5 Hz, 2H), 4.95 (s, 2H), 4.76 - 4.73 (m, 1H), 4.68 - 4.65 (m, 1H), 4.23 - 4.19 (m, 1H), 4.18 - 4.14 (m, 1H).

<Example 6> (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((4-phenylthiophen-2-yl)methylene)indolin-2-one

(Z)-3-((4-phenylthiophen-2-yl)methylene)indolin-2-one ( 20 ) was dissolved in 0.6ml DMF, and then 60% NaH (1.5 eq) was added at 0°C. After 15 minutes, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene ( 12 , 1.2 eq) was added and stirred for 1 hour. Thereafter, extraction was performed using EtOAc, washed with water and brine, _{water was removed using MgSO 4} , and separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1) as a yellow solid (Z)-1- (4-(2-fluoroethoxy)benzyl)-3-((4-phenylthiophen-2-yl)methylene)indolin-2-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.42 (d, J = 1.6 Hz, 1H), 8.33 (d, J = 14.4 Hz, 1H), 8.28 (s, 1H), 8.24 (s, 1H) , 7.81 (dd, J = 8.3, 1.2 Hz, 1H), 7.79 - 7.73 (m, 2H), 7.47 (t, J = 7.7 Hz, 2H), 7.35 (dq, J = 8.4, 7.4 Hz, 1H), 7.30 (d, J = 8.9 Hz, 2H), 7.09 - 7.03 (m, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 4.95 (d, J) = 8.6 Hz, 2H), 4.77 - 4.71 (m, 1H), 4.69 - 4.64 (m, 1H), 4.24 - 4.18 (m, 1H), 4.18 - 4.13 (m, 1H).

<Example 7> (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one

Step 1: Preparation of 6-phenyl-2-pyridine-carboxaldehyde

6-Bromo-2-pyridine carboxaldehyde ( 23 ), phenylboronic acid ( 18 , 1.2 eq), K ₂ CO ₃ (3.0 eq), Pd(PPh ₃ ) ₄ (0.04 eq) were prepared in 2 parts under argon gas. Place in a round bottom flask and degas. 10ml each of dioxane and water was added to the flask using a syringe and stirred at 100°C for 17 hours. After that, the mixture was cooled to room temperature, extracted with EtOAc, and water was removed using _{MgSO 4 .} After separation by column chromatography (5% EtOAc in hexane), 6-phenyl-2-pyridine-carboxaldehyde ( 21 ) as a pale yellow solid is obtained.

Step 2: Preparation of (Z)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one

2-oxindole ( 9 ), 6-phenyl-2-pyridine-carboxaldehyde ( 21 , 1.0 eq) was dissolved in 1.3ml acetic acid and 0.03ml 37% HCl and stirred at reflux for 3 hours. After basifying with 5 ml of 10% NaOH, 13 ml of water is added and the resulting solid is filtered. Then column chromatography (CH ₂ Cl ₂ : EtOAc = 100: 3, v/v) to give (Z)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one ( 22 ) as a yellow solid.

Step 3: Preparation of (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one

(Z)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one ( 22 ) was dissolved in 0.6ml DMF, and then 60% NaH (1.5 eq) was added at 0°C. After 15 minutes, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene ( 12 , 1.2 eq) dissolved in 0.1ml DMF was added and stirred for 1 hour. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using MgSO 4} , and column chromatography (hexane: CH ₂ Cl ₂ : EtOAc) = 10: 10: 1, v/v/v) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((6-phenylpyridin-2-yl)methylene ) to obtain an indolin-2-one.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.95 (dd, J = 7.7, 1.3 Hz, 1H), 8.17 - 8.10 (m, 2H), 8.10 - 8.05 (m, 1H), 8.03 (dd, J = 8.0, 1.1 Hz, 1H), 7.90 (dd, J = 7.7, 1.1 Hz, 1H), 7.79 (s, 1H), 7.57 (t, J = 7.3 Hz, 2H), 7.54 - 7.50 (m, 1H) , 7.31 (d, J = 8.7 Hz, 2H), 7.28 (td, J = 7.7, 1.3 Hz, 1H), 7.02 - 6.98 (m, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.86 ( td, J = 7.6, 1.1 Hz, 1H), 4.95 (s, 2H), 4.76 - 4.73 (m, 1H), 4.68 - 4.65 (m, 1H), 4.23 - 4.20 (m, 1H), 4.18 - 4.14 ( m, 1H).

<Example 8> 4-((E)-3-((Z)-1-(4-(2-fluoroethoxy)benzyl)-2-oxoindolin-3-ylidene)prop-1 -en-1-yl)benzonitrile

Step 1: Preparation of 4-cyano cinnamaldehyde

4-Cyanobenzaldehyde ( 26 ) was dissolved in 20 ml of THF, and (triphenylphosphoranylidene)acetaldehyde ( 27 , 1.08 eq) was added and stirred at 50° C. for 8 hours. Then, the mixture was concentrated in vacuo and separated by column chromatography (hexane: EtoAc = 10: 1) to obtain 4-cyano cinnamaldehyde (24) as a white solid.

Step 2: Preparation of 4-((E)-3-((Z)-2-oxoindolin-3-ylidene)prop-1-en-1-yl)benzonitrile

2-oxindole ( 9 ), 4-cyano cinnamaldehyde ( 24 , 1.0 eq) was dissolved in 6ml acetic acid and 0.11ml 37% HCl and stirred at reflux for 3 hours. After that, 50 ml of water is added, the generated solid is filtered, and recrystallized using methanol. Then column chromatography (CH ₂ Cl ₂ : EtOAc = 100: 10, v/v) to 4-((E)-3-((Z)-2-oxoindolin-3-ylidene)prop-1-en-1-yl)benzonitrile ( 25 ) is obtained.

Step 3: 4-((E)-3-((Z)-1-(4-(2-fluoroethoxy)benzyl)-2-oxoindolin-3-ylidene)prop-1-ene Preparation of -1-yl) benzonitrile

After dissolving 4-((E)-3-((Z)-2-oxoindolin-3-ylidene)prop-1-en-1-yl)benzonitrile ( 25 ) in 1.5ml DMF, 60% NaH (1.5 eq) is added at 0°C. After 15 minutes, 1-(bromomethyl)-4-(2-fluoroethoxy)benzene ( 12 , 1.2 eq) dissolved in 0.1ml DMF was added and stirred for 5 minutes. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using NaSO 4} , and column chromatography (hexane: CH ₂ Cl ₂ : EtOAc) = 10: 10: 3, v/v/v) to 4-((E)-3-((Z)-1-(4-(2-fluoroethoxy)benzyl)-2-oxoin Dolin-3-ylidene)prop-1-en-1-yl)benzonitrile is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.65 (dd, J = 15.7, 11.5 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H) , 7.70 (dd, J = 11.5, 1.0 Hz, 1H), 7.67 (d, J = 7.4 Hz, 1H), 7.33 - 7.26 (m, 3H), 7.23 (td, J = 7.7, 1.2 Hz, 1H), 7.02 (td, J = 7.5, 0.9 Hz, 1H), 6.97 (dd, J = 7.9, 0.9 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 4.89 (s, 2H), 4.76 - 4.73 (m, 1H), 4.68 - 4.64 (m, 1H), 4.22 - 4.19 (m, 1H), 4.17 - 4.14 (m, 1H).

<Example 9> (Z)-1-(2-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one

Step 1: Preparation of (Z)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one

Dissolve 2-oxindole ( 9 ) and 4-nitrocinnamaldehyde ( 7 , 1.0 eq) in 6 ml acetic acid and 0.11 ml 37% HCl, followed by stirring at reflux for 3 hours. Then, 50 ml of water was added, the resulting solid was filtered, and column chromatography (CH ₂ Cl ₂ : EtOAc = 100: 5, v/v) to give (Z)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one ( 28 ).

Step 2: Preparation of (Z)-1-(2-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one

(Z)-3-((E)-3-(4-nitrophenyl) allylidene)indolin-2-one (28 ) was dissolved in 0.7ml DMF and then 60% NaH (1.5 eq) was added at 0°C add After 15 minutes, 2-fluorobenzyl bromide (1.2 eq) was added and stirred for 1 hour. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using NaSO 4} , and separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1) (Z)-1-(2- Fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.67 (dd, J = 15.7, 11.5 Hz, 1H), 8.28 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H) , 7.74 (d, J = 11.5 Hz, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.35 (d, J = 15.8 Hz, 2H), 7.29 - 7.21 (m, 4H), 7.15 (t, J = 7.4 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.93 (d, J = 7.9 Hz, 1H), 5.02 (s, 2H).

<Example 10> (Z)-1-(3-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one

(Z)-3-((E)-3-(4-nitrophenyl) allylidene)indolin-2-one (28 ) was dissolved in 0.7ml DMF, and then 60% NaH (1.5 eq) was added at 0°C. add After 15 minutes, 3-fluorobenzyl bromide (1.2 eq) was added and stirred for 30 minutes. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using NaSO 4} , and separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1) (Z)-1-(3- Fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.68 (dd, J = 15.7, 11.5 Hz, 1H), 8.28 (d, J = 8.9 Hz, 2H), 7.86 (d, J = 8.9 Hz, 2H) , 7.74 (dd, J = 11.5, 1.1 Hz, 1H), 7.70 (dd, J = 7.6, 1.2 Hz, 1H), 7.41 - 7.32 (m, 2H), 7.25 (td, J = 7.7, 1.2 Hz, 1H) ), 7.22 - 7.16 (m, 2H), 7.13 - 7.08 (m, 1H), 7.05 (td, J = 7.6, 1.0 Hz, 1H), 6.98 (d, J = 7.7 Hz, 1H), 4.98 (s, 2H).

<Example 11> (Z)-1-(4-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one

(Z)-3-((E)-3-(4-nitrophenyl) allylidene)indolin-2-one (28 ) was dissolved in 0.7ml DMF and then 60% NaH (1.5 eq) was added at 0°C add After 15 minutes, 4-fluorobenzyl bromide (1.2 eq) was added and stirred for 30 minutes. Then, extraction was performed using EtOAc, washed with water and brine, _{water was removed using NaSO 4} , and separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1) (Z)-1-(4- Fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one is obtained.

¹ H NMR (600 MHz, DMSO- d ₆ ) δ 8.69 (dd, J = 15.8, 11.5 Hz, 1H), 8.31 - 8.26 (m, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.73 ( dd, J = 11.6, 1.0 Hz, 1H), 7.69 (dd, J = 7.7, 1.2 Hz, 1H), 7.41 (dd, J = 8.6, 5.6 Hz, 2H), 7.35 (d, J = 15.7 Hz, 1H) ), 7.25 (td, J = 7.7, 1.2 Hz, 1H), 7.17 (t, J = 8.9 Hz, 2H), 7.04 (td, J = 7.5, 0.9 Hz, 1H), 6.98 (d, J = 7.7 Hz) , 1H), 4.95 (s, 2H).

<Example 12> Phenyl (Z)-3-((E)-3-(4-nitrophenyl)allylidene)-2-oxoindoline-1-carboxylate

(Z)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one ( 28 _{) was dissolved in 1.8ml CH 2} Cl ₂ at 0° C. and phenyl chloroformate (1.5 eq), triethylamine (2.0 eq) was added and stirred for 30 minutes. After that, the organic layer was extracted with EtOAc, _{water was removed using NaSO 4} , and separated by column chromatography (hexane: CH ₂ Cl ₂ = 1:1), followed by phenyl (Z)-3-((E)-3-(4). -Nitrophenyl)allylidene)-2-oxoindoline-1-carboxylate is obtained.

1H NMR (600 MHz, DMSO-d6) δ 8.60 (dd, J = 15.6, 11.6 Hz, 1H), 8.32 (d, J = 7.8 Hz, 1H), 8.30 - 8.26 (m, 3H), 8.13 (d, J = 8.8 Hz, 1H), 8.08 - 8.02 (m, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.90 - 7.83 (m, 3H), 7.68 (d, J = 15.1 Hz, 1H), 7.56 - 7.41 (m, 7H), 7.36 (dd, J = 8.1, 1.5 Hz, 6H), 7.32 - 7.28 (m, 1H).

<Example 13> 3-(3-(4-nitrophenyl)allylidene)-1-(phenylsulfonyl)indolin-2-one

60% NaH (1.1 eq) in 0.4 ml THF is added at 0° C. under argon gas. After 10 minutes, (Z)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one ( 28 ) dissolved in 0.4ml of anhydrous THF was slowly added while maintaining 0 ℃ Benzenesulfonyl chloride (1.1 eq) dissolved in 0.1 ml of anhydrous THF was added, and the temperature was gradually lowered to room temperature, followed by stirring for 3 hours. Thereafter, _{water in which NH 4} Cl was dissolved was added to terminate the reaction. After extraction using _{CH 2} Cl _{2 , moisture was removed using MgSO 4} , and separation was performed by column chromatography (hexane: CH ₂ Cl ₂ = 1:1) to 3-(3-(4-nitrophenyl)allylidene). )-1-(phenylsulfonyl)indolin-2-one is obtained.

1H NMR (600 MHz, DMSO-d6) δ 8.37 (dd, J = 15.6, 11.6 Hz, 1H), 8.30 - 8.24 (m, 4H), 8.12 - 8.03 (m, 5H), 7.97 - 7.87 (m, 4H) ), 7.87 - 7.82 (m, 2H), 7.82 - 7.77 (m, 3H), 7.72 - 7.61 (m, 5H), 7.50 (td, J = 8.4, 8.0, 1.2 Hz, 1H), 7.47 - 7.39 (m) , 3H), 7.33 (td, J = 7.6, 1.0 Hz, 1H), 7.28 (td, J = 7.6, 1.0 Hz, 1H).

<Comparative Example> (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indol-2-one

A comparative example compound was prepared by the method disclosed in the prior literature (J. Med. Chem. 2015, 58, 6002-6017).

The chemical structures of the compounds prepared in Examples 1-13 are summarized in Table 1 below.

Example chemical structure Example chemical structure One

8

2

9

3

10

4

11

5

12

6

13

7

<Experimental Example 1> Measurement of binding affinity

<1-1> Preparation of recombinant alpha-synuclein fibers (α-Syn Fibrils)

Recombinant α-syn monomer (1 mg/mL) was incubated in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl for 14 days at 37° C. with shaking at 300 rpm. The fiber concentration was analyzed by BCA protein assay to determine the monomer concentration of the supernatant obtained by centrifuging the fiber reaction mixture at 15000 g for 20 min to separate the fiber from the monomer. The measured decrease in α-syn monomer concentration was used to analyze the fiber concentration in the 14-day fibrosis reaction mixture.

<1-2> Preparation of alpha-synuclein fibers for binding and competition analysis

The prepared fiber mixture was centrifuged at 15000 g for 20 minutes to prepare fibers for binding analysis. The supernatant was discarded and the fiber pellet resuspended in PBS buffer, 0.1% BSA to achieve the desired fiber concentration for use in the assay.

<1-3> Binding affinity of the comparative compound to alpha-synuclein fibers (IC ₅₀ ) analysis of

10 μM of α-syn fibers were incubated at 37° C. for 2 hours with a concentration gradient of Thio-T in PBS buffer, 0.1% BSA in a reaction volume of 150 μL. Non-specific binding was determined in a set of overlapping binding reactions containing the same concentration of Thio-T in the absence of fibers. Fluorescence was measured in a Biotek plate reader using a 440/30 excitation filter and a 485/20 emission filter. _{The binding affinity (IC 50} ) values of the comparative compound (Compound 1) were analyzed by fitting the data to the equation Y = Bmax × X / (X + Kd) by non-linear regression using Graphpad Prism software (version 4.0). , the results are shown in FIG. 1 .

As shown in FIG. 1 , the IC ₅₀ binding affinity value of the Comparative Example compound (Compound 1) was confirmed to be 5.118 nM.

<1-4> Binding affinity analysis with Thio-T under competitive conditions

The competition assay used 25 μM Thio-T and a fixed concentration of 10 μM α-syn fibrin, and the competition reaction was diluted with PBS buffer, 0.1% BSA, and the reaction was added in a 5 μM solution. Reactions to five inventive Example 1-5 compounds were incubated at room temperature for 8 h and fluorescence was measured with a Biotek plate reader using a 440/30 excitation filter and a 485/20 emission filter at 6, 7 and 8 h time points, respectively. measured. The data were analyzed by calculating the percent inhibition at each 3 time points (6, 7 and 8 hours) compared to the control solution of Thio-T and a-syn fibers in PBS buffer, and the results are shown in FIG. 2 . .

As can be seen in FIG. 2 , it was confirmed that all of the compounds of Examples 1-5 of the present invention compared to the compound of Comparative Example (Compound 1) had better binding affinity for α-syn fibers.

Claims (12)

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

(In Formula 1,
X is C, O, or S,
when X is -C-, -C- is _{joined together with R 1} to form phenyl;

R ₁ is =S, or
when X is -C-, R ₁ is joined together with -C- to form phenyl;

R ₂ is an unsubstituted or substituted C _2-10 straight or branched chain alkenyl, or an unsubstituted or substituted 5 to 6 membered hetero atom including one or more heteroatoms selected from the group consisting of N, O and S aryl,
wherein said substituted alkenyl and substituted heteroaryl are substituted with unsubstituted phenyl or phenyl substituted with one or more substituents selected from the group consisting of cyano and nitro; and

R ₃ is unsubstituted or substituted _{phenylC 1-3} alkyl, unsubstituted or substituted phenoxycarbonyl or unsubstituted or substituted phenylsulfonyl,
The substituted phenylalkyl, substituted phenoxycarbonyl and substituted phenylsulfonyl are halogen, hydroxy, C _1-5 straight or branched chain alkyl, unsubstituted or substituted C _1-5 straight chain with one or more halogens. or substituted with one or more substituents selected from the group consisting of branched chain alkoxy).
According to claim 1,
R ₂ is an unsubstituted or substituted C _2-4 straight or branched chain alkenyl, or an unsubstituted or substituted 5 to 6 membered group comprising at least one heteroatom selected from the group consisting of N, O and S heteroaryl;
wherein said substituted alkenyl and substituted heteroaryl are substituted with unsubstituted phenyl, or phenyl substituted with cyano or nitro; and
wherein R ₃ is unsubstituted or substituted benzyl, phenoxycarbonyl or phenylsulfonyl,
wherein the substituted benzyl is substituted with one or more substituents selected from the group consisting _{of halogen, unsubstituted or C 1-5 straight or branched chain alkoxy substituted with one or more halogens,}
A compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
According to claim 1,
wherein X is O or S; and
Wherein R ₁ Is =S A compound, a stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that.
According to claim 1,
wherein X is -C-, and R ₁ are joined to form phenyl; and
wherein R ₂ is a substituted 5-6 membered heteroaryl containing one or more heteroatoms selected from the group consisting of N, O and S, wherein the substituted heteroaryl is substituted with phenyl, a compound thereof, A stereoisomer or a pharmaceutically acceptable salt thereof.
According to claim 1,
wherein X is -C-, and R ₁ are joined to form phenyl; and
Wherein R ₂ is a substituted C _2-4 straight-chain or branched alkenyl, the substituted alkenyl is a compound, characterized in that substituted with phenyl substituted with cyano or nitro, a stereoisomer thereof, or a pharmaceutical thereof as acceptable salts.
According to claim 1,
The compound is any one compound selected from the group consisting of the following compounds, a stereoisomer or a pharmaceutically acceptable salt thereof:
(1) (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxothiazolidine -4-one;
(2) (Z)-3-(4-(2-fluoroethoxy)benzyl)-5-((E)-3-(4-nitrophenyl)allylidene)-2-thioxoxazolidine -4-one;
(3) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylfuran-2-yl)methylene)indolin-2-one;
(4) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylthiophen-2-yl)methylene)indolin-2-one;
(5) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((5-phenylpyridin-2-yl)methylene)indolin-2-one;
(6) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((4-phenylthiophen-2-yl)methylene)indolin-2-one;
(7) (Z)-1-(4-(2-fluoroethoxy)benzyl)-3-((6-phenylpyridin-2-yl)methylene)indolin-2-one;
(8) 4-((E)-3-((Z)-1-(4-(2-fluoroethoxy)benzyl)-2-oxoindolin-3-ylidene)prop-1-ene -1-yl)benzonitrile;
(9) (Z)-1-(2-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one;
(10) (Z)-1-(3-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one;
(11) (Z)-1-(4-fluorobenzyl)-3-((E)-3-(4-nitrophenyl)allylidene)indolin-2-one;
(12) phenyl (Z)-3-((E)-3-(4-nitrophenyl)allylidene)-2-oxoindoline-1-carboxylate; and
(13) 3-(3-(4-nitrophenyl)allylidene)-1-(phenylsulfonyl)indolin-2-one.
A composition for diagnosing neurodegenerative diseases, comprising the compound of claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
8. The method of claim 7,
The diagnostic composition for diagnosing a neurodegenerative disease, characterized in that it binds to alpha-synuclein.
8. The method of claim 7,
The neurodegenerative disease is a neurodegenerative disease diagnosis composition, characterized in that the degenerative brain disease.
8. The method of claim 7,
The neurodegenerative disease is Parkinson's disease (PD), Parkinson's disease dementia (PDD), Lewy body dementia (DLB), Alzheimer's disease Lewy body variant (LBVAD), multiple system atrophy (MSA), Alzheimer's disease, A composition for diagnosing a neurodegenerative disease, characterized in that it is Pick's disease or tau disease.
administering the composition for diagnosis of claim 7 to a subject; and
Including; measuring a signal for alpha-synuclein;
A method for providing information for diagnosing neurodegenerative diseases.
12. The method of claim 11,
The signal measuring step is a method of providing information for diagnosing a neurodegenerative disease, characterized in that the signal is measured and imaged through a positron emission tomography (PET) device.

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